https://orcid.org/0000-0003-4448-0600
Figures
Abstract
Background
Urogenital schistosomiasis caused by Schistosoma haematobium affects approximately 110 million people globally, with the majority of cases in low- and middle-income countries. Schistosome infections have been shown to impact the host immune system, gene expression, and microbiome composition. Studies have demonstrated variations in pathology between schistosome subspecies. In the case of S. haematobium, infection has been associated with HIV acquisition and bladder cancer. However, the underlying pathophysiology has been understudied compared to other schistosome species. This systematic review comprehensively investigates and assimilates the effects of S. haematobium infection on systemic and local host mucosal immunity, cellular gene expression and microbiome.
Methods
We conducted a systematic review assessing the reported effects of S. haematobium infections and anthelmintic treatment on the immune system, gene expression and microbiome in humans and animal models. This review followed PRISMA guidelines and was registered prospectively in PROSPERO (CRD42022372607). Randomized clinical trials, cohort, cross-sectional, case-control, experimental ex vivo, and animal studies were included. Two reviewers performed screening independently.
Results
We screened 3,177 studies and included 94. S. haematobium was reported to lead to: (i) a mixed immune response with a predominant type 2 immune phenotype, increased T and B regulatory cells, and select pro-inflammatory cytokines; (ii) distinct molecular alterations that would compromise epithelial integrity, such as increased metalloproteinase expression, and promote immunological changes and cellular transformation, specifically upregulation of genes p53 and Bcl-2; and (iii) microbiome dysbiosis in the urinary, intestinal, and genital tracts.
Conclusion
S. haematobium induces distinct alterations in the host’s immune system, molecular profile, and microbiome. This leads to a diverse range of inflammatory and anti-inflammatory responses and impaired integrity of the local mucosal epithelial barrier, elevating the risks of secondary infections. Further, S. haematobium promotes cellular transformation with oncogenic potential and disrupts the microbiome, further influencing the immune system and genetic makeup. Understanding the pathophysiology of these interactions can improve outcomes for the sequelae of this devastating parasitic infection.
Author summary
The parasitic trematode S. haematobium affects 110 million people worldwide. Many studies have described the effects of schistosome infections on humans and animals, but data focusing solely on S. haematobium infections, which cause urogenital schistosomiasis are scarce. Our goal was to evaluate, in a systematic manner, how S. haematobium infection affects the immune system, gene expression and microbiome of the host. These effects are important because they could lead to increased risk of infections, such as HIV, and bladder cancer. We screened 3,177 studies for potential relevance and included 94 of them in this review. Our analysis showed that S. haematobium infection profoundly alters the immune system with a mixed pro-inflammatory and anti-inflammatory response, though with a predominant type 2 immune phenotype and increased regulatory cells. We further found consistent evidence that it impairs local mucosal epithelial barrier integrity, promotes cellular transformation with pro-oncogenic changes in the host, and is associated with microbial alterations in urine, stool, and genital tracts. We discuss how these findings might be interpreted, and the additional research needed, to improve our understanding of S. haematobium pathophysiology and ameliorate the potential sequelae of S. haematobium infection, such as increased viral infections and cancer.
Citation: Mertelsmann AM, Bowers SF, Wright D, Maganga JK, Mazigo HD, Ndhlovu LC, et al. (2024) Effects of Schistosoma haematobium infection and treatment on the systemic and mucosal immune phenotype, gene expression and microbiome: A systematic review. PLoS Negl Trop Dis 18(9): e0012456. https://doi.org/10.1371/journal.pntd.0012456
Editor: Hamed Kalani, Golestan University of Medical Sciences and Health Services, ISLAMIC REPUBLIC OF IRAN
Received: February 7, 2024; Accepted: August 13, 2024; Published: September 9, 2024
Copyright: © 2024 Mertelsmann et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All extracted data used for this systematic review are available in the tables that accompany this manuscript.
Funding: This work was supported by grants for Research Training in Infectious Diseases (5T32 AI 007613; AMM) as well as K24 AI182638 (JAD) and R01 AI 168306 (JAD) from the National Institute of Allergy and Infectious Diseases, the Burroughs Wellcome Fund (BWF 1020043; AMM) and by the Fogarty International Center (D43 TW 011826; JKM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Schistosoma haematobium (Sh) is a parasitic trematode affecting over 110 million people worldwide, disproportionately infecting the world’s poor, who suffer from limited access to clean water and adequate healthcare [1–4]. Adult Sh worms reside predominantly in the veins surrounding the genitourinary tract and release eggs that can become entrapped in genitourinary and reproductive tract organs, causing urogenital schistosomiasis. Schistosome eggs are highly immunogenic and provoke local irritation, inflammation, including granulomatous disease, and fibrosis in the affected tissue leading to chronic symptoms, morbidity, and even organ failure and death. Sh eggs that become lodged in the genital tract can result in female or male genital schistosomiasis, while those in the urinary bladder are categorized as Class A carcinogens, contributing to the development of squamous cell carcinoma in the bladder [2].
Overall, Sh infection leads to approximately 1.5 million disability-adjusted life years (DALYs) lost annually [5]. Not included in these DALY calculations is the increasing evidence suggesting that Sh may additionally contribute to DALYs lost through its association with increased susceptibility to secondary infections, including HIV, HPV, and other sexually transmitted infections [6–12]. Moreover, a previously infected host is not immune against reinfection. Hence, individuals living in endemic areas frequently become reinfected with Sh after successful treatment with praziquantel (PZQ) [13] and can experience additional DALYs lost after reinfection. It remains unknown why the human host does not generate a sterilizing immune response upon reencountering Sh. To date, there is no active vaccine against schistosome infections.
Sh is the leading risk factor for bladder cancer in Sh endemic countries with the highest incidence in the Middle East and Africa [14–16]. Carcinogenesis is a complex process resulting from the accumulation of multiple genetic and epigenetic changes including activation of oncogenes, inactivation or loss of tumor suppressor genes, and alterations in apoptotic gene products. These changes can alter cell proliferation of the urothelium and ultimately lead to urothelial transformation [14]. Sh-associated bladder cancer is largely thought to be due to the combination of changes in gene expression plus the chronic inflammation caused by Sh eggs lodged within the bladder tissue.
Schistosome infection also has been reported to affect composition of the human microbiome across a variety of sites, including genitourinary and gastrointestinal tracts [17]. In addition to infections, other factors that influence the composition of the microbiome include inheritance, mode of birth, diet, including malnutrition and breastfeeding, environment, geographical locations, hormonal changes, antimicrobial exposures, and age-related changes throughout child- and adulthood, among many others [17–23]. A mounting number of studies demonstrate that the microbiome influences the immune system, gene expression, and the development of human pathology including cancer and immune mediated diseases [17,24]. Infections, including parasitic infections such as Sh, may directly affect the composition of the microbiome, and thereby then indirectly affect the host immune phenotype [25,26]. Identifying the links between the microbiome and Sh infection may provide prophylactic or therapeutic tools to improve human health [17].
Critically, within schistosome studies, most knowledge to date is derived from studies focusing on S. mansoni and other Schistosoma species despite Sh being the most prevalent schistosome species worldwide. The pattern of immune response, alterations in gene expression or microbiome likely differ from that in Sh infection because the bladder and reproductive tract represent immunologic, genetic and microbiome environments distinct from that of the gastrointestinal tract or liver, which are affected during infections with other Schistosoma spp. A comprehensive analysis of the pathophysiology leading to increased secondary infections or bladder cancer in the setting of Sh infection has not been performed. Gaining a deeper comprehension of these factors could provide valuable insights into the fundamental processes involved and could provide novel targets for treatment and prevention.
To fill this gap, we conducted a rigorous systematic review that aimed to summarize concisely the current knowledge on immune, gene, and microbiome alterations of the host in the setting of Sh infection. We sought to consolidate data to enhance our understanding of the specific pathology occurring during this parasitic infection, bringing together disparate immunological, molecular, and microbial findings from an array of human and animal studies.
Results
Titles and abstracts of 3,177 studies were screened after which 398 full-text papers were assessed for inclusion. A total of 105 studies were included for extraction, and 11 of the 105 were excluded based on the quality assessment using the Downs and Black checklist (Fig 1) [27]. The 11 studies that did not meet Downs and Black criteria of “fair” or better quality are reported in S1 Table. The remaining 94 studies examined effects of schistosome in 3 categories: immune system (N = 66), gene expression (N = 26), and microbiome (N = 6). Each category is discussed in the following sections. Of note, 4 studies reported both on effects of Sh infection on the immune system and gene expression, and in these instances the studies were each cited in the relevant section.
The host’s immune response to Sh infection is dynamic with an initial Th1 predominate response to the acute Sh infection followed by a shift to a Th2 predominate response that also includes T regulatory cells (Tregs), B regulatory cells (Bregs), and alternatively activated macrophages (AAM) [28]. This shift to a Th2 predominate response is thought to explain why individuals infected with Sh have poorer immune responses to viruses, as well as higher risk of developing bladder cancer [29,30]. Most studies included in this review focused on the consequences of chronic Sh infection in humans living in Sh-endemic regions. We begin with studies focused on the host’s immune system, followed by those on gene expression, and lastly microbiome. Table 1 outlines the organization of the results section.
S. haematobium infection and the host immune system
We begin by examining studies of the systemic immune responses to Sh infection in humans categorized into studies focusing on cytokines (N = 27), innate immunity (N = 12), and adaptive immunity (N = 26). We then review studies of local tissue immune responses to Sh infection in humans separating studies on humans without (N = 6) and with bladder cancer (N = 3), and end with animal and other experimental models (N = 8), and how these fit with observations from humans. For studies that investigated multiple aspects of the immune response, such as cytokines and cellular changes, each is referenced twice in the relevant section.
Systemic Immune Responses to Sh Infection in Humans
The immune response to schistosome infections is multifaceted and, as with other pathogens, is mediated by both cytokines and chemokines. These molecules orchestrate both innate and adaptive immune responses, serving both as regulators and amplifiers, and may directly or indirectly be involved in pathophysiology (Box 1). CD4+ T helper (Th) cells produce a variety of cytokines that can be grouped based on the cells they are associated with, including Th1, Th2, Th17, and regulatory T cell cytokines. The type 2 immune response associated with chronic schistosome and other helminth infections is marked by the release of Th2-linked cytokines, which promote wound healing and worm expulsion while dampening inflammation [31–35].
References to box 1 right below Box 1: [36–56]
Box 1 Overview of cytokines and chemokines
Cytokines and chemokines are a diverse group of molecules that play a crucial role in coordinating both innate and adaptive immune responses to pathogens. They can be categorized into two functional groups: pro- or anti-inflammatory cytokines, although some overlap exists. Cytokines primarily originate from immune cells but can also be generated by non-hematopoietic cells including epithelial cells (36).
Th1 cytokine family:
- Mediate pro-inflammatory responses important for immune responses to pathogens.
- Main cytokines are IFN-γ and TNF- α.
- IFN-γ is a key mediator of inflammation in response to invading pathogens, particularly bacteria and viruses, and is produced by Th1 cells and other cell types including Natural Killer (NK) cells.
Th2 cytokine family:
- Mediate host protection during parasitic infections through enhancing tissue repair, control of inflammation, and helminth expulsion.
- Play significant roles in allergic-inflammatory immune responses, inducing eosinophils, basophils, mast cells, and macrophages.
- Can counteract the Th1 cytokines since they are important for anti-inflammatory response and have a role in dampening excessive inflammation which can lead to uncontrolled tissue damage and/ or autoimmune disease.
- A Th2 dominant immune phenotype counteracts Th1 microbicidal action, and therefore a balance between Th1 and Th2 response is critical.
- Main cytokines are IL-4, IL-5, IL-10, IL-13, and IL-33.
- IL-4 and IL-13 can additionally stimulate fibroblasts to produce collagen.
- IL-13 plays a pro-fibrotic role in the liver during S. mansoni infection.
Overlapping Th1 and Th2 cytokine IL-2:
- IL-2 is predominantly produced by activated CD4+ T cells of either the Th1 or Th2 subset.
- IL-2 is pertinent for the differentiation of CD4+ and CD8+ T cells into effector and memory T cell subsets following antigen-mediated activation, and it is also crucial for the induction and maintenance of Tregs.
- IL-2 can have pro- or anti-inflammatory properties depending on the immune milieu.
Immunosuppressive cytokines:
- IL-10 and TGF-β mediate the immune suppressive function of Tregs and can suppress immune responses of any cell type.
Pro-inflammatory cytokines and chemokines, not part of the Th1 family:
- Important for immune activation against invading pathogens.
- The IL-1 cytokine family, such as IL-1-α and IL-1-β, regulate inflammation by controlling a variety of innate and adaptive immune processes, including acting as leukocytic endogenous mediator and inducing acute-phase proteins and lymphocyte activation factor.
- IL-6 is central to host defense and is a potent inducer of inflammation and acute phase proteins.
- IL-15 is pertinent in anti-pathogen defense by promoting NK and NK T cell development and memory CD8+ T cell function.
- IL-18 is a pleiotropic cytokine functioning as both a potent inducer of inflammation and an inducer of host defense against pathogens regulating innate and acquired immunity.
- IL-17 plays a central role in protective immunity against a variety of bacterial and viral pathogens, and promotes pathogenic cytokines in T cell-mediated autoimmune disease pathology. IL-17 can be produced by multiple cell types, including Th17 cells, CD4+ and CD8+ T cells, NK and NK T cells.
- IL-21 plays a key pro-inflammatory role in stimulating differentiation of B cells, promoting anti-viral and anti-tumor effects of CD8+ T cells, and inducing Th17 cells which secrete IL-17, recruit neutrophils, and link innate and adaptive immunity. It is produced by T cell subsets and NK T cells.
- IL-23, produced by inflammatory myeloid cells, stimulates the development of Th1 cell and Th17 cell responses.
- Monocyte chemoattractant protein-1 (MCP-1) is a key chemokine that regulates migration and infiltration of monocytes/macrophages.
- Monocyte chemoattractant protein-3 (MCP-3) is a pluripotent chemokine and can activate all types of leukocytes.
- Macrophages secrete the chemokine macrophage Inflammatory Protein-1a (MIP-1a) to elicit effects including recruiting inflammatory cells, promoting wound healing, suppressing stem cells, and sustaining effector immune responses.
S. haematobium infection and systemic host chemokine and cytokine immune responses in humans
Systemic cytokine responses to Sh infection.
In total, 16 studies assessed systemic cytokine changes in Sh infections (Table 2), with an additional 10 studies investigating cytokine responses following PZQ treatment (Table 3). Many of these studies used various antigens (Box 2) to stimulate cells and quantify responses.
Box 2 Overview of immune cell stimulants
Peripheral blood mononuclear cells (PBMCs) release cytokines through a receptor-mediated cascade, which depends on both the cell type and the specific antigen encountered. Researchers frequently utilize the potential to activate PBMCs using different antigens to examine the cytokine response following antigen stimulation in cells from study participants or in experimental models. This assessment can help ascertain the immune system’s functional response to stimulation by specific or general pathogens.
Using different schistosome antigens from different stages of the parasite life cycle, such as worm antigen or egg antigen, is important because each antigen can elicit a different immune response in the host. This variety helps researchers and clinicians better understand the dynamics of the infection and develop more effective diagnostic tools and treatments. Different antigens may also have varying levels of sensitivity and specificity, making them useful in different contexts or stages of the disease.
Schistosome antigens
- Soluble egg antigen (SEA)
- Adult worm antigen (AWA)
- Soluble adult worm antigen preparation (SWAP)
- Schistosome phosphatidylserine (PS)
Mycobacterium tuberculosis antigen
- Purified protein derivate (PPD)
Generic antigens
- Glycolipid (GL)
- Lipopolysaccharide (LPS)
- Phytohemagglutinin (PHA)
- Pokeweed mitogen (PWM)
- Concanavalin A (ConA)
- TLR-2 stimulants: FSL-1 and Pam3
- TLR-9 stimulants: CpG and R848
In an earlier study examining human peripheral blood mononuclear cells (PBMCs) from two Zimbabwean children with Sh eggs in urine, stimulation with schistosome antigens AWA and SEA led to elevated IL-4 and IL-5 levels and higher ratios of these cytokines to IFN-γ, compared to 2 children without Sh infection [57].
In a similar study, whole blood of 40 children with Sh infection responded to schistosome antigen AWA and SEA stimulation with production of higher levels of Th1 and Th2 cytokines including IFN-γ, IL-2, IL-5, IL-10, and TNF-α, compared to 39 children without Sh infection [58]. PZQ treatment increased the production of IFN-γ, IL-2, IL-5, IL-10, and TNF-α in whole blood of previously infected children in response to schistosome antigens AWA and SEA and also increased production of the immunosuppressive cytokine, IL-10, but not the other cytokines, in response to generic antigen stimulation [58].
Circulating IL-10 and INF-γ were elevated in serum of Malian children with Sh infection, similarly to the stimulation study above alongside IL-4 and IL-6, compared to individuals without Sh infection. In contrast to the stimulation study above, there were no differences seen in IL-2, TNF-α, and IL-5 [59].
A PBMC stimulation study in Gabonese children with Sh infection revealed increased macrophage-released chemokines (MCP1-MCAF/MIP-1α) and Th2-related cytokines such as IL-5, IL-10, and IL-13, with a simultaneous decrease of pro-inflammatory cytokines (IFN-γ, TNF-α) compared to children without Sh infection [60]. Factors like soil-transmitted helminths (STH) [59] and filarial co-infections [60] were similarly distributed among the groups. Nevertheless, co-infections in these two studies might have influenced the results.
A factor analysis study in Zimbabwean children similarly found that a composite variable representing IL-5, IL-10, and IFN-γ increased with age and was positively associated with current Sh infection as well as with past Sh exposure determined by the presence of Sh-specific antibodies in serum [61]. Of note, pro-inflammatory IFN-γ results were not reported separately.
A study from an area of Gabon by Van der Kleij et al [62] found varying responses of IL-8 and IL-10 depending on the specific antigen used for stimulation, with lower levels after lipopolysaccharide (LPS) and schistosomal phosphatidylserine stimulation in individuals with Sh infection versus individuals without Sh infection, but higher IL-8 in response to the schistosome glycolipid (GL) antigen. Notably, PBMC stimulation in the presence of a toll-like receptor 2 (TLR2)-blocking antibody decreased the cytokine responses induced by schistosomal phosphatidylserine for IL-10, IL-8, and IL-6 [62]. Similarly, Meurs et al [63] found elevated IL-10 and TNF-α levels in Gabonese children with Sh infection after stimulation of PBMCs with TLR2-specific and schistosome antigens SEA and AWA, compared to children without Sh infection [63]. Meurs et al [63] selected children with Sh infection and children without infection from the same area, while the participants without Sh infection in van der Kleij’s study were recruited from a nonendemic neighboring semi-urban area and were more likely truly Sh-uninfected [62]. Both studies suggest that the immune response to Sh operates via TLR ligand signaling [62,63].
Five studies demonstrated higher systemic levels of IL-6, which integrates the immune defense against infections [47], in individuals with Sh infection versus individuals without Sh infection [59,62,64–66]. Serum IL-6 levels were also higher among those with active infections than those with positive Sh serologies without active infection [64] and in individuals with Sh infection with urinary tract pathology [67], supporting a likely role of IL-6 in the pathogenesis of urogenital schistosomiasis.
IL-10 has both immunomodulatory and anti-inflammatory properties [43] that appear to be important during Sh infection. Two studies showed that blockade of IL-10 with monoclonal antibodies of isolated PBMCs led to increased IFN-γ production and enhanced lymphocyte proliferation following stimulation with schistosome antigens SEA and AWA in people with Sh infection [68,69]. A Zimbabwean study assessing the cytokine response to AWA stimulation of PBMCs revealed that IL-10 was most strongly correlated with Sh infection intensity, and that participants with high intensity infection also tended to produce lower IL-5 levels [70]. The addition of IL-10 blocking antibodies to PBMCs isolated from individuals with Sh infection led to an increase in IL-5 response [70], suggesting that IL-10 may be exerting its modulatory effect by down-regulating IL-5 production. In contrast, in a large Zimbabwean cohort, IL-10 levels were lower in plasma of those with Sh infection, and subsequently increased when measured 6 weeks after PZQ treatment [71]. Similarly, serum of pre-school aged children with Sh infection had lower IL-10 levels compared to individuals without Sh infection, and had higher pro-inflammatory IL-6 and TNF-α [66]. In conclusion, the diverse findings on IL-10 in Sh infection highlight its complex role in the immunopathogenesis of Sh infection, with studies indicating both modulatory and anti-inflammatory effects on cytokine responses, and dynamic changes in systemic levels following treatment.
In general, Sh infection appears to result in systemic alterations in an array of cytokines and chemokines, with most studies describing an increase in the Th2 cytokines IL-4, IL-5, and IL-10, as well as Th1 cytokines IL-6 and IFN-γ (Fig 2). This mixed pro-inflammatory and anti-inflammatory response observed in Sh infection highlights the complexity of immune responses to this parasite. While the prevailing notion of a type 2 immune state is confirmed here, this synthesis further identifies a co-existing pro-inflammatory environment in chronic Sh infection.
Overview of results of the 14 studies assessing systemic cytokine trends at baseline or after antigen stimulation in individuals with Sh infection individuals of the total 27 studies investigating systemic cytokines in this review. Data on counts of the reported cytokines were collected. Sh infection was associated with an increased release of systemic Th2 cytokines (IL-4, IL-5, Il-10): Four out of 5 studies (80%) reported an increase in IL-4, 4 out of 5 studies (80%) reported an increase in IL-5 and 8 out of 12 studies (67%) showed an increase in IL-10 in the setting of Sh infection. The regulatory cytokine IL-2, which is pertinent for induction and maintenance of T cell subsets including Tregs, was elevated in 2 out of 3 studies (67%) but also unchanged in 2 out of the 3 studies (67%) in individuals with Sh infection depending on which specific antigen was used. Increased proinflammatory cytokines (IL-6, IFN-γ, IL-23) were associated with Sh infection: Five out of 6 studies (83%) reported increased IL-6 levels, 4 out of 6 (67%) reported an increase in IFN-γ and one study reported an increase in IL-23 in the setting of Sh infection. One study (100%) reported an increase in macrophage related inflammatory cytokines MCP1-MCAF and 2 studies (100%) an increase of MIP-1α in individuals with Sh infection. Graph developed using GraphPad Prism.
Variation in cytokine responses to Sh by age.
A Zimbabwean study found elevated circulating cytokines IL-2, IL-10 and IL-23 and lower IL-4, IL-13, and IL-21 in individuals with Sh infection aged 6–60 years compared to individuals without Sh infection [72]. Cytokine patterns varied with age, with increasing IL-4 and IL-10 levels, and decreasing IL-5 levels in plasma, in those with Sh infection as age increased [72]. Notably, all participants had detectable parasite-specific antibodies, particularly IgM reflecting recent schistosome exposure, but only those with Sh eggs in urine were considered individuals with Sh infection [72]. Given the suboptimal sensitivity of urine microscopy, particularly among females, some infected participants may have been misclassified as individuals without Sh infection [73]. In agreement with studies above, this cross-sectional study shows that individuals with Sh infection have mixed Th1 /Th2 systemic cytokines across the lifespan, while Th2 cytokines dominated in uninfected people who were Sh-seropositive, indicating past exposure to Sh [72].
In another Zimbabwean study, responses to antigen stimulation varied by age of the person with Sh infection. Adolescents with Sh infection aged 15–16 years showed the highest IL-4 and IL-5 responses to SWAP stimulation of whole blood, and similarly IL-5 peaked in this age group after ConA stimulation [70]. IFN-γ levels peaked at age 13–14 years in response to generic antigen stimulation, but not SWAP stimulation. IL-10 was highest in serum of children in response to SWAP but was lower in participants older than 14 years and decreased further with age. In contrast, stimulation with a ConA led to IL-10 levels that were lowest in children aged 6–12 years with an increase starting in aged 13–14 years, peak at age 15–16 years, and a decline in older participants though those over age 16 years still had higher levels than children aged 6–12 years [66].
In younger children, TNF-α was higher in serum of pre-school aged children with Sh infection, while IL-6 was only found to be higher in children aged 11–12 years in a second study of children aged 2–19 years [65]. Consistent with the Mutapi study above, Bustinduy et al also reported increased IL-10 in 13–14-year-olds with Sh infection compared to their age-matched counterparts without Sh infection [65]. These studies uniformly emphasize the age-related variability in cytokine response.
Variation in cytokine responses to Sh infection by genitourinary pathology and infection intensity.
A study in Kenyan individuals with Sh infection and concomitant bladder pathology assessed by ultrasonography reported a 12-fold higher serum TNF-α production at baseline and 13-fold higher production in response to schistosome and purified protein derivative antigen stimulation than in persons with Sh infection without bladder pathology [74]. This implies that pro-inflammatory TNF-α may contribute to the genitourinary damage during Sh infection [74]. Conversely, in a Gabonese cohort, Sh urinary egg burden correlated inversely with IFN-γ levels in PBMCs after AWA or SEA stimulation from individuals who developed Sh reinfection two years after PZQ treatment, suggesting a lower pro-inflammatory immune environment in those with the highest Sh burden [75].
Variation in cytokine responses to Sh infection by sex.
Sex hormones affect immune function and lead to differences in the immune phenotype and disease response between women and men [76,77]. Furthermore, sex differences in immune response also vary over the lifespan and with reproductive status, which is particularly pronounced in women with fluctuations due to the menstrual cycle as well as during pregnancy, breastfeeding, and menopause [78–82]. In a Senegalese cohort, women with Sh infection produced lower levels of TNF-α and IFN-γ following SEA stimulation of PBMCs, while they produced more TGF-β and IL-10 and had higher expression of the apoptosis marker nuclear matrix protein, compared to men with Sh infection with comparable egg burdens and schistosome circulating anodic antigen (CAA) levels [83]. Although this was a small study consisting of 23 participants, these findings suggest that women with Sh infection produce less pro-inflammatory Th1 cytokines, and more anti-inflammatory IL-10 than men. Women’s higher production of TGF-β, which is mainly produced by fibroblast and epithelial cells, may have either pro- or anti-inflammatory immune effects [44,45].
S. haematobium infection and the systemic host cytokine immune response in humans after praziquantel therapy.
Wilson et al conducted two cohort studies comparing cytokine response in plasma of Malian individuals with Sh infection at time-points 24 hours after PZQ treatment and 9 weeks post-treatment [84,85]. In one cohort, systemic IL-5 increased 24 hours post-PZQ, around the time of adult worm death, but decreased at 9 weeks, while IL-33 was higher at 9 weeks post-treatment [84]. Further, intracellular eosinophil IL-5 and -13 both increased at 9 weeks, and sST2 (IL-33 receptor) was associated with intracellular IL-13, suggesting a role of IL-33 in the human response to Sh infection [84]. In the second Malian cohort, IL-5 increased 24 hours post-treatment, as did IL-13 in plasma [85]. Eosinophils increased at 9 weeks, associated with an IL-5 boost and pre-treatment infection intensities. Of note, 13.9% of a subset of participants who provided stool were found to have concomitant S. mansoni infection [85]. Together, these studies suggest a time dependent cytokine response post-PZQ treatment likely due to an initial surge of Sh antigen release from dying adult worms which diminishes over time [86].
Cytokine production after PZQ treatment also appears to differ between children and adults and with specific antigen stimulation. In those with prior Sh infection, stimulation of whole blood cultures with egg-specific antigen SEA led to a pronounced pro-inflammatory cytokine pattern with elevated TNF-α, IL-6, IL-8, IFN-γ, IL-12p70, IL-21, and IL-23 6 weeks post-treatment, while cercariae-specific antigen CAP led to a mixed pattern of pro- and anti-inflammatory cytokines with elevated IL-8, IL-13, and IL-10 and lower IL-23 [87]. Stimulation with adult worm-specific antigen WWH evoked the lowest inflammatory responses, reflected by low IL-6 and TNF-α and elevated IL-21 which can have pro- and anti-inflammatory properties [87], likely reflecting the low immunogenic potential of adult worms while they are living in the bloodstream [1]. In the same study, a schistosome egg-specific pro-inflammatory cytokine profile and adult worm-specific Th-2 and Th-17-associated cytokine profiles were associated with lower risk of Sh reinfection. Overall, Bourke et al report increased inflammatory, Th1 and Th17 cytokines after PZQ after egg-specific stimulation, while Th2 cytokines were unchanged [87].
Mduluza et al [88] assessed cytokine responses in PBMCs in response to schistosome antigens SEA and SWAP in two Zimbabwean cohorts residing in a high or low Sh endemic area and with or without Sh infection. Individuals with Sh infection from both areas produced higher levels of IFN-γ while IL-4 and IL-5 were only elevated in individuals with Sh infection from the high endemic area compared to those without Sh infection [88]. PZQ treatment decreased the IFN-γ response, while IL-4 and IL-5 remained elevated in both cohorts compared to persons without Sh infection. At enrollment there was no difference in cytokine ratios, while ratios increased towards a Th2 phenotype 12 and 18 months post-PZQ therapy in those who remained uninfected versus those who became reinfected in areas of both high and low endemicity. Specifically, those who became reinfected showed a more pronounced Th1 phenotype [88]. A weakness of this study was lack of data analysis by host sex, despite its potential impact on the immune response. This study highlights a mixed immune response to Sh infection and the putative role of Th2 immunity in Sh resistance.
The pro-inflammatory chemokine macrophage inflammatory protein-1 (MIP-1α) was elevated in plasma of individuals with Sh infection compared to Zimbabwean adults without infection, correlated with urinary Sh egg counts, and decreased 3-months post-PZQ therapy [89]. While MIP-1α can enhance expression of HIV entry molecules CCR5 and CXCR4 on HIV target cells such as CD4+ T cells [90], MIP-1α did not vary by HIV infection status in this Zimbabwean study. This is pertinent since some studies have shown associations between HIV and Sh infections [6–8,12].
In a Gabonese cohort, Sh reinfection was associated with lower IL-4, IL-5, and IFN-γ levels after schistosome stimulation with AWA and SEA of PBMCs compared to those who remained uninfected 2-years after PZQ treatment, suggesting that these lower cytokine levels could possibly predict risk for reinfection [75]. Of note, this study did not document sex distribution, nor was age clearly defined. Similarly, PBMCs of Egyptian male children with previous Sh infection who had become reinfected 12–18 months post-PZQ had lower IL-4 and IL-5 production in response to schistosome antigen SWAP, compared to male children who were not reinfected. Depletion of CD4+ T cells abrogated this response, suggesting that they are the source of these cytokines [91]. These two studies, combined with the Mduluza study discussed above, consistently suggest that an impaired type 2 response post-treatment is associated with susceptibility to reinfection. In contrast, in an Egyptian cohort of male children and adults, higher IL-4 levels were found in individuals who became reinfected compared to those who did not. Further, IL-4 was associated with higher egg burden and remained persistently elevated even after treatment [92].
A study in Gabon showed that plasma cytokine levels varied in individuals with prior Sh infection after two years depending on if they received treatment and how many PZQ treatment courses they received [93]. Individuals who were untreated and those who received a single dose of PZQ had higher IL-5 levels and a trend towards higher IL-13 levels in response to schistosome antigen AWA compared to people without infection at the two-year follow up who had not been treated. Individuals who cleared infection after a single dose of PZQ had the highest IFN-γ, suggesting a potential protective effect of this cytokine. Moreover, repeated treatment led to high IL-5 and low IFN-γ production in response to AWA in comparison to no or single treatment, but was not protective against Sh reinfection. The authors concluded that overall cytokine response to AWA remained altered in previously persons with Sh infection despite treatment [93]. On further analysis, the authors also reported that elevated levels of schistosome-specific IL-10 preceded Sh reinfection, and high levels of IL-5 were associated with hematuria.
Overall synthesis of cytokine studies in Sh infection.
Overall, Sh appears to evoke aspects of both pro- and anti-inflammatory responses in the host. Nine Sh studies align with the prevailing hypothesis that a type 2 immune response predominates in most helminth infections [33–35,94], accompanied by an increase in Th2 related cytokines and decrease in Th1 cytokines [57,60,62,63,70,75,92].
Notably, ten other studies showed evidence of pro-inflammatory responses in individuals with Sh infection compared to individuals without Sh infection, including higher IL-6 [59,62,64–66,87], IFN-γ [59,70,88], and pro-inflammatory chemokine MIP-1α [89]. Supporting the complexity of this area, seven other studies have documented a hybrid response which includes pro- and anti-inflammatory cytokine responses [58,59,61,63,66,70,72].
Cytokine responses to PZQ treatment are also complex. Taken together, most studies support a conclusion that a strong Th2 immune response is triggered by dying Sh worms [85]. In the ensuing time after treatment, most studies indicate further immune changes including a continued increase of Th2 cytokines [71,84,85,88,93] and decrease in pro-inflammatory cytokines and chemokines including IFN-γ [88,93] and MIP-1α [89]. It remains to be determined how long this Th2 predominant immune phenotype persists and if it ever reverts to a more balanced Th1/ Th2 phenotype in the absence of further helminth infections. Importantly, higher levels of Th2 cytokines have been linked to reduced likelihood of reinfection, while lower levels correlated with reinfection in two separate studies [75,91]. Additional studies to understand the protective role and duration of Th2 cytokines could have high relevance for infection prevention and elimination.
A caveat to these overall cytokine studies in Sh is the complexity of assessing immune responses in Sh infection. It is not possible to elucidate how long the individual has been infected, nor to account fully for other contributing factors, including age [65,72], sex [83], and infection history both past and current [95]. Further complicating comparisons across studies, varied chemokine and cytokine responses to stimulation by different antigens in different environments may also affect results [62,87]. Indeed, many studies included participants with other parasitic infections, mostly other helminths [59,60,62,69,89] and malaria [63]. These other infectious stimuli likely alter the individual’s immune response, and therefore many findings cannot be attributed to Sh infection alone.
Finally, there are likely significant differences between systemic and local mucosal cytokine production or secretion during Sh infection in the genitourinary tract. It will be critical to increase our understanding of the local tissue immune response to genitourinary tissue damage, which also might increase the risk of acquiring other infections such as HIV [96] or establishing a permissive environment for the development of bladder cancer [30]. The single study that correlated cytokines with organ damage found higher systemic levels of the pro-inflammatory cytokine TNF-α in children with Sh infection with genitourinary pathology assessed by ultrasonography [74]. Additional studies to link cytokine activity with genitourinary pathology are critically needed to understand the tissue immune milieu and strategies to prevent mucosal pathology. It remains to be determined whether a pro-inflammatory immune response occurs in reaction to Sh-induced tissue damage or if it plays a causative role in tissue damage. Longitudinal studies to assess associations between organ pathology and immune responses, as well as effects post-treatment, would provide much needed additional insight.
Accessing tissue samples is more challenging and costly than obtaining peripheral blood, which is likely why most studies concentrate on systemic cytokine responses while trying to relate them to local mucosal processes in the genitourinary tissue, instead of studying cytokine responses directly at the affected organ. Some of these mucosal studies do exist and are discussed in more detail below.
S. haematobium infection and the host innate immune response in the systemic circulation in humans
A total of 12 studies have assessed changes in innate immune cells seen in Sh infections (Table 4).
Dendritic cells.
Antigen-presenting dendritic cells (DCs) are among the first immune cells to contact invading parasites, and they respond by sending specific or generic signals that polarize T cell responses [97,98]. DCs can be divided into CD11c+ myeloid DCs (mDCs) and CD123+ plasmacytoid DCs (pDCs) [99], both of which are immature DCs with distinct roles. MDCs are believed to preferentially induce T cell responses to invading pathogens while pDCs are presumed to contribute to innate antiviral responses and self-antigen tolerance [100,101]. Since DCs are the first to encounter the parasite Sh in all its forms, including eggs that migrate into tissue, it has been hypothesized that they may play a part in the known T cell hyporesponsiveness occurring in chronic Sh infections [102].
Two studies have reported that individuals with Sh infection had lower mDCs [102,103] and pDCs [102] than individuals without Sh infection. In adults in Gabon, both mDCs and pDCs had lower HLA-DR expression in blood [102], while in children and adults in Zimbabwe no differences were seen in activation marker expression (HLA-DR or CD86) [103]. Interestingly, when the Zimbabwean analysis was restricted only to children aged 5–10 years, those with Sh infection had more mDCs and lower levels of HLA-DR expression than children without Sh infection [103]. On further analysis, both pDCs and mDCs from the Gabonese adults with Sh infection had reduced capacity to respond to toll-like-receptor (TLR) ligands with decreased effector marker expression (CD40, CD80, HLA-DR, and CCR7) compared to adults without Sh infection, which was more pronounced in mDCs with decreased cytokine production (IL-6, IL-12, TNF-α, IL-10) and mitogen-activated protein kinase signaling [102]. Co-culture of mDCs isolated from these adults with Sh infection with naïve T cells led to lower production of IL-4 and IFN-γ and expression of the activation marker CD25, emphasizing a potential role of DCs functional impairment in mediating T cell hyporesponsiveness in chronic Sh infection [102].
Overall, there appears to be an age-related pattern in DCs immune response to Sh infection which may reflect the chronicity of the infection, with lower levels of mDCs particularly in adults with Sh infection. These lower circulating mDCs levels might correlate with impaired mDCs response in chronic Sh infection, and/or could represent increased recruitment of this cell population to the target sites of Sh infection. Such a decrease in systemic mDCs in blood could have further implications such as an impairment of immune response against concurrent bacterial or viral infections, for which the host immune response which relies on antigen presenting cells to induce pertinent T cell responses [104].
Monocytes.
Monocytes are circulating white blood cells with phagocytotic capabilities. They can differentiate into macrophages or dendritic cells after migration from blood into tissue, and can express the activation marker, CD16 [105], linking humoral and innate cellular immune responses [106]. Three studies in total have examined circulating monocytes in Sh infection.
In South African women with Sh infection, circulating monocytes were increased and had higher expression of the HIV entry molecule CCR5, compared to women without Sh infection. However, no difference was observed in monocyte CCR5 expression in cells collected from the cervix in the same women [107].
In two other studies, no difference was seen in circulating monocytes between individuals with Sh infection and participants without Sh infection [62,108], but one reported lower CD16 expression in systemic monocytes of participants with Sh infection aged 16 and above from Zimbabwe [108]. This lower CD16 expression could lead to weaker innate immune responses in those with Sh infection. Further, healthy participants without Sh infection had a positive correlation between CD16 monocyte expression and IgG and IgG1 levels against schistosome antigen SEA and WWH, which has been associated with resistance to infection [109,110].
The disparity in monocyte findings of the above studies could be attributed to variations in sex distributions among the two study populations. The study in Zimbabwe consisted of both sexes (60% females) with a mean age 16.4 years, while all 44 female participants were above 14 years of age in the South African study. Given evidence of sex-dependent immune responses to schistosome infection [83], these differences in study populations could explain the higher monocyte levels seen in the Kleppa study.
Granulocytes.
Eosinophils have been well established to play a critical effector cell role in the immune response against helminth infections and are a cardinal sign of parasitic infection [111]. In S. mansoni infections, eosinophils can kill cercariae and adult worms in a degranulation- and IgE-dependent manner [112]. Five studies examined eosinophils in Sh infection.
People with Sh infection in Senegal who had sonographic genitourinary pathology exhibited higher eosinophil and granulocyte levels compared to individuals with Sh infection without pathology and/or people without Sh infection [113]. This might indicate the heightened immune responses in the presence of ongoing Sh-mediated genitourinary pathology, implying a pathogenic role of eosinophils in chronic inflammation and tissue damage. No differences were reported in neutrophils between Senegalese adults with and without Sh infection and children in this study [113] nor in Zimbabwean pre-school aged children [66].
The effect of PZQ treatment on eosinophil counts is inconclusive. One study reported no change 9 weeks post-treatment in 47 Malian children and adults [84] while another study by the same group showed increased eosinophils 9 weeks post-treatment in 326 Malian children and adults [85]. Higher eosinophil counts post-treatment were positively associated with reinfection 2 years after treatment [85]. In contrast, a study from The Gambia reported that children with prior Sh infection who remained uninfected one year after PZQ treatment had higher circulating eosinophils than children who became reinfected [114]. Of note, the study in Mali did not report eosinophil counts at the 2-year follow up, and hence used the preceding eosinophilia as a predictive factor for reinfection. In contrast, the study in The Gambia reported the association between Sh reinfection and low eosinophil count at 12 months after PZQ-treatment and therefore assessed the association of low eosinophils and Sh infection at the same time point.
Innate lymphoid cells.
Innate lymphoid cells (ILC) are a newly discovered immune population that mainly reside at mucosal sites and play a critical role in mucosal immunology. ILCs can be categorized into three distinct groups that mirror the Th1, Th2, and Th17 cell subsets. ILC1, mirroring Th1, produce IFN-γ upon activation to promote host defense against pathogens, particularly of viral and bacterial origin. ILC3s, similar to Th17 cells, promote tissue repair, contain commensal bacteria, and protect against pathogenic bacteria [115,116].
Similar to Th2 cells, ILC2s are elicited during parasitic infections and regulate tissue immunity and inflammation by producing Th2 cytokines (IL-4, IL-5- and IL-13) to recruit and activate eosinophils and alternatively activated macrophages. In this way, ILC2s mediate anti-helminth immunity and can also be associated with allergic reactions with increased presence in bronchial secretions of asthmatic patients [116].
Thus far only Nausch et al [117] have investigated the impact of Sh infection on ILCs. People with Sh infection had lower circulating ILC2 frequencies versus people without Sh infection, a difference that was most profound in the youngest aged 6–9 years. ILC2 frequencies also negatively correlated with infection intensity assessed by urinary egg burden. Upon re-evaluation of 12 of the 36 previously participants with Sh infection 6 weeks after PZQ therapy, ILC2 frequencies had increased to frequencies comparable to those reported in participants without Sh infection. Th2 cytokines (IL-4, IL-5 and IL-13) were only associated with infection intensity in the oldest age group aged 14–18 years of age [117]. This observed decrease in systemic ILC2 in individuals with Sh infection may be due to increased migration to affected tissues, initiating a local immune response leading to fewer ILC2s detectable in blood. Conclusions about the effects of PZQ are limited by the follow-up to one third of individuals who were initially infected.
Systemic adaptive immunity
In total, 25 studies have assessed changes in adaptive immune cells seen in Sh infections (Tables 5–7).
S. haematobium infection and host systemic lymphocytic proliferation in humans
In three studies, lymphocyte proliferation was similar among individuals with Sh infection and without Sh infection in response to stimulation by generic antigens [57,88,118] and by schistosome antigens AWA and SEA before and after PZQ chemotherapy [119]. In contrast, in a Gabonese cohort, lymphocyte proliferation was reduced after schistosome antigen stimulation with AWA but not egg antigen SEA [75] in peripheral blood from those with versus without Sh infection. Further, a cohort study in Zimbabwe showed changes in lymphocyte proliferation in individuals with Sh infection depending on Sh endemicity levels in their area of residence. Those living in a low-endemic Sh area had higher lymphocyte proliferation in comparison to those without Sh infection from the same area, while the opposite was seen in participants living in a Sh high-endemic area. These proliferative responses did not change in either population 12 and 18 months after PZQ treatment and no difference was seen in response to generic antigen stimulation with PHA [88]. This study reflects the complexity of assessing immune responses from different populations living in different environments and again highlights the multiple factors that evidently influence an individual’s immune response to Sh infection. These factors may include the individual’s exposure history beyond parasitic infections which can impact their immune status and memory, as well as overall health, microbiome, sex, and age.
S. haematobium infection and host T cell immune response in the systemic circulation in humans
T cells play a crucial role in the immune response against invading pathogens, including schistosome infections [28]. Both CD4+ and CD8+ T cells are critical in protecting the host against pathogens, with CD8+ T cells being particularly important in fighting intracellular pathogens including viruses and bacteria, as well as in eliminating malignant cells in cancer [120].
Some effector CD4+ and CD8+ T cells will develop into memory T-cells, which after antigen encounter can rapidly expand upon re-encountering the pathogen. Memory T cells can be further subdivided into multiple subpopulations including central memory and effector memory T cells [121]. Central memory T cells reside mainly in lymphoid tissue and blood, while effector memory T cells are present in blood and traffic through peripheral organs [122].
CD4+ T cells have a wide array of functions that rely on specialization through functional polarization. CD4+ T cell subsets addressed in manuscripts included in this review include Th1, Th2, Th17 and Tregs. The characteristic immune alteration induced by helminth infections is an increase in Th2 cells and a decrease in Th1 cells [33]. Th17 cells provide protection and immunity against extracellular pathogens, especially at mucosal tissue, and play a role in the pathogenesis of multiple chronic inflammatory diseases [52]. Tregs, characterized by high expression of IL-2 receptor α chain, CD25, and Foxp3+, are important in the maintenance of peripheral immune tolerance [36]. Each of these subsets will be discussed in detail in this section.
CD4+ and CD8+ T cells.
Overall frequencies of T cells largely do not appear to differ between individuals with and without Sh infection [62], though multiple studies have reported altered composition of T cell subsets. Analysis of T cell subsets in people with Sh infection compared to individuals without infection showed lower CD4+ (257 versus 681) and higher CD8+ (210 versus 114) T cells/μL in a Nigerian cohort without HIV infection [123]. Notably, no information was given on where the 44 negative control subjects were recruited from, which might shed light on the major differences in CD4+ T cell numbers between the groups. Further, decreased CD4+ to CD8+ T cell ratios were associated with heavy Sh infection in children and adults (urine eggs >50/mL) compared to ratios in those with light infections. The authors reported that lowest ratios were found in infected adults, followed by children with Sh infection, as compared to the control group without infection, demonstrating an age dependent pattern in host immune response to Sh [123]. Contrastingly, Nausch et al [103] did not find a difference in circulating CD4+ or CD8+ T cell frequencies in persons with or without Sh infection in their HIV-uninfected Zimbabwean study population which at least is partially attributable to lower infection intensity in comparison to the Nigerian cohort. A South African study also did not find a difference in mean CD4 cell count in women with or without Sh infection or clinical evidence of FGS, regardless of HIV status [124].
In a cohort of 235 men and women who did not have HIV infection in Tanzania, 82.1% were helminth infected and only 9.3% were individuals with Sh infection [125]. No difference was seen in HLA-DR+ T cell frequencies comparing individuals without Sh infection, which included participants with and without other helminth infections, to individuals with Sh infection. Notably, the small number of individuals with Sh infection in this cohort warrants caution when interpreting these results.
Memory T cells.
Observed associations of Sh infection with CD4+ and CD8+ T cell memory subsets vary. In Gabonese schoolchildren, the frequency of CD4+ effector memory T cells increased in children with Sh infection post-PZQ treatment, but there was no difference at baseline between CD4+ effector or memory T cells [58]. In contrast, Nausch et al found that children and adults with Sh infection at baseline had lower CD4+ memory and effector memory T cells than uninfected [126]. Stimulation of CD4+ memory T cells with generic antigen led to increased activation, assessed by HLA-DR+ expression, and increased IFN-γ secretion in individuals without but not in individuals with Sh infection, highlighting possible functional impairment of CD4+ memory T cells in the setting of Sh infection. Neither group produced Th2 cytokines (IL-4, IL-5) in response to stimulation [126]. In the same study, PZQ treatment of those with Sh infection led to decreased CD4+ T cells and CD4+ memory T cells, increased proliferation of naïve thymic CD31+CD4+ T cells, and no changes in CD8+ T cell and CD8+ memory T cell frequencies at 6 weeks [126]. From these studies together, it appears that CD4+ memory T cells are an important source of IFN-γ production that are lower in persons with Sh infection and may further decrease at 6 weeks after treatment [126]. Follow-up beyond 6 weeks was not performed, rendering longer-term effects of PZQ on CD4+ memory T cells unknown.
Th1 and Th2 cells.
Two studies examined Th1 and Th2 CD4+ T cell populations in Sh infection. PBMCs in response to stimulation with AWA or SEA of individuals with Sh infection in Gabon had lower T cell proliferation and lower IL-4 production, both of which were reversed after PZQ treatment [127]. Notably, IL-4 production was abolished by CD4+ T cell depletion, but not by CD8+ T cell depletion [127].
Further supporting these findings, PBMCs of children with Sh infection from Swaziland stimulated with Mycobacterium tuberculosis (MTB) antigens developed a lower Th1:Th2 cytokine ratio, determined by the ratio of IFN-γ and TNF-α to IL-4, than children without Sh infection, and their CD4+ T cells produced greater MTB specific IL-4, with a decrease in MTB specific IFN-γ and TNF-α [128]. Six months after chemotherapy, the Th1:Th2 ratio increased and MTB specific IFN-γ and TNF-α producing CD4+ T cells were detected, but all of these remained lower than in children without Sh infection [128]. Overall, this study suggested an Sh-associated decrease in MTB specific Th1 CD4+ T cell function, with associated increases in Th2 cells, raising concern for a compromised immune response to MTB in individuals with Sh infection which may not be entirely reversed after treatment.
Th17 cells.
Only one study to date reported on Th17 cells in the setting of Sh infection. In Senegalese children, Th17 cells (CD3+CD4+RORγt+) were elevated in individuals with Sh infection with sonographic evidence of genitourinary pathology than individuals with Sh infection without pathology and matched individuals without Sh infection [113]. Although IL-17 producing CD4+ T cells tended to be higher overall in individuals with versus without Sh infection, the greatest differences was noted between individuals with Sh infection with genitourinary pathology compared to those with Sh infection but without pathology [113]. Investigators also reported that both individuals with and without Sh infection with genitourinary pathology had higher ratios of IL17+ to IL-10+ CD4+ T cells compared to those individuals with Sh infection without genitourinary pathology. These cytokine findings accord with their finding that participants with pathology had a higher ratio of Th17 cells to Tregs, primarily due to the presence of higher levels of Th17 cells [113]. Though a single study, this work importantly highlights the potential role of Th17 cells in Sh-induced tissue pathology, a role that has been suggested in S. mansoni and S. japonicum infection as well [129–131].
T regulatory cells.
A total of 4 studies investigated Treg populations in Sh infection. Most studies (3 out of 4) demonstrated an increase in systemic Tregs individuals with Sh infection compared to uninfected [58,119,132], and both studies that assessed the effect of PZQ treatment reported that Tregs decreased after anti-helminthic treatment [58,119].
Mbow et al, described above, found no differences in Tregs between those with Sh and genitourinary pathology, Sh without pathology, and without Sh infection [113].
In addition to finding that Tregs normalized after PZQ treatment in Gabonese children infected with Sh, Schmiedel and colleagues also reported that both Th1 (IL-17, IFN-γ, TNF) and Th2 (IL-5, -10, -13) cytokine responses to schistosome and MTB antigen stimulation increased post-treatment [119]. Furthermore, ex vivo depletion of Tregs led to an increase in both Th1 and Th2 cytokine responses to both schistosome and MTB antigen stimulations in samples of individuals with Sh infection before treatment and after treatment. This demonstrates the persistent suppressive capacity of Tregs, even following a decrease in their numbers post-PZQ [119].
Of note, Nausch et al found that Tregs increased with infection intensity in those below 14 years of age, but the opposite was seen in individuals above age 14 with decreasing Tregs in those with higher Sh burden [132]. Overall children with Sh infection (< 14 years of age) had higher Treg proportions than children without infection, while Treg proportions were lower in the people with versus without infection older than 14 years. Notably, in these older patients, the burden of Sh was lower, possibly reflecting the development of acquired anti-schistosome immunity with age that could contribute to a diminished regulatory response [132].
Together, these studies suggest that Tregs play a major and potentially age-dependent role in the immune response to Sh infection. During youth, when individuals have less acquired immunity, higher Treg proportions may be crucial in dampening excessive inflammation and downstream pathology. As host protective acquired immunity develops with age, the necessity for an enhanced regulatory phenotype that was seen in children may decrease.
T cell marker expression.
A total of four studies examined T cell surface markers in Sh infection. CD4+ T cells that expressed the inhibitory protein Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) were decreased in individuals with Sh infection compared to persons without infection. Notably, CTLA-4+CD4+ T cells increased with age in those individuals with Sh infection but did not change with age in those without Sh infection [126].
In a study in Zimbabwe, expression of the regulatory molecule CD200 receptor (CD200R) [133], was higher on CD4+ T cells of persons with Sh infection. Further, CD200R-expressing cells appeared to belong predominantly to the Th2 lineage, with IL-4 secreting CD4+ T cells almost all expressing CD200R while IFN-γ secreting CD4+ T cells expressed significantly less CD200R [134]. CD200R expression on CD4+ T cells also correlated positively with infection intensity [134]. These studies indicating increased expression of immune regulatory proteins CTLA-4 and CD200R suggest a state of immune suppression, potentially influencing the effectiveness of the immune response to other pathogens, particularly viral.
A study in South Africa reported a negative correlation between CD3ζ expression on T cells, which promotes downstream immune responses including cell proliferation [135], and infection intensity, while noting a positive correlation between CD3ζ expression and PBMC proliferation in response to schistosome antigen stimulation with SWAP [136]. The authors hypothesized that CD3ζ downregulation could be a mechanism for immune dysregulation that is observed during chronic Sh infection in humans without changes in the absolute numbers of T cells. It is thought that immune dysregulation in addition to local mucosal damage may contribute to the increased risk of HIV acquisition seen in some studies of women with Sh infection [8,12,137]. In accordance with this, women with FGS (defined as presence of sandy patches on colposcopy exam) were found to have increased expression of the HIV entry molecule CCR5 on both systemic circulating and local cervical CD4+ T cells and CD14+ monocytes, which decreased on both cell populations post-PZQ treatment in both compartments [107].
Overall synthesis of T cell studies in Sh infection.
Together, these 14 studies indicate that systemic T cells play a crucial role in immunity against Sh infections. It appears that Sh infection leads to increases in T cell populations which augment suppressive and regenerative functions on the immune system, particularly upregulating Th2 immune responses, Tregs, and Th17 cells. These increases appear to be accompanied by decreases in CD4+ memory and effector, Th1, and IFN-γ producing T cell populations. These systemic immune changes may further explain why individuals with Sh infection may be more susceptible to a variety of different pathogens [29].
S. haematobium infection and host B cell immune response in the systemic circulation in humans
In contrast to the abundance of data examining T cells in Sh infection, only 6 studies have investigated B cells. One study showed no difference in B cell frequencies between individuals with and without Sh infection [62]. Two others reported that activated B cells, memory B cells, and Bregs are higher during Sh infection. Sh infection has been associated with an increase in memory B cells in response to schistosome antigens SWAP and SEA in a Malian cohort [138] and at baseline in a Gabonese cohort [139] compared to the uninfected individuals in each cohort. At a functional level, B cells were shown to have increased expression of the activation marker CD23, similar to the rising soluble CD23 levels with Sh infection intensity described by Rujeni et al [140]. B cells from children with Sh infection further had lower proliferation and intracellular B cell TNF-α production compared to children without Sh infection [139]. Notably, PZQ treatment led to an increase in naive B cells, decrease in memory B cells apart from TNF-α producing memory B cells, and restoration of B cell responsiveness [139].
In a different study in Gabon, children with Sh infection had elevated CD1dhi B cells, a Breg subpopulation, compared to children without Sh infection, with elevated IL-10 production by B cells after stimulation, which was reduced post-PZQ treatment [141]. Stimulation of B cells with antigens PMA, ionomycin, brefeldin A (PIB) and LPS from individuals with Sh infection led to induction of multiple different Breg subpopulations compared to individuals without Sh infection. Co-culture of CD1hi Bregs from persons with Sh infection together with CD4+ T cells led to a decrease in IFN-γ, IL-4 and IL-17 production by CD4+ T cells, and increased frequencies of Tregs and IL-10+ T cells [142]. Depletion of CD1hi Bregs in the CD4+ T cell co-culture restored IL-10+ T cell numbers, while Tregs remained the same [142].
Together, these data suggest that Sh infection induces changes in the B cell compartment including an increase in Bregs, which may play a key role in inducing T cell hyporesponsiveness and altering memory B cell subsets. Bregs have been shown to suppress Th1 immune response [143] and suppress viral specific CD8+ T cell responses in individuals with HIV, hepatitis B, and hepatitis C [144,145]. Increases in Bregs and alterations in memory B cells subsets might offer at least one mechanism by which Sh infection is associated with increased acquisition of certain viral infections and worse outcomes in individuals with Sh and viral co-infections.
S. haematobium infection and the host local genitourinary tissue immune response in humans
Six studies have examined local mucosal immune responses in Sh infection in humans (Table 8).
Schistosome eggs are highly immunogenic and cause a strong local immune response [94]. Eggs migrating through tissue can become entrapped in organs of the genitourinary system, including not only the bladder but also reproductive organs, causing female and male genital schistosomiasis (FGS, MGS) [35,146] with symptoms dependent on egg location [147]. In MGS eggs become entrapped in male genital organs including seminal vesicles, prostate gland, and vas deferens causing local inflammatory responses with subsequent tissue damage and scarring [146]. Similarly, in FGS eggs become entrapped in female reproductive organs, including the cervix and vagina, leading to tissue damage which can cause a wide array of symptoms including discharge, pain, and infertility [35].
A recent study in Zambia by Sturt et al [148] evaluated cervical immune changes in women with definite FGS (positive vaginal or cervical Sh PCR), probable FGS (urinary Sh eggs or CAA with one of four typical cervical findings), and those without FGS (absence of these findings) [148]. In the primary analysis, cervical lavage (CVL) specimens of women with definite FGS demonstrated elevated IL-5 compared to women without FGS, without differences in other genital cytokines. In exploratory analyses, women with high Sh FGS burden, defined as two or more positive genital Sh PCRs, had higher concentrations of Th2 cytokines IL-4, IL-5, and IL-13 as well as inflammatory cytokines IL-1 α and IL-15 compared to women without FGS [148]. In a second exploratory analysis, after adjustment for multiple comparisons, cervical Th2 cytokines IL-5 and pro-inflammatory cytokine TNF-α were associated with FGS. Of note, a study in Tanzania found decreased IL-15 levels in CVL samples of women infected with Sh as diagnosed by presence of elevated CAA plus Sh eggs in urine, but no difference in any of the other 26 measured cytokines [149].
The diverse results of local cervical cytokine expressions might reflect the different study designs among women with Sh infection. While the Tanzania study evaluated cervical cytokines in all women with Sh infection (therefore possibly including some women who had parasitic infection but without genital tract involvement), the Zambia study’s strict requirement of PCR-confirmed Sh infection in the genital tract likely restricted that study population to a younger age group and/or only those with the greatest genital tract abnormalities. It is also possible that the presence of FGS plus STIs, particularly trichomoniasis, which tended to be higher in women with Sh infection (p = 0.08), could explain why women with FGS in Zambia had both type 2 and pro-inflammatory immune responses since trichomoniasis can elicit a pro-inflammatory response [150]. Furthermore, analysis of the local mucosal cervical immune phenotype is challenging since there are numerous additional potential confounders, including physiologic changes occurring throughout the menstrual cycle [78,151,152], effect of recent pregnancy [153] or current breastfeeding [79], recent sexual contact, and vaginal cleansing practices [154,155], which were not consistently assessed in the above studies. Nevertheless, studies investigating local mucosal changes are critically important to enhance our understanding of the local cervical immune changes occurring during Sh and why women with FGS or Sh infections may have impaired ability to control genital viral infections such as HIV and HPV [6,11].
In a study of blood and cervical samples of South African women, monocytes were increased in the systemic circulation of women with FGS defined as sandy patches seen on the cervix [107]. While no difference was found in cervical CD4+ T cells or monocytes or their CCR5 expression of the same cohort of women between women with and without FGS, PZQ treatment led to a significant decrease in CD14+ monocytes in both blood and cervical samples. PZQ treatment also led to a decrease in CCR5 expression on CD4+ T cells in blood and cervical samples [107]. The findings of this study provide evidence in favor of the hypothesis that FGS may raise the risk of HIV acquisition, potentially by altering local mucosal cell populations to increase HIV target cell populations.
Jourdan et al [156] performed cervical biopsies to evaluate HIV target cell populations in the female genital tract of Malawian women with Sh infection who had Sh eggs in urine [156]. This study found that the density of CD4+ T cells was higher in cervical biopsies containing viable or calcified Sh ova, compared to cervical biopsies without ova from both endemic and non-endemic Norwegian controls. No difference was observed in the density of CD4+ T cells in tissue comparing viable ova (N = 2) and calcified ova (N = 11), or in Malawian women without presence of Sh ova on biopsies. Importantly, there was no difference in densities of CD4+ T cells in tissue with calcified ova and in biopsies from Norwegian women with chronic cervicitis. The density of CD68+ macrophages was higher in specimens with viable ova than specimens with calcified ova, specimens from Malawian women without ova, and specimens from Norwegian women with healthy cervical tissue [156].
Relatively complementary findings have been reported from one study in men. Men with Sh infection, determined by presence of Sh eggs in urine or semen, were found to have increased leukocytospermia compared to men without Sh infection, which was more pronounced in men with moderate to high egg burden. Similarly, increased seminal eosinophils and IL-10 were seen in men with high egg burden when compared to men without Sh infection [157]. Even though only 5 of the 240 individuals in this study had detectable IL-4 in semen, all had Sh infection with high burden of infection, as measured by concentration of seminal eggs. In the same study, younger men had higher egg secretion in both urine and semen compared to men above age 39 [157], possibly reflecting partial immunity to the helminth with increasing age.
Similarly, Sh disease burden was reported to impact urinary cytokine expression in children with higher Sh urinary egg burden, who had higher urinary levels of IL-6 and lower IL-10, compared to children with lower disease burden [67]. Both these cytokine changes were also associated with hematuria and were not seen in peripheral blood. Furthermore, when the same children were assessed by ultrasound for possible genitourinary pathology, those found to have genitourinary pathology had increased geometric mean intensity (GMI) of IL-6 and lower GMI of TNF-α [67]. This study by Njaanake et al complements genital tract findings and demonstrates that the burden of disease, measured here by concentration of urinary eggs, appears to be a key mediator of extent of tissue pathology and the local mucosal immune milieu.
Overall synthesis of immune studies in Sh infection on local tissue.
Collectively, the above studies demonstrate that chronic Sh infection with ova in genitourinary tissues leads to a local inflammatory reaction consisting of Th2 associated cytokines, pro-inflammatory cytokines, and an increased cellular immune response to viable ova comprised of CD4+ T cells and CD68+ macrophages. The intensity of Sh infection appears to be important, with a higher burden of disease triggering a stronger local immune response. Furthermore, individuals with local Sh mediated damage, such as hematuria or pathology seen on genitourinary ultrasound or cervical exam, appear to have higher rates of immune changes compared to individuals without such tissue damage.
S. haematobium infection and the local tissue host immune response in patients with bladder cancer
Three studies focused on tissue immune responses in people with bladder cancer and Sh infection (Table 9).
There are many studies investigating the local gene changes that occur in patients suffering from Sh-associated bladder cancer [158], but only three analyzing changes in the immune phenotype.
In one study, immunohistochemical analysis of bladder biopsies showed increased numbers of cells expressing: (1) signal transducer and activator of transcription 4 (STAT4), which plays a key role in the differentiation of Th1 cells [159], and (2) Foxp3, a marker for Tregs, in patients with Sh-associated bladder cancer, compared to patients with bladder cancer without Sh infection. Cancer patients with Sh infection also had fewer cells expressing GATA3, which is important to generate an optimal Th2 immune response [160]. Two other studies similarly reported that Sh-associated bladder cancer was associated with fewer CD8+ T cells on bladder biopsy specimens, compared to patients with bladder cancer without Sh infection [161,162].
PBMCs from individuals with Sh-associated bladder cancer had lower CD3+ T cells compared to Sh-unassociated bladder cancer, while CD4+ T cells, Tregs, and Bregs were higher in Sh-unassociated bladder cancer compared to healthy controls who did not have Sh infection or bladder cancer [162]. Moreover, investigators reported fewer total CD3+ T cells and T helper cells in these individuals and further demonstrated that individuals with Sh-associated bladder cancer had higher bladder tissue levels of IL-1β, IL-6, and TNF-α, which correlated positively with egg burden, compared to individuals with bladder cancer but without Sh infection [162]. In contrast to this, El-Salahy et al did not find a difference in IL-6 between bladder specimens of patients with bladder cancer or chronic cystitis with or without Sh infection, although they did not report the findings in Sh-associated bladder cancer versus cystitis separately, which could obscure potential differences between the two groups [163].
Overall synthesis of Sh infection and the local tissue host immune response in patients with bladder cancer.
The finding that two of these three studies that individuals with Sh infection with bladder cancer have a shift towards a Th2 predominant phenotype, even compared to individuals with bladder cancer without Sh infection, suggests a possible role for Sh-induced immune alterations the carcinogenesis of Sh. Parasite-induced immune dysregulation that leads to a compromise in Th1 immunity might enable malignancy to thrive more easily, and explain at least partially why Sh infection is associated with bladder cancer, since a Th1 immune response is required for cancer control [164].
Experimental models to study effects of Sh infection on immune response
Eight studies have assessed host immune response to Sh using experimental ex vivo organ culture and animal models (Table 10).
Animal models that recapitulate natural Sh egg deposition during infection must be achieved by injection of eggs and/or cercariae into tissue, which is at least a partial limitation, given that this technique disrupts the normal epithelium in a way that does not occur during natural human infection. To date, no animal model exists that mimics the human cycle of Sh infection, which is likely due to its specificity to humans, unlike S. mansoni which can infect rodents to provide a useful animal model [165]. Sh infection models in mice have shown that the response to Sh is dynamic and involves multiple different immune cell populations as well as a broad range of cytokines and chemokines. All support the well-established type 2 hypothesis in helminth infections, marked by an increase in Th2 type cytokines such as IL-4, IL-5, and IL-13 in mucosal tissue of the bladder in response to Sh eggs [166–168] as well as systemically and in lymphoid tissue [166,168,169].
IFN-γ levels appear to vary depending on tissue type and time since infection. Specific differences between murine models of Sh likely affect the observed immunological consequences. For example, introduction of Sh cercariae via intraabdominal injection only leads to egg disposition in the liver and intestines, as opposed to human infections where egg disposition is mainly in the genitourinary system [169]. Studies have reported an elevation of IFN-γ in lymphoid tissue [169] in the first weeks after intraabdominal cercarial injection, followed by a decline, and an increase in systemic IFN-γ [168] when Sh eggs were directly injected into the bladder wall in combination with intraabdominal cercarial injection. In contrast, Fu et al found decreased systemic IFN-γ levels after Sh bladder egg injection alone without additional cercarial infection [170]. Interestingly, leptin was increased in the local bladder tissue in mice injected with both Sh eggs and cercariae, which has immunomodulatory properties to shift the immune response towards a Th1 phenotype, which is observed during the first weeks of infection before cercariae have developed into adult worms [168,171].
Local Sh egg injection into the bladder or vaginal walls of mice leads to a change in local immune cell populations, with subsequent rises in macrophages, B cells, neutrophils, and eosinophils [166,167,172]. Accompanying these rises, overall local tissue reductions of T cells as well as NKT cells have been reported, with NKT cell specific IFN-γ production and decreased expression of CD1d on antigen presenting cells [167]. These findings align with the decrease in IFN-γ levels seen by Fu et al [170] after direct injections of Sh eggs into the bladder wall.
In this experimental mouse model, Fu et al used intraperitoneal injections to cause a local depletion of macrophages and thereby demonstrated their profound role in the immune response to Sh eggs [170]. After macrophage depletion, mice that underwent direct injection of Sh eggs into the bladder wall developed disrupted cellular and tissue architecture of the resulting granulomata and decreased percentages of macrophages, AAMs, eosinophils, B cells, and T cells overall compared to mice whose macrophages were not disrupted. These mice also developed lower bladder tissue levels of eotaxin, and higher levels of IL-1a, IL-3, MCP-1, MCP-3, and MIP-1a. Furthermore, macrophage depletion led to decreases in systemic levels of numerous cytokines, including cytokines associated with leukocyte recruitment (RANTES, MIP-1a, and eotaxin), Th1 (IFN-γ), Th2 (IL-4 and IL-13), and Th17 (IL-17 and IL-23) T cell responses, and vascular endothelial growth factor (VEGF). Of note, this increase in VEGF in Sh-infected mice was consistent with findings from a second study [166]. Overall, this work demonstrated the wide-ranging role of macrophages in inducing a breadth of local and systemic immune responses during Sh infection in mice [170]. This is not unexpected since different macrophage subtypes are involved distinct types of immune responses. Notably, this study of macrophage depletion depleted all subtypes simultaneously and did not examine differential effects of various subtype depletions [173].
In another murine model of FGS, mice were injected with Sh eggs into the vaginal wall which led to an influx of different cell populations at different time points after Sh egg injections. In the first two weeks post-injection, vaginal mucosa exhibited a rise of T cells as well as RANTES. This was followed by a decline in T cells and RANTES, while macrophages became elevated starting 4 weeks after infection and persisting through week 8. Both T cells and macrophages in the vaginal wall were found to have increased expression of HIV entry molecules including CXCR4 and CCR5 from week 2 onward [172].
Nair et al used a murine knockout (KO) model to demonstrate a role of Metastasis-associated protein (MTA1) in Sh infection after direct percutaneous inoculation by immersion of the tail in water containing cercariae which typically leads to egg disposition in the liver [174]. MTA1 is a chromatin-bound protein that plays a role in mediating host inflammatory responses to components of virus and bacterial infectious products [175]. Mta1 KO mice had lower Sh infectious burden with fewer worms and eggs in the liver compared to wild-type mice and lower circulating CD4 mRNA, TNF-α and eosinophils. Mta1 KO mice also had a distinct cytokine profile in the liver, with an aberrant Th1/ Th2 cytokine profile marked by elevations in both Th1 and Th2 cytokines (IFN-γ, IL-2, IL-4, IL-5, IL-10) [174]. According to these findings, MTA1 appears to play a significant role in the egg mediated inflammatory response to murine Sh infection in the liver.
Acute infection with Sh cercariae was found to cause local immune response within hours of cercarial skin penetration, as evident in organ cultures of circumcised foreskin of healthy men infected with Sh cercariae by direct injection [176]. The analyzed cytokine expression in the foreskin from 1 to 8 hours after infection demonstrated an increase of IL-1a and IL-10 mRNA levels 4 and 8hrs after Sh infection versus controls, while macro gene array analysis showed decreased IL-6, IL-8, IL-15 and IL-18, elevated IL-1β, IL-2, IL-10 and TNF-α, and no change in IL-1 α in infected foreskin tissue.
Overall synthesis of experimental immune studies in Sh infection.
Together, these experimental models demonstrate that there is a complex and dynamic immune response to Sh infection, consisting of an initial mixed Th1 and Th2 response followed by a dominant Th2 response in the local tissue and systemically [168,170]. These animal findings largely cohere with human data and provide greater insight into the immune response. These studies extend insights into the response over time after initial infection and allow for an enhanced understanding of the biological mechanisms through in vivo experiments, such as the involvement of Mta1 and macrophages in the immunopathology of Sh, and through heightened control in comparison to human studies. However, it is important to highlight that there is still no animal model of Sh infection that completely resembles human Sh infection. To mimic human disease in which worms reside in the genitourinary tract and eggs are deposited in the genitourinary system, eggs must be directly injected into the genitourinary tissue since infection with cercariae only leads to egg disposition in liver and intestines in animals. Despite the importance of animal studies, it is highly likely that the presence of adult worms in humans with egg secretion and proteolytic passage of the eggs through tissue to reach their destination in the bladder cause a far more complex immune response that so far cannot be replicated in animal models.
S. haematobium infection and host gene expression
In the next section, we turn to described effects of Sh infection on gene and protein expression. A total of 26 studies are included in this section. Twelve studies investigated the local tissue host gene and protein expression changes in Sh-associated bladder cancer and three studies investigated noncancerous Sh infection. Four studies focused on changes in systemic gene expression in individuals with Sh infection and seven studies focused on Sh experimental techniques in cell cultures and animal models.
S. haematobium infection and host mucosal gene and protein expression in S. haematobium associated bladder cancer
In total, 12 studies have compared gene expression in people with bladder cancer with and without Sh infection (Table 11).
Bladder carcinoma typically involves alterations in several genes including the tumor suppressor genes p53, p63, retinoblastoma (Rb), and p16, as well as oncogenes c-myc and epithelial growth factor (EGFR), which are all associated with aggressive and invasive bladder carcinomas. Antiapoptotic protein BCL-2 [177] and the proliferative protein ki-67 [178] have also been linked to aggressive bladder carcinoma.
Three studies have quantified p53 expression in bladder carcinomas. Two studies reported higher expression of p53 in individuals with Sh-associated bladder cancer compared to Sh-unassociated bladder cancer, in bladder specimens [179] and urine [180], suggesting tumor aggressiveness [177,181,182]. In contrast, p53 was not more overexpressed in Sh-associated bladder cancer specimens than in Sh-unassociated bladder cancer specimens in patients from Egypt [183].
In addition to increased p53 expression, Abdulamir et al reported more frequent expression of BCL-2, c-myc, and Rb in bladder biopsy specimens in Sh-associated bladder cancer in Jordan and Syria [179]. Further, Sh-associated cancer exhibited higher grade and increased invasiveness compared to Sh-unassociated bladder cancer [179]. Bernardo et al found, in addition to increased p53 expression, increased estrogen receptor (ER) α expression in Angolese Sh-associated bladder cancer specimens, which was absent in Sh-unassociated bladder cancer specimens from Angolese and Portuguese patients without Sh exposure. Furthermore, ERα expression was positively correlated with numbers of Sh eggs found in bladder cancer specimens [180]. This increased ERα expression in Sh-associated bladder cancer appears to be unique to the association with Sh infection.
Bladder tissue obtained from radical cystectomies of patients with squamous cell carcinoma in Egypt showed higher expression of the proto-oncogene c-KIT in carcinomas associated with Sh, compared to those not associated with Sh [184]. Furthermore, in the same study, c-KIT expression was also associated with increased tumor size and distant metastasis, arguing that Sh-associated squamous cell carcinoma is more aggressive than Sh-unassociated squamous cell carcinoma [184]. Another Egyptian study found increased p63 in bladder specimens from people with Sh infection with either chronic cystitis or bladder cancer, suggesting that p63 may be linked more generally to Sh-associated tissue disease rather than specifically to cancer [185]. The above four studies highlight that there are distinct differences in the genetic alterations occurring in Sh-related and unrelated bladder cancer [179,180,184,185].
The death receptor Fas (Apo1/CD95) and FasL system is recognized as a major pathway for the induction of apoptosis in vivo, and its blockade plays a critical role in carcinogenesis and progression in several malignancies [186]. Expression of both Fas and FasL was higher in Sh-associated bladder cancer samples than in non-Sh-associated bladder cancer [187]. A potential mechanism is that the acquisition of the functional FasL may induce apoptosis of anti-tumor T lymphocytes during tumor progression. Neither this study nor another found a change in cytokeratin-19 or -20 expression, which have also been associated with bladder cancer, in Sh-associated versus non-associated bladder cancers [163,187].
The enzymes glutathione S-transferases (GST) and glutathione reductase (GR) are a group of inducible enzymes that support detoxification of many different xenobiotics in mammals. Decrease in GST activity and depletion of glutathione (GSH) levels might potentiate the harmful effects of environmental toxins and carcinogens. Consequently, inducers of GSTs are generally considered to be protective compounds against malignancy [188]. A study conducted in Egypt showed that bladder biopsies of individuals with Sh-associated bladder cancer had higher levels of GST as well as decrease in GR and GSH levels. All of these changes would contribute to poorer ability to attenuate environmental toxins and carcinogens, thereby increasing multiple carcinogen-metabolizing enzymes and free radicals and enhancing carcinogenic potential [189].
Cyclooxygenase-2 (COX-2) is an important pro-inflammatory mediator that is present only at low levels during normal conditions but is robustly induced after an insult such as infection or injury [190]. COX-2 expression, together with inducible nitric oxide synthetase (iNOS) which is associated with oxidative stress [191], was higher in bladder biopsies of Sh-associated versus non-Sh-associated bladder cancer patients and in Sh-associated versus non-Sh-associated benign cystitis [192]. A second study similarly reported higher iNOS in Sh-associated bladder cancer tissue [193,194]. Further, this second study found that levels of 8-hydroxy-2-deoxyguanosine (8-OHdG), a marker for DNA damage [195], were increased in tissue of Sh-associated bladder cancer [194]. Biopsies from Sh-associated cancers also had higher expression of DNA-repair genes, including 8-OHdG and apurinic/apyrimidinic endonuclease, and higher formation levels of single strand DNA [194]. Together, these two studies suggest a strong correlation between Sh infection and increased levels of oxidative stress accompanied by continuous DNA damage, necessitating increased DNA repair.
Proteome profiling of urine samples of 9 participants with either Sh-associated bladder inflammation or cancer, or Sh-unassociated bladder cancer, showed an increase in proteins involved in immunity and inflammation as well as negative regulation of endopeptidase activity in Sh-associated bladder cancer. Samples from participants with Sh infection, whether inflammation or cancer, had higher urinary proteins associated with oxidative stress and immune defense systems responsible for microbicide activity, in comparison to Sh-uninfected participants with bladder cancer [196].
Overall synthesis of gene expression studies in Sh infection and bladder cancer.
Together, the above studies highlight the unique genetic changes associated with immune activation, chronic inflammation, and fibrosis in Sh infection. These changes in gene expression may both predispose to carcinogenesis, and then be further modulated by carcinogenesis, resulting in high-grade and aggressive bladder cancer.
S. haematobium infection and mucosal gene expression of the host
A total of three studies have compared local mucosal gene expression in people with and without Sh infection, without cancer (Table 12).
110 genes were differently expressed in cervical cytobrush samples from Tanzanian women with or without Sh infection. In this study, the transcript count showed that 7 out of 8 genes had lower expression in women with Sh infection. The top altered canonical pathway was “inhibition of matrix metalloproteinases”, with increased expression of metalloproteinases (MMP) MMP-2 and MMP-16 as well as decreased expression of tissue inhibitor MMP-3 (TIMP-3), followed by: “colorectal cancer metastasis signaling”, “glioma invasiveness signaling”, and “bladder cancer signaling.” Together, these results suggest that Sh infection is associated with compromised integrity of the genital mucosal barrier in women with Sh infection, and with genetic changes that could promote malignancy [149].
In a study of genetic changes in liver biopsies of patients with Sh and/or S. mansoni infection, biopsies with fibrosis were compared to normal liver biopsies without schistosomiasis. People with Sh mono-infection or Sh/ S. mansoni co-infection had elevated aflatoxin B1 adducts, known hepatic carcinogens [197], and those with Sh mono-infection had more p53 mutations than S. mansoni mono-infected or healthy controls [198]. Together, this suggests that entrapped Sh eggs can lead to pro-carcinogenic changes in the liver in addition to the bladder, while S. mansoni eggs appear to be less carcinogenic.
Methylated RASSF1A and TIMP3 genes, both tumor suppressor genes that have been documented to be elevated in bladder cancer and damage [199], were found to be increased in the urine of individuals with Sh infection who had ultrasound-confirmed bladder damage compared to individuals without Sh infection from the same endemic region in Ghana. DNA methylation of two tumor suppressor genes in urine samples that correlated with Sh induced bladder damage suggested that these might be early signs of bladder transformation that occurred in advance of bladder cancer. If this is correct, these methylated genes could serve as predictive biomarkers in individuals with Sh infection for genitourinary damage and cancer risk.
S. haematobium infection and systemic gene expression in blood
Four studies compared systemic gene expression changes by examining whole blood transcriptional profiles in people with and without Sh infection (Table 13).
In a study conducted in Ghana, mRNA expression of TLR2 was downregulated in whole blood of children with Sh infection compared to those without infection [200]. TLR2 recognizes a wide range of pathogenic molecules and is able to induce a Th1 or Th2 immune response [201] and suppressor of cytokine signaling 3 (SOCS-3), which leads to increased Th2 differentiation and altered IL-2 and IFN signaling. The children infected with helminths, including Sh, had lower frequencies of reactions to dust hermits tested by a mite allergen skin prick test [200] in line with prior studies describing decreased allergic reactions in S. mansoni [202] and helminth infected individuals [203]. Further, the lower allergic reactions in those with Sh were accompanied by lower expression of genes associated with the innate immune system, particularly TLR2 and SOCS-3, which trigger allergic inflammation and IFN response. The impaired IFN response in Sh infection may connect Sh infections not only to a reduced risk of atopy but also to an increased risk of viral infections. This research underscores the unique genetic modulation in Sh infection compared to other helminth infections, emphasizing the importance of understanding the unique characteristics of Sh infection apart from other helminths.
In the previously mentioned study by Labuda et al [58], analysis of the whole blood transcriptome identified 320 differentially expressed genes between 8 Gabonese schoolchildren with Sh infection and 9 without Sh infection, while after PZQ treatment only 4 genes remained differently expressed between the two groups. Gene ontology analysis showed increase in processes associated with cell adhesion in those with Sh infection, whereas PZQ treatment resulted in downregulation of immune responses and cell cycle genes [58]. Furthermore, children with Sh infection had reduced gene signatures for NK cells, DCs, and monocytes, while T cell activation, miotic cell cycles, and pathways involved in oxidative phosphorylation and the citrate/TCA cycle were upregulated. All these oncologic changes suggest induction and maintenance of type 2 immunity [204]. The differences in NK cell signatures and mitotic cell cycle were reversed, and immune activation was overall reduced post-PZQ therapy [58]. Chemotherapy with PZQ also led to upregulation of oxidative phosphorylation and downregulated citrate/TCA cycle, and transcriptional factors for the induction of T cell anergy, including early growth response genes 2 and 3 [58]. Overall, this study indicated multiple changes occurring on a molecular level during Sh infection, consistent with increased cell adhesion and type 2 immunity, which was only partly reversed by PZQ treatment. A limitation of this study is that schoolchildren in both groups also had different prevalence of co-infections with other parasites, which included Plasmodium falciparum, Ascaris lumbricoides, Trichuris trichiura, and hookworm. These co-infections may have influenced the transcriptome profiling of the individuals.
Transcriptional profiles of PBMCs in a Tanzanian cohort showed 383 differentially expressed genes between those with and those without Sh infection, of which 270 genes correlated with CAA values. Interestingly, 29 genes were differentially expressed in men with Sh infection, and 2,142 genes women with Sh infection compared to men and women without infection, respectively [205]. Ingenuity pathway analysis identified altered networks associated with development, cell death and survival, cell signaling, and immunologic disease pathways. Genes which were differentially expressed were associated with p53 signaling, including BCL-2, as well as with histone transcription. Furthermore, expression of multiple genes involved in Notch signaling were increased. This study emphasizes not only changes in genetic expression occurring during Sh infection, but furthermore the importance of host factors, such as sex, that impact this host response to Sh.
Individuals with Sh infection (n = 15) who had lived in Spain for 6 months after immigrating from West Africa had increased VEGF expression on whole blood ELISA testing, which has been associated with bladder cancer, compared to those without Sh infection (n = 18) [206]. While the study’s size limits interpretability, the reported rise in VEGF aligns with findings by Fu et al in Sh-infected mice [166], despite the two prior studies not reporting changes in VEGF expression.
Overall synthesis of systemic gene expression studies in Sh infection.
Together, these gene expression data triangulate and substantiate immune changes observed in studies described above. Both groups of studies indicate changes in type 2 immune phenotype, cell signaling, and survival. Besides the known role of sex differences in the immune response, this literature demonstrates that they also affect genetic modulation in response to Sh infection [205]. This underscores the importance of evaluating host response to infection, including host gene expression, by sex.
Experimental models to study S. haematobium infection and host gene, protein expression, and cell cycle dynamics
Seven studies assessed changes in gene expression associated with Sh utilizing experimental models (Table 14).
A study that assessed activity of GST and GR, and levels of GSH, in multiple organs of transdermally infected hamsters with Sh cercariae with successive sampling for up to 10 weeks post infection showed substantial variation depending on the specific organ as well as the time point after infection [207]. There was no change in uninfected hamsters used as a control at each time point. Activity of GST and GR, as well as GSH levels, increased in the studied organs (liver, bladder, spleen, and lung) at some time point post-infection followed by a partial decrease in the liver and bladder. Only the bladder was found to have a decrease in all three (GST and GR activity, and GSH levels) at the last two stages post-infection. Free radicals remained elevated throughout only in the bladder and kidneys. This demonstrated reduction, particularly of GST activity in the bladder during the later stages of Sh infection, and the associated high levels of free radicals, could provide new insights on Sh induced damage to the bladder and other organs [207].
A mouse study investigated effects of injection of Sh eggs directly into the bladder, versus administration of carcinogen N-butyl n-(4-hydroxylbutyl) nitrosamine through drinking water, which is a known carcinogen, versus eggs plus carcinogen. Mice injected with eggs alone had higher expression of activation marker Ki-67 in lymphocytes and p53 after 4 weeks, and Ki-67 remained elevated at 12 weeks. The eggs plus carcinogen group was the only group with increased expression of Ki-67 in urothelial cells, which was found at week 20. Additionally, this group had elevated p53 expression and vimentin at week 20, while E-cadherin was decreased [208]. This suggests that Sh eggs in bladder tissue led to upregulation of lymphocyte proliferation, yet led to genetic changes worrisome for urothelial transformation only when combined with a known carcinogen.
In a study by Botelho and colleagues [209], half of the mice that were exposed to Sh total antigen, infused directly into the bladder through the urethra, developed histologically dysplastic bladder lesions. Almost all Sh-exposed mice were found to have inflammatory infiltrates and elevated lymphocytes in the bladder tissue, compared to control mice. Furthermore, 20% of the dysplastic bladder lesions were found to have a KRAS gene mutation [209], which is commonly found in bladder cancers [210]. This implies that Sh presence may upregulate KRAS, increasing the risk of developing bladder cancer. It is unclear if this is specific to Sh or a general response in bladder epithelium to carcinogens, including Sh antigens. A control group with a non-Sh carcinogen-induced bladder transformation would have added insight into differences between Sh-mediated and other carcinogenic mediated bladder lesions.
Proteome profiling of bladders of mice that underwent local injection of Sh eggs into the bladder walls showed differential expression of proteins important for immune and inflammatory responses, increased protein translation or turnover, oxidative stress responses, reduced epithelial barrier integrity, and carcinogenic pathways 7 days after eggs were administered [211]. Overall, the bladders of mice with Sh egg injection were found to have 45 differentially expressed proteins (25 upregulated and 20 downregulated) compared to mice that were injected with uninfected hamster liver and intestine tissue extract into their bladder walls as controls.
Microarray-based comparisons of Sh egg versus control vehicle-injected bladders demonstrated differential gene transcription changes over time with 301 changes at 1 week, a peak of 1571 changes at 3 weeks, and 1012 genetic changes by 5 weeks post egg injection [212]. The presence of Sh eggs in the bladder of mice prompted an increase of genes related to inflammation, particularly cytokine-cytokine receptor pathways dominated by Th2 cytokines, and macrophage function-associated gene transcription. Simultaneously, extracellular matrix remodeling-related gene transcription was differentially modulated over time, and pathways analysis pointed to differential transcription of multiple genes implicated in carcinogenesis [212]. Sh eggs also dampened transcription of epithelial barrier genes, such as tight junctions and uroplakins, which may cause compromises in the urothelial barrier [212]. These findings indicate that the presence of Sh eggs in bladder tissue not only provoke a strong local, largely Th2 immune response, but also enhance genes associated with extracellular matrix remodeling suggesting fibrosis development, compromised epithelial barrier integrity, and carcinogenesis.
Sh egg bladder injection in wild-type mice increased mitotic cells, with a higher proportion of urothelial cells in S phase and G2/M phase [213], which was also seen after Sh total antigen administration to a Chinese hamster ovary cell line [214]. This pro-miotic effect was diminished in IL-4 receptor-deficient mice [213], indicating an IL-4-dependent mechanism. In addition to alterations in cell cycle phases, Sh total antigen administration to the Chinese hamster ovary cell line resulted in decreased apoptosis with increases in anti-apoptotic gene Bcl-2, and in tumor suppressor gene p27. These studies suggest that Sh may lead to bladder cell cycle skewing in an IL-4-dependent fashion.
Overall synthesis of experimental gene expression studies in Sh infection.
Taken together, these experimental studies conducted in animal models and cell lines demonstrate that Sh infection mimicked by the presence of Sh eggs or antigens can lead to changes in enzyme and protein expression, and cell cycle changes, which are linked to an environment favorable for inflammation, compromised epithelial barrier, and carcinogenesis. These studies emphasize not only the role of Sh infection in the development of bladder cancer, but also provide insight into the pathophysiologic changes that underlie Sh-linked bladder cancer, as well as possibly facilitate entry of additional pathogens through a compromised epithelial barrier.
S. haematobium infection and the host microbiome
A total of six studies compared microbiome changes in gut, urine, and cervicovaginal microbiota during Sh infection (Table 15).
In four of these, Firmicutes, Proteobacteria, and Bacteroidetes were the most abundant bacteria phyla in both gut and urine microbiota in individuals with and without Sh infection [26,215–217].
Three studies focused on differences in the gut microbiome through analysis of stool samples of individuals with and without Sh infection. Sh adult worms reside in the vasculature of the genitourinary system, and eggs migrate preferentially into the genitourinary system to be excreted in the urine. Though not common, Sh eggs can be occasionally identified in stool of infected individuals as well [218,219]. Sh could affect the fecal microbiome through the presence of eggs in the gastrointestinal tract or through a systemic effect. Ajibola et al [26] showed higher Proteobacteria and lower Firmicutes proportions in the gut microbiota by analysis of stool samples of adolescents with and without Sh infection. Furthermore, beta diversity, the measure of similarity or dissimilarity between microbiome groups, differed between people with and without Sh infection, while alpha diversity, the diversity within a microbiome, remained the same [26]. This study also emphasized how changes in the gut microbiome of adolescents with Sh infection led to a dysbiotic state. Kay et al [216] found a difference in the abundance of 5 Prevotella operational taxonomic units (OTUs) in the gut microbiome in fecal samples of children with Sh infection ages 6 months to 13 years in comparison to children without infection [216]. Osakunor et al [217] studied children with Sh infection ages 1–5 and reported that Sh infection status and intensity affects bacteria and fungi genera in stool [217]. These studies consistently demonstrate associations of Sh infection with altered microbial composition in different compartments outside of the genitourinary tract in infected adolescents and children. Changes in fecal microbiota in individuals with Sh infection may have multiple downstream consequences for the host, including altering the host’s mucosal immune system, which could impact local response to intruding pathogens as well as cancer. Furthermore, these changes in the fecal microbiota appeared to be refractory to praziquantel treatment [216,217,220].
Studying the urine microbiota in Sh infection is pertinent since the genitourinary system is the site where Sh eggs mainly become entrapped, subsequently inducing local inflammation, fibrosis, and granuloma formation [212,221]. Changes in the urine microbiome have been postulated to contribute to the pathogenesis of the development of bladder cancer in individuals with Sh infection [211,212]. Despite this, Adebayo and colleagues are the only study thus far to report urine microbial composition in individuals with Sh infection. Their study found higher Firmicutes and lower Proteobacteria proportions in the urine of individuals with Sh infection, compared to individuals without Sh infection [215]. The presence of distinct microbial species and varying levels of Firmicutes and Proteobacteria in individuals with Sh infection could induce inflammation, with less protection for the infected host. Altered Firmicutes and Proteobacteria levels have been connected to gastrointestinal dysbiosis, which can alter bile acid composition, leading to gastrointestinal tract inflammation and increased gut permeability [222–225].
Two studies investigated the cervicovaginal microbiome using swabs collected from cervicovaginal mucosa, the site of Sh egg deposition. Changes in the cervicovaginal microbiome, particularly increased diversity and loss of Lactobacilli, are associated with increased susceptibility to some infections, including HIV [226,227]. Women with high intensity Sh infections, defined as a CAA value of above 3000 pg/ ml, were found to have higher alpha diversity compared to women with lower intensity infections or women without infection [228]. After PZQ treatment, an increase in alpha diversity in participants that continued to test positive for Sh was found [228]. In contrast, Sturt et al [229] did not find differences in presence or concentration of key cervicovaginal species in women with and without FGS. Interestingly, individuals in whom two or more genital samples had detectable Sh DNA had a significantly higher risk of T. vaginalis [229]. T. vaginalis has been shown to influence HIV-1 acquisition and transmission [230,231], with some studies showing a decline in HIV-1 shedding rates after metronidazole treatment [232–234]. These two studies underscore how alterations in the cervicovaginal microbiome due to Sh infection could be a mechanism that increases host susceptibility to HIV and other infectious diseases.
Overall synthesis of microbiome studies in Sh infection.
Together, the above studies demonstrate that Sh infection can lead to changes of the microbiome both locally in the genitourinary tracts where egg disposition mainly occurs, as well as in other mucosal compartments such as the gastrointestinal tract. These changes in microbial populations may have far reaching consequences on both immune responses and acquisition and transmission of diseases. To date, no study has examined the relationship between microbiota and mucosal immune populations in Sh infection, which would be an important area for future investigation.
Discussion
In this systematic and comprehensive review, a total of 89 of 94 studies (95%) demonstrated associations of Sh infection with considerable changes in the host immune response, gene expression, and/or microbiome composition (Fig 3). These changes underlie several major public health impacts of Sh infection. This chronic parasitic infection, which lasts for an average of 3–5 years when untreated [235], shifts the host’s immune phenotype to a predominate type 2 immune phenotype, provokes increases in regulatory cell populations, such as Tregs and Bregs, and inflammatory cytokines, and compromises the epithelial barrier and microbiome diversity. Each of these major changes could contribute to the impaired defenses against viruses, the increased risk of carcinogenesis, or both that are seen in Sh infection. Of further importance, the concordance of many studies that treatment with praziquantel only partially resolves these immune, gene expression, and microbial changes emphasizes the urgent need to prevent and aggressively manage this infection in the first place.
Overview of the main changes in immune, gene and microbiome with S. haematobium infection reported in the 94 studies included in this review. Tregs = T regulatory cells; MBC = Memory B cells; Bregs = B regulatory cells; Th = T helper cell. Black font indicates two or more studies have demonstrated this relationship. Light grey indicates a finding that has only been reported by a single study. Image created with BioRender.com.
The studies assessed reported multiple immune alterations in Sh infection, which are summarized in Fig 4. In addition to the type 2 and regulatory phenotype mentioned above, numerous studies provided evidence that Sh infection hinders an adequate cellular immune response. This includes functional impairment of various immune cell subsets crucial for host defense against invading pathogens, such as DCs, B cell subsets, CD4+ memory, and EM T cells. Interestingly, the role of CD8+ in infection remains to be determined. One study showed no difference, while another indicated an increase in this pertinent T cell population systemically [103,123]. A number of studies also associated Sh infection with increased pro-inflammatory cytokines, such as IFN-γ, IL-6, and MIP1-α, in both blood and the genital tract [236].
Overview of immune changes associated with S. haematobium infection. Th = T helper cells; Tregs = T regulatory cells; Bregs = B regulatory cells; DCs = dendritic cells; ILCs = innate lymphoid cells; CVL = cervicovaginal lavage; FGS = female genital schistosomiasis. Black font indicates two or more studies have demonstrated this relationship. Light grey indicates a finding that has only been reported by a single study. Image created with BioRender.com.
When applied to the reproductive tract, where the tissue burden of Sh eggs is frequently high, these immune changes may increase the risk of acquisition and transmission of viral infections, particularly in women. It is well established that female genital tract inflammation caused by other infections creates a conducive environment for HIV acquisition, both via recruitment of HIV target cells to mucosal surfaces and via damage to epithelial integrity. Only one study in this review assessed HIV target cells in the female genital tract, and found decreases in CD4+ T cells expressing CCR5 in the genital tract following PZQ treatment [107]. Two others identified genital tract cytokine changes consistent with increased genital tract inflammation and/or decreased antiviral immunity [148,149]. It is also possible that IL-6, which one study reported to be elevated in the urinary bladder in Sh infection and which is associated with increased HIV viral shedding in the genital tract, could play a role [237].
A second putative mechanism for increased viral susceptibility in the genital tract could be Sh-induced alterations in mucosal gene expression and microbiota. The first line of host defense against pathogens is the epithelial barrier. Alterations in epithelial barrier proteins have been associated with impaired epithelial barrier function, allowing easier pathogen translocation [227,238]. In this review, many studies demonstrated lower expression of epithelial barrier proteins and higher expression of proteases, which can cause epithelial barrier destruction, in Sh infection. Interestingly, changes in the epithelial barrier can also impact the microbiome and vice-versa [239]. The reported microbial shifts associated with Sh infection, such as altered Firmicutes and Proteobacteria levels, may further inflammation. Furthermore, reported cervicovaginal findings of increased microbial diversity, abundance of taxa, and low Lactobacillus in Sh infection have all been associated with increased risk of HIV acquisition [226]. Together, these Sh infection gene expression and microbiome data corroborate and may potentiate the Sh-associated immune alterations, which collectively align with enhanced potential for increased viral susceptibility.
Of note, it has been reported that that schistosome infection might alleviate disease severity in some chronic viral infections such as HIV, HTLV-1, and respiratory viruses [29]. These observations underscore the importance of a thorough examination of immune effects of Sh infection in endemic settings where viral and Sh, as well as other helminth infections, intersect. It is possible that the mixed picture of type 2, regulatory, and pro-inflammatory immune responses in Sh infection may contribute to these sometimes seemingly paradoxical findings. Moreover, a strong Treg environment in schistosome infection has been linked to reduced hepatic damage from HBV [240], improved HTLV outcome [241] and decreased susceptibility to HIV-1 R5 virus [242]. Further research is essential to understand the reasons that underlie disparate viral presentations during Sh infection, to explore effects of Sh on other important viruses including HPV, and potentially to guide future immunoregulatory interventions that harness beneficial effects of Sh infection.
In Sh endemic countries, bladder cancer is commonly linked to past or current infections with Sh [14,158]. Across an array of human and animal studies of Sh infection, articles in this review report a Th2-skewed environment with concomitant downregulation of Th1 response, which may favor carcinogenesis. Furthermore, chronic inflammation is a well-known risk factor for the development of malignancy [243] and elements of a pro-inflammatory immune environment, in addition to type 2 findings, were identified in a substantial portion of studies included in this review. Other studies demonstrated distinctive genetic, epigenetic, protein, and cell cycle alterations in bladder cancer associated with Sh infection when compared to Sh-unassociated bladder cancer. These multiple alterations link Sh infection to an environment conducive to malignant growth. It is also possible that the noted shifts in the microbiome during Sh infection, particularly in urine, impact bladder carcinogenesis by affecting the immune response or by inducing changes in gene expression in the urothelium. Given the emerging possibility of manipulating microbiota to prevent other cancers [244], this is another important area for future research.
The variation of reported findings among the microbiome studies highlights the challenges inherent in these types of analyses. Further adding to the complexity, microbiota can impact the immune system and gene expression [245,246] and the microbiome is highly diverse, with different body sites hosting distinct microbial communities that are impacted by multiple factors, beginning from birth. The microbiome is also dynamic, with variations not only throughout the individual’s lifespan, but sometimes daily within the same individual [17,20,247]. Geographic location also impacts microbiome composition. The six microbiota-focused studies in this review investigated different compartments and spanned four different countries, with age groups ranging from 13-month-year-olds to 65-year-old adults. This could explain why the specific changes seen in one bodily compartment do not completely overlap with the findings of another study, despite investigating the same compartment. What is clear from this review is that Sh was associated with alterations in the microbiome in all three of these compartments. The scarcity of these studies highlights the need for additional microbiome analyses in Sh infection across geography, age, and assessing associations of microbiota with both baseline and provoked immune responses.
Our synthesis of articles suggests that anti-schistosome treatment results in the partial, but often incomplete, restoration of Sh-associated changes in immune phenotype, gene expression, and microbiota. The single study investigating genetic changes post- versus pre-PZQ treatment showed a reduction, but not complete normalization, of genetic alterations. The two studies investigating microbiome changes before versus three months after successful treatment did not observe any significant changes. It is possible that the microbiome may require longer time to recover after Sh infection than the three month follow up periods in these studies. Together, the study results discussed in this review argue that PZQ could be beneficial, at least in reverting some immune alterations associated with Sh infection back to a pre-infection state, but that PZQ may be less able to reverse the effects of Sh infection on gene expression and microbiota. For these reasons, it will be important to investigate whether resolution of Sh infection truly diminishes the risk of viral acquisition and the onset of bladder cancer.
This review has strengths and limitations. We included 94 studies that documented active schistosome infection by microscopy, antigen testing, PCR, serologies, or clinical exam. Serologies to diagnose Sh infection were included despite antibodies remaining positive after clearance of infection, and the possibility that some cases might not reflect active infection. Nevertheless, we included these studies which identified Sh infection based on positive serologies to ensure we did not underestimate effects of Sh infection, and in order to assess possible long-term effects of past Sh infections. The included studies assessed a broad range of human populations, sometimes leading to heterogenous results. Beyond age, sex, co-infections, and areas of residence with different Sh endemicity, a large array of other factors may influence the individual’s immune response to Sh infection. These include environmental, sociodemographic, and economic aspects of Sh infection, which could not be ascertained. Furthermore, the effects of timing, duration, and infectious burden of schistosome infection likely affect the manifestation of schistosome-associated immune, gene and microbiome changes. Finally, publication bias could have led to the non-publication of studies that showed no impact of Sh on immune system, gene expression, and microbiome and thereby would not have been included.
Despite these limitations, our rigorous analysis provides, to our knowledge, the first comprehensive synthesis of immune, genetic, and microbial factors associated with Sh infection. Our synthesis can help to guide understanding of potential supplementary therapeutic approaches to reverse consequences of Sh infection. This body of data may also pave the way for novel immunoregulatory, genetic, or microbiome interventions and guide global strategies related to prevention of infections and cancer.
Methods
We conducted a systematic review to assess the effects of Sh infection has on the immune system, gene expression and microbiome. This study was reported in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines and was registered prospectively in PROSPERO (CRD42022372607).
Search strategy
Comprehensive literature searches were developed and performed by a medical librarian [DW]. The initial search was performed December 22nd, 2022 via OVID MEDLINE ALL (December 22nd, 2022). The Cochrane Library (Cochrane Database of Systematic Reviews, Cochrane Central Register of Controlled Trials (CENTRAL), Cochrane Methodology Register, Technology Assessments (HTA)), LILACS (Latin American and Caribbean Health Sciences Literature), and OVID EMBASE (1974-March 28, 2019) were searched on December 22nd, 2022; Scopus (Elsevier) on December 22nd, 2022. Follow-up searches were performed on August 9th, 2023. Search terms included all subject headings and/or keywords associated with our research question, clustered as:
- Parasite or proxy for parasitic infection (e.g. Schistosoma haematobium, Schistosomiasis, Bilharzia, Bilharziasis)
- Immune-related headings (e.g. Immunity, Immunomodulation, Immune response, Mucosal immunity, Immune changes, Cellular immunity)
- Microbiome related headings (Microbiome, Microbiota, Cervicovaginal microbiota, Genitourinary microbiota)
- Gene expression-related headings (e.g. Gene expression, Mucosal gene expression, Transcriptomics)
Boolean operators ‘OR’ and ‘AND’ were used as appropriate. Grey literature and bibliographies of included articles were also searched; see full search strategy in the Supplementary Appendix (S1 Text). Publication date restrictions were not imposed. Only papers written in the English language were included. Databases were searched from inception. Authors were not contacted for further information. Studies were uploaded to the Covidence platform (Melbourne, Australia) for conduct of the screening and extraction phases. Included study types were randomized clinical trials, cohort, cross-sectional, and case-control studies. Both human, animal and experimental ex vivo studies were included. S1 Text summarizes our study’s predefined search terms, keywords, and study types included. S2 Text summarizes the decision rules for data selection and extraction.
Study selection and evaluation process
- Study selection
After excluding duplicates, two investigators independently screened titles and abstracts to identify studies eligible for inclusion using the above criteria, and a third investigator independently resolved discrepancies. Discrepant analyses of study quality were resolved by discussion and author consensus. Due to the heterogeneity of study designs, including human, animal and ex vivo studies, a meta-analysis was not possible. We included studies describing the effects of Sh on the immune system, gene expression or microbiome. Pre-defined exclusion criteria included: not available in English, and study design of case study or case review, conference proceedings/meeting abstracts, dissertations/theses, newspaper/magazine articles and research in progress. We prespecified that studies must test for Sh infection by serologies, Sh egg detection by tissue or urine microscopy, or antigen testing. For the scope of this review, we prespecified that we would exclude studies describing humoral immune responses and solely focus on cellular immunity, cytokine and chemokine signaling and immune cell interactions. Full-text review followed the initial title and abstract screening phase, with data extracted into Microsoft Excel. - Data extraction
Data extracted included study type; country of origin; sample size; immunology, gene, or microbiome outcomes; key findings; effects of anthelmintic therapy on outcomes, if applicable; and study limitations. - Quality assessment
Quality assessment of data was performed using the Downs and Black checklist [27]. Studies were categorized as “poor,” “fair,” “good,” or “excellent” based on a summative score derived from this checklist, and only those of “fair” quality or above were included in the final analysis.
Supporting information
S1 Table. List of excluded studies during quality assessment using the Downs and Black Checklist.
https://doi.org/10.1371/journal.pntd.0012456.s001
(DOCX)
S2 Table. Extracted data of included studies.
https://doi.org/10.1371/journal.pntd.0012456.s002
(XLSX)
S1 PRISMA Checklist. For more information, visit: http://www.prismastatement.org/.
https://doi.org/10.1371/journal.pntd.0012456.s003
(DOCX)
S2 Text. Decision Rules for Data Selection and Extraction Processes.
https://doi.org/10.1371/journal.pntd.0012456.s005
(DOCX)
Acknowledgments
We thank the librarian team at Weill Cornell Medicine for their assistance with searching and retrieving study records for this systematic review. Furthermore, we want to thank Drs. Mary Charleston and Carol Mancuso who lead the Master’s program Clinical Epidemiology & Health Services Research, and Dr. Kyu Rhee, who leads to Burroughs Wellcome Physician Scientist Program at Weill Cornell, who all have been continued supporters and offered instrumental guidance for this review.
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