Figures
Abstract
Background
Severe leptospirosis is challenging as it could evolve rapidly and potentially fatal if appropriate management is not performed. An understanding of the progression and pathophysiology of Leptospira infection is important to determine the early changes that could be potentially used to predict the severe occurrence of leptospirosis. This study aimed to understand the kinetics pathogenesis of Leptospira interrogans strain HP358 in the hamster model and identify the early parameters that could be used as biomarkers to predict severe leptospirosis.
Methodology/Principal findings
Male Syrian hamsters were infected with Leptospira interrogans strain HP358 and euthanized after 24 hours, 3, 4, 5, 6 and 7 days post-infection. Blood, lungs, liver and kidneys were collected for leptospiral detection, haematology, serum biochemistry and differential expression of pro- and anti-inflammatory markers. Macroscopic and microscopic organ damages were investigated. Leptospira interrogans strain HP358 was highly pathogenic and killed hamsters within 6–7 days post-infection. Pulmonary haemorrhage and blood vessel congestion in organs were noticed as the earliest pathological changes. The damages in organs and changes in biochemistry value were preceded by changes in haematology and immune gene expression.
Author summary
As the severe form of leptospirosis could progress rapidly and be potentially fatal if not treated earlier, deciphering the pathophysiology kinetics of infection is crucial to determine the parameters of disease severity. To understand this, we challenged hamsters with the highly virulent Leptospira interrogans strain HP358. Pulmonary haemorrhage was observed as the earliest pathological change followed by liver and kidneys damages. The increased expression of IL-1β, CXCL10/IP-10, CCL3/MIP-α, high neutrophils and low lymphocytes and platelets production observed in the present study indicate that these parameters could serve as a cumulative panel of biomarkers in severe leptospirosis.
Citation: Philip N, Priya SP, Jumah Badawi AH, Mohd Izhar MH, Mohtarrudin N, Tengku Ibrahim TA, et al. (2022) Pulmonary haemorrhage as the earliest sign of severe leptospirosis in hamster model challenged with Leptospira interrogans strain HP358. PLoS Negl Trop Dis 16(5): e0010409. https://doi.org/10.1371/journal.pntd.0010409
Editor: Tao Lin, Baylor College of Medicine, UNITED STATES
Received: January 31, 2022; Accepted: April 10, 2022; Published: May 18, 2022
Copyright: © 2022 Philip 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 relevant data are within the manuscript and its Supporting Information files.
Funding: This work was supported by the Ministry of Education, Malaysia through the Fundamental Research Grant Scheme (FRGS/1/2019/WAB13/UPM/02/2) with vot number: 5540259. The funding was received by Associate Professor VKN. 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
The severe manifestation of leptospirosis could be either Weil’s disease, a triad of jaundice, renal impairment and haemorrhages; or severe pulmonary forms of leptospirosis (SPFL) without distinct renal and hepatic impairments [1]. The multi-organs involvement often appears as a sudden onset of clinical manifestations, rapidly progressive and associated with high mortality rates [2–5]. This severe manifestation of leptospirosis could be either due to infecting strain of Leptospira, the load of leptospiral inoculum and the age and immune status of the infected host.
Despite the existence of the disease for many years, the evolution and factors determining the development of severe leptospirosis in the infected host are still not well defined. Clinical features and pathological changes in severe leptospirosis are described and suggested its association with cytokine storm [6–9]. Several studies have been focused on identifying the factors associated with severe leptospirosis [10–14]. However, the majority of these studies were performed on samples collected at a single time point. For a detailed understanding of the parameters that could be monitored to prevent the illness from progressing to a severe form, a kinetics study of the pathogenesis is vital.
In our earlier study, we isolated and identified a new genotype of Leptospira interrogans strain HP358 (L. interrogans strain HP358) with Sequence Type (ST) 238 in rodents trapped from the hotspot of human leptospirosis in the forest area of Hulu Perdik, Selangor, Malaysia [15]. We performed an in vivo pathogenesis screening for the strain HP358 in the hamster model and found that this strain is highly pathogenic manifesting pulmonary haemorrhage, liver and kidneys damages and death as early as six days of post-infection (p.i.) [16]. The evidenced life-threatening clinical manifestations prompted us to investigate and understand the kinetics of the pathophysiology of severe leptospirosis. Therefore, this study was carried out to decipher the progression of the illness by monitoring the clinical manifestation of infected hamsters, histopathological changes in tissues (lungs, liver and kidneys), the leptospiral load, hemogram and serum biochemistry and the cytokines and chemokines expression profiles. We hypothesised that understanding the virulence severity and the time course progression of the disease development may identify factors that are expressed or altered during the early stage of infection which could be recruited for further evaluation and subsequently utilized as biomarkers in severe leptospirosis.
Methods
Ethics statement
All experiments were conducted following the guidelines of the Code of Practice for the Care and Use of Animals for Scientific Purposes, Universiti Putra Malaysia. Male golden Syrian hamsters aged between four and six weeks purchased from Monash Universiti Malaysia, Bandar Sunway, Selangor were housed (three per cage) with sterile sawdust bedding, fed with commercial feed and given water ad lib in sterile bottles during the study course. The hamsters were acclimatized for 14 days prior to the experiment. All animal procedures carried out in this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC), Universiti Putra Malaysia with Animal Use Protocol (AUP) number: UPM/IACUC/AUP-R044/2018. This study also is in compliance with the ARRIVE guidelines.
Infection, monitoring and euthanization of hamsters
Upon completion of two weeks of acclimatization, the hamsters (n = 21) were infected intraperitoneally (IP) with 2 x 108 of L. interrogans strain HP358 in 500μl Ellinghausen-McCullough-Johnson-Harris (EMJH) medium. The bacterial load (to develop infection) to be inoculated were selected based on our earlier investigation [16] and in previous studies [17,18]. Control hamsters (n = 7) were injected intraperitoneally with 500μl sterile EMJH medium (without any Leptospira). The infectivity study was carried out for seven days. The hamsters were monitored throughout the study for clinical signs such as progressive loss of weight, loss of appetite, reduced physical activity and dyspnea. One control and three infected hamsters were euthanized from day 1 to 7 p.i. except for day 2 to study the pathological events. Due to unforeseen reasons, we were not able to sacrifice the hamsters on day 2. The hamsters were anaesthetized with 100 mg/kg ketamine and 5 mg/kg xylazine injected intraperitoneally and subsequently whole blood was collected by cardiac puncture. Blood was collected for (1) direct culture in EMJH medium, (2) detection of leptospiral DNA and haematological analysis in EDTA tube, (3) biochemistry analysis in plain tube and (4) detection of immune genes in RNAprotect animal blood tube. Hamsters were euthanized by atlanto-occipital dislocation and following dissection, lungs, liver and kidneys were harvested and examined macroscopically for any morphological changes. Twenty-five milligrams of each lung, liver and kidney tissues were collected and transferred into tubes containing absolute ethanol for leptospiral DNA detection and RNAlater for immune genes expression study. The remaining parts of the organs were fixed in 10% neutral buffered formalin for histopathological investigations.
Macroscopic and microscopic examinations of infected organs
Formalin-fixed organs (lung, liver and kidney) were processed for light microscopy and stained with hematoxylin and eosin (H&E) using the standard protocol. Lesions and changes in the target organs were graded according to previously reported criteria [17,19,20].
Leptospira growth and DNA quantification in blood and organs
Portions of the kidneys of all hamsters were cultured in EMJH medium and observed for up to two months for the growth of leptospires. Leptospiral DNA from blood, lungs, liver and kidneys were extracted using the DNAeasy Blood & Tissue Kit (Qiagen, German) according to the manufacturer’s instructions. The 242bp lipL32 (primers: LipL32-45F: 5′-AAGCATTACCGCTTGTGGTG-3′, LipL32-286R: 5′-GAACTCCCATTTCAGCGATT-3′, probe: LipL32-189P: FAM-5′-AAAGCCAGGACAAGCGCCG-3′-BHQ1) [21] gene amplification was performed for detection of leptospires in blood and organs. A serial dilution of pure culture of L. interrogans strain HP358 was used as a standard curve to determine the leptospiral counts, linear range, efficiency and reproducibility of the qPCR assay.
Haematology and serum biochemistry analyses
Blood samples taken from the hamsters were sent to the Haematology and Biochemistry laboratory, Faculty of Veterinary Medicine, Universiti Putra Malaysia for complete blood counts and biochemical analysis. The parameters for biochemical analysis were selected based on their association with organs damage in human leptospirosis.
Expression of pro-inflammatory and anti-inflammatory markers
Total RNA extraction.
Total RNA from blood (RNeasy Protect Animal Blood kit, Qiagen, German), lungs, liver and kidneys (HiYield Total RNA Mini Kit) were extracted following the manufacturers’ instructions. The extracted RNA was eluted in 20μl of RNase-free water. Before storage at -80°C, the quantity and quality of the purified RNA were measured using the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific) at OD 260/280 and OD 260/230 ratios. The integrity of RNA was verified using gel electrophoresis.
Reverse-transcription.
DNA-free total RNA extracted from blood, lungs, kidneys, (1μg) and liver (0.5μg) was reverse transcribed into cDNA using the Quantinova Reverse Transcription kit (Qiagen, German). Genomic DNA from the RNA samples was removed using gDNA removal mix (2μl). The total volume of 20μl reverse transcription (RT) reaction mix contained RT enzyme (1μl), RT mix (4μl) and template RNA (entire gDNA elimination reaction, 15μl). RT was conducted on a BioRad machine and consisted of annealing (3 min, 25°C), RT step (10 min, 25°C) and inactivation step (5 min, 85°C). The transcribed cDNA was diluted in 1:2.5 with RNase-free water and kept at -40°C until used.
Real-time PCR and amplification program.
Primers for immune genes were synthesized (MyTACG Bioscience Enterprise, Malaysia) utilizing sequences from the previous studies (Table 1). These immune genes were selected based on the association of these genes with leptospirosis. For every sample, the amplification (real-time PCR) was carried out in duplicates containing 1μl cDNA in a 20μl final volume for each cytokine and chemokines (Table 1) using Quantinova SYBR green I master kit (Qiagen, German). The amplification was performed on the Eppendorf instrument using Realplex software. The amplification program consisted of an activation step at 95°C for 2 min followed by amplification cycle of the target cDNA for 40 cycles (95°C for 5 s and a combined annealing/extension at 55.7°C for 10 s). Negative control with RNase-free water was included in each run. The specificity of the amplification was verified by analysis of the melting curves of the PCR products.
Gene expression analysis.
The level of expression of each gene was normalized to the levels of glyceraldehyde-3-phosphate dehydrogenase (GADPH) housekeeping gene using a comparative delta delta CT method (ΔΔCT Method). The average Ct values of genes tested obtained from the control and infected hamsters for blood and organs (lungs, liver and kidneys) were directly normalized to the reference gene. Then, the difference between the Δct value of infected and control hamsters was calculated to arrive at the double delta Ct value. Finally, the value of 2-ΔΔCt was calculated to obtain the expression fold change.
Results
Clinical response to infection
The earliest clinical sign observed in the infected hamsters was weight loss which occurred as early as day 3 p.i. Body weight continued to decrease over the days, with average weight loss of 0.3% (D3), 2.2% (D4), 4.6% (D5), 7.3% (D6) and 7.7% (D7). On day 5 p.i., all hamsters showed loss of appetite, reduced physical activity and developed dyspnea. On day 6 p.i., three hamsters died and one hamster died on day 7 p.i.. All control (non-infected) hamsters euthanized on days 1 and 3 to 7 p.i. showed normal behaviour and progressive weight increase (16.6% increase at the end of the study).
Macroscopically pulmonary haemorrhage occurred earliest
All three hamsters euthanized on day 1 p.i. showed normal morphology of the liver and kidneys, however, few focal haemorrhagic areas were observed in the lungs. Beginning from day 3 p.i., haemorrhage in the lung continued to spread (Fig 1). The kidneys appeared pale from day 6 p.i. while the liver did not show any marked changes. Another notable finding observed was yellowish discolouration of adipose tissues in some of the dead and euthanized hamsters on days 6 and 7 p.i. (Fig 2). The lungs, liver and kidneys harvested from the control hamsters showed no gross changes.
Petechial haemorrhages were observed in the lungs from day 1 p.i. and became severe from day 3 p.i. onwards. Kidneys became progressively pale from day 6 p.i. onwards. No notable changes were observed in the liver. C: Control; D1-D7: Day 1 to Day 7. The arrow shows a petechial haemorrhage.
The yellowish discolouration was observed on adipose tissue on day 6 p.i. in infected hamsters. C: control; D6: day 6 p.i.
Microscopically all organs showed progressive damages
The earliest pathological changes observed were congestion of the lung and liver on day 1 p.i. and kidneys on day 3 p.i. (Figs 3–5; Table 2). Haemorrhage was observed in the lung as early as on day 1 and day 4 p.i. in kidneys. In the lung, apart from congestion and haemorrhage, septal thickening and collapsed alveoli were observed from day 1 p.i. while mild alveoli dilation was noted from day 5 p.i. Inflammatory cells infiltration appeared in the liver and kidneys from day 4 p.i. henceforth. In the liver, disorganized hepatic cords and enlargement of hepatocytes were observed from day 4 p.i. and progressively deteriorated. Marked pathological changes were also observed in the kidney. Shrinkage of glomerulus capillaries leading to dilation of Bowman’s space and renal tubular damages characterized by tubular dilation and degeneration of epithelial cells lining of the proximal and distal convoluted tubules were observed in kidneys on day 4 p.i. and henceforth increased in severity. Hamsters that died earlier before day 7 p.i. showed similar pathological changes which were haemorrhagic lungs and kidneys and congested liver. No lesions or any pathological changes were observed in the organs of control hamsters.
Congestion and haemorrhage occurred as early as day 1 p.i. while dilated and collapsed alveoli occurred on days 6 and 7 p.i. and in dead hamsters. CG: Congestion, H: Haemorrhage, CA: Collapsed alveoli, ST: Septal thickening, DA: Dilated alveoli. C: Control, D1-D7: Day 1- day 7 and DD: Dead. Magnification: x100, bar: 100 μm.
Congestion occurred as early as day 1 p.i. while infiltration of inflammatory cells, disorganized hepatocyte cords and swelling of hepatocytes occurred on day 4 p.i. onwards. SC: Sinusoid congestion, IC: Infiltration of inflammatory cells, DHC: Disorganized hepatocyte cords, SH: Swollen hepatocytes and H: Haemorrhage. C: Control, D1-D7: Day 1- day 7 and DD: Dead. Magnification: x100, bar: 100 μm.
Congestion occurred as early as day 3 p.i., haemorrhage, infiltration of inflammatory cells, dilation and collapse of Bowman’s space on day 4 p.i. and dilation of tubule and degeneration of epithelial cells in proximal and distal tubules occurred on day 5 p.i. onwards. CG: Congested glomerulus, CB: Collapse of Bowman’s space, DB: Dilated Bowman’s space, DT: Dilated tubule, DPD: Degeneration of epithelial cells in proximal and distal tubules, IC: Infiltration of inflammatory cells, and H: Haemorrhage. C: Control, D1-D7: Day 1- day 7 and DD: Dead. Magnification: x100, bar: 100 μm.
Leptospiral load in blood and organs
Blood and kidneys samples of infected hamsters from day 1 to day 7 p.i. cultured in the EMJH medium yielded positive growth for leptospires while no growth was observed in the control hamsters. Likewise, qPCR also showed positive amplification for all samples (blood, lungs, liver and kidneys) collected from the infected hamsters from day 1 to day 7 p.i. (Fig 6). No cultures were performed for the lungs and liver. The leptospiral load (qPCR) in blood and organs showed a progressive increase from day 1 to day 5 p.i. for blood, lungs and kidneys and until day 6 for the liver. On day 7 p.i., the leptospiral load was lower than day 1 p.i. in blood, lungs and liver while the load remained unchanged in the kidneys.
D1-D7: day 1 to day 7; DD: dead hamsters. The leptospiral copies number in blood and organs can be found in S1 Table.
Haematological and serum biochemical changes
Haematological changes.
White blood cells (WBC), neutrophils and monocytes counts in infected hamsters showed an increase from as early as day 1 p.i. (p-value = <0.05) (Fig 7) while lymphocytes and platelets showed a decreasing trend (p-value = <0.05).
D1-D7: Day 1 to day 7. *P≤0.05, **P≤0.01, ***P≤0.001.
Serum Biochemical changes.
The level of total bilirubin (TB), direct bilirubin (DB) and creatinine kinase (CK) in infected hamsters showed a significant increase (p-value<0.05) compared to that of control hamsters from day 5 p.i. onwards (Fig 8). Similarly, alanine transaminase (ALT) and aspartate aminotransferase (AST) levels rose significantly (p-value = <0.05) beginning on day 5 p.i. The levels of creatinine and urea in infected hamsters began to increase beginning from day 3 p.i. and 5 p.i. respectively.
The biochemical measurements convey the function state of the liver and kidney. D1-D7: Day 1 to Day 7. *P≤0.05, **P≤0.01, ***P≤0.001.
Expression of immune mediators in infected animals
Pro-inflammatory mediators.
Among the pro-inflammatory cytokines and enzymes (interleukin-1beta: IL-1β, interleukin-6: IL-6, Tumor necrosis factor alpha: TNF-α, interferon gamma: IFN-γ, cyclooxygenase-2: COX-2 and inducible nitric oxide synthase: iNOS) tested, IL-1β was found to be significantly expressed in blood and all organs (Figs 9–12) from day 3 onwards. Expression of IL-6 and TNF-α was only observed in the lungs and kidneys. IL-6 was significantly higher in the lungs on days 1 and 7 p.i. while in kidneys, it demonstrated a progressive increase from day 3 p.i. TNF-α showed increased expression in the lungs from day 1 to day 4 p.i. while in kidneys, it showed a progressive increase from day 3 p.i. IFN-γ was higher until day 4 p.i. in blood and lungs. COX-2 was higher than the control in blood until day 5 p.i. while in kidneys, it increased progressively from day 3 p.i. In the lungs, COX-2 was found to be downregulated beginning on day 1 p.i. IL-1β, TNF-α and COX-2 were found to be highly expressed in the liver of dead hamsters. iNOS was downregulated in lungs and kidneys and not detected in blood and liver.
Total RNA was extracted from whole blood on day 1 and days 3 to 7 p.i.. D1-D7: Day 1 to day 7. *P≤0.05. The fold gene expression value can be found in S2 Table.
Total RNA was extracted from lungs on day 1 p.i. and days 3 to 7 p.i. D1-D7: Day 1 to day 7. DD: dead. **P≤0.01, ***P≤0.001. The fold gene expression value can be found in S2 Table.
Total RNA was extracted from liver on day 1 p.i. and days 3 to 7 p.i. D1-D7: Day 1 to day 7. DD: dead. *P≤0.05, **P≤0.01, ***P≤0.001.The fold gene expression value can be found in S2 Table.
Total RNA was extracted from kidneys on day 1 p.i. and days 3 to 7 p.i. D1-D7: Day 1 to day 7. DD: dead. *P≤0.05, **P≤0.01, ***P≤0.001. The fold gene expression value can be found in S2 Table.
Chemokines CXCL10/IP-10 and CCL3/MIP-α.
Increased expression of C-X-C motif chemokine ligand 10 (CXCL10/IP-10) in the blood (from day 1 to 4 p.i), liver (day 1 to 5 p.i.) and kidneys (day 1 to 7 p.i.) (Fig 13). Likewise, chemokine (C-C motif) ligand 3 (CCL3/MIP-α) expression increased in blood, liver and kidneys.
D1-D7: Day 1 to day 7. DD: dead *P≤0.05, **P≤0.01, ***P≤0.001. Control. The fold gene expression value can be found in S3 Table.
Anti-inflammatory mediators.
The expression of transforming growth factor-beta 1 (TGF-β1) showed an increasing pattern in the liver and kidneys (Fig 14) beginning on day 4 p.i. and significantly high on days 6 and 7 p.i. TGF-β1 was found to be downregulated in blood while in the lungs it was slightly higher on days 3 and 4 p.i. Two hamsters showed amplification of IL-10 in lungs, liver and kidneys with a ct value of > 35 on days 5 and 6 p.i. One dead hamster showed amplification of IL-10 in the lung with a ct value of 33.63.
D1-D7:Day 1 to day 7. DD: dead. *P≤0.05, **P≤0.01, ***P≤0.001.The fold gene expression value can be found in S4 Table.
Association between clinical manifestations and pathophysiology in infected hamsters: Identification of biomarkers for severe leptospirosis.
As summarized in Table 3, the pathophysiological presentations observed for severe leptospirosis in the hamster model included: (1) occurrence of pulmonary haemorrhage earlier than liver and kidney damages (before any clinical manifestations), (2) increased WBC, monocytes and neutrophils and decreased lymphocytes and platelets (before severe signs and symptoms), (3) serum biochemistry parameters changes were concurrent with the apparent clinical manifestations and (4) earlier expression of pro-inflammatory mediators IL-1β, CXCL10/IP-10 and CCL3/MIP-α in all organs (blood, lungs, liver and kidneys) prior to observable damages. The early expression of IL-1β, CXCL10/IP-10 and CCL3/MIP-α, increase of neutrophils and decrease of lymphocytes and platelets suggesting that these parameters could be used as a cumulative panel for potential biomarkers in severe leptospirosis.
Discussion
Leptospirosis presents a protean clinical manifestation and most cases (90%) are mild. Severe cases account for 5 to 15% and usually occur in the immune phase of illness [12,24,25]. Severe leptospirosis also presents with a fulminant monophasic illness [26–28]. In both conditions, the evolution of the disease is rapid and potentially fatal if not treated. Hence, early management of the disease is vital. Given the fact that the prompt diagnosis of this illness is challenging and the sudden progression to severe leptospirosis is life-threatening; understanding the sequence of disease progression and determination of early prognosis markers are of utmost importance for a favourable outcome.
In the present study, the first investigation was focused on the clinical manifestation developed in the hamsters when infected with the L. interrogans strain HP358. Hamsters reproduce the severe form of human leptospirosis [29] thereby suitable to be used as a model for studying the progression of severe leptospirosis. As seen from the results, loss of body weight started from day 3 p.i. and from day 5 p.i., all hamsters showed loss of appetite, reduced physical activities and difficulty in breathing (dyspnea). Similar clinical signs were also reported in several previous studies [19,30,31].
To relate the above clinical manifestations with the sequence of events occurring within the body during the infection, four hamsters (one control and three infected) were euthanized on days 1 (24 hours post-infection), 3, 4, 5, 6 and 7 p.i. The earliest pathological changes (macroscopically and microscopically) observed in the infected hamsters were pulmonary haemorrhage and blood vessel congestion in the lungs, liver and kidneys. The earliest sign of haemorrhage was observed in the lungs from day 1 p.i. while in kidneys on day 4 p.i. Marked organ damages (Table 3) were detected beginning from day 4 p.i concurrent with the clinical manifestations. These were followed by the death of four hamsters on days 6 and 7 p.i. while progressive moribund conditions were observed in the remaining hamsters. A similar observation was reported in a recent study where pulmonary haemorrhage appeared much earlier followed by liver and renal damages prior to the animal’s state of moribund and death [31]. In human leptospirosis, pulmonary haemorrhage is the severe form of the illness, though it occurs only in a small number of cases, mortality is seen higher among these patients (more than 70%) [32,33]. The ability of the leptospires to invade multiple organs also depends on the Leptospira species or strain [16]. It could be postulated that in patients with severe leptospirosis, the patients might be infected with a highly virulent Leptospira strain invading multiple organs. As observed in the present study, the lungs were the first organ showing damage, hence it is important to monitor the respiratory problems or bleeding in leptospirosis patients as a prognostic factor for severe leptospirosis. As reported in previous studies, pulmonary haemorrhage could occur prior to jaundice and renal failure and led to severe disease and fatality in human leptospirosis [34,35].
Leptospiral DNA was detected in the blood and all organs on day 1 p.i. indicating rapid dissemination and successful colonization of L. interrogans strain HP358 in the hamster as reported in other studies [17,36]. The bacterial load in blood and all organs continued to increase until day 5 p.i. denoting the replication of leptospires. We observed a decrease in the leptospiral load from day 6 p.i. onwards in blood, lungs and liver while it was maintained in kidneys. Hamsters that died before the completion of the study had a high load of leptospires in all organs (lungs, liver and kidneys) compared to those euthanized on day 7 p.i.
Changes in haematological parameters were observed to occur as early as on day 1 p.i. indicating the response of the innate immunity of hamsters against the invading leptospires. Monocytes and neutrophils continued to increase while lymphocytes and platelets showed decreasing trends. Although neutrophils and monocytes could recognize leptospires, both have limited capacity to control the pathogen [37–42]. It was reported that pathogenic Leptospira spp. could bind to platelets and induce cytotoxic effects resulting in dysfunction and clearance of platelets [43–47]. A low level of platelets noticed in the present study could also play important role in the haemorrhagic presentation as observed in both animal [46] and human leptospirosis [48–50]. Significant neutrophilia and lymphocytopenia had also been reported in severe and fatal cases of human leptospirosis [6,51–55]. The significant changes in total and direct bilirubin, AST, ALT, creatinine, urea and CK appeared much later than the haematological parameters which were on days 4 and 5 p.i. henceforth concurrent with the appearance of damage in the liver and kidneys. These changes in the liver and kidney function tests were in agreement with changes in human leptospirosis [51,55–59].
Both pro-inflammatory cytokines and chemokines were expressed in the infected hamsters. Pro-inflammatory cytokines and enzymes which were IL-1β, IL-6, TNF-ɑ, IFN-γ and COX-2 and chemokines CXCL10/IP-10 and CCL3/MIP-α showed upregulation as reported in both human and animal leptospirosis [6,17,18,23,60–62]. Pro-inflammatory enzyme iNOS was downregulated in lungs and kidneys and not expressed in blood and liver which was similarly reported in a previous study [63]. Anti-inflammatory cytokine TGF-β1 showed expression from day 3 p.i. while IL-10 was slightly induced in some of the dead hamsters on days 5 and 6 p.i. as similarly reported in the previous study [64].
Overall, two main manifestations of pathophysiology in severe leptospirosis were observed; haemorrhage and organ damage where pulmonary haemorrhage appeared as the earliest pathological event. The mechanism of pulmonary haemorrhage is still poorly understood and could result from multiple factors [65]. Direct injury by leptospires or their circulating products (leptospiral outer membrane proteins, glycoproteins, hemolysins and lipopolysaccharides) and indirectly by the host’s immune dysregulation have been proposed to contribute to the haemorrhagic manifestation in leptospirosis [66–69]. Pathogenic leptospires could bind to the endothelial lining of the blood vessels [50,67,70–72] and potentially disrupts the endothelial cell layer [69,73]. IL-6 has been associated with severe pulmonary haemorrhage [6]. In the present study, IL-6 was significantly high in the lung on day 1 p.i. and while in the kidney, it only appears on day 4 p.i. concurrent with the haemorrhagic presentation, thereby could support the role of this cytokine in the haemorrhagic presentation in leptospirosis.
Haemorrhagic manifestation of leptospirosis could also be due to vascular cell damage by reactive oxygen species (ROS) and arterial hypertension [74]. Neutrophils could produce ROS [75], thus the high production of neutrophils in the infected animals may indirectly contribute to the haemorrhagic manifestation observed in this study. Nitric oxide (NO) production catalyzed by the enzyme iNOS [76,77] functions as a vasodilator [78] and is also able to control the production and activity of ROS [79,80] in inhibiting the replication of the pathogen [81–83]. The down-regulation of iNOS influences the release and activities of NO [76,77]. Low or specific inhibition of iNOS is associated with pulmonary haemorrhage [63], increased mortality, bacterial load in the kidney and reduced specific humoral response [84] in the hamster model and patients with severe disease [85].
Mild alveoli dilation observed on day 5 p.i. is a contributing factor for dyspnea which is in agreement with a report in a recent study [86]. The dilated alveolus is a characteristic of chronic obstructive pulmonary disease (COPD) with dyspnea as the cardinal symptom. Enlargement of the alveolus destructs the alveoli walls through inflammation [87]. Cytokine IL-1β has been shown to exert airway inflammation and emphysema in the COPD mice model [88–92]. The raised level of serum IL-1β in patients with mild alveolar dilation is in agreement with the present investigation where expression of IL-1β was significantly high from day 4 p.i. onwards [93].
The progression of damage in the liver and kidneys is associated with the changes in the serum biochemistry and increased expression of inflammatory cytokines and chemokines. The liver and kidney functions test markers (total bilirubin, direct bilirubin, ALT, AST, creatinine and urea, CK) showed a significant increase from day 4 p.i. The progressive upregulation of inflammatory cytokines and chemokines in the kidney (IL-1β, IL-6, TNF-ɑ, COX-2, TGF-β1, CXCL10/IP-10 and CCL3/MIP-α) and liver (IL-1β, TGF-β1, CXCL10/IP-10 and CCL3/MIP-α) beginning from day 3 p.i. support the possibility that damage in these organs is associated with the increased inflammatory response. CXCL10/IP-10 and CCL3/MIP-α are known to mediate the migration of T cells, monocytes, neutrophils and natural killer (NK) cells from the bloodstream to tissues in response to inflammation [94–97].
A severe manifestation of leptospirosis is comparable to sepsis that occurs due to an imbalance in the inflammatory responses in the host infected with pathogens. The infected hosts release inflammatory mediators in an attempt to neutralize the pathogenic effect. The occurrence of a sustained and increased expression of pro-inflammatory cytokines characteristic of a “cytokine storm” will lead to persistent inflammation [9] and this is followed by a massive and systemic production of anti-inflammatory mediators resulting in a state of “immunoparalysis” and tissue oedema [98,99]. Tissues oedema could impair the local organ perfusion leading to loss of organ function and endothelial permeabilization [98,100]. In asymptomatic or mild leptospirosis and mice animal models, homeostasis between pro-inflammatory and anti-inflammatory is maintained where both are produced early and strictly regulated [9,17]. In severe leptospirosis in humans, two scenarios have been reported; either high IL-10 and low TNF-α [6,61] or low IL-10 and high TNF-α [17,101,102]. In this present study, we saw a sudden surge of the pro-inflammatory mediators (cytokines and chemokines) beginning from day 3 p.i.without the prominent expression of anti-inflammatory IL-10. A low expression of IL-10 (ct value of >33 cycles) and early (day 3) expression of TNF-α, TGF-β and IP-10 in hamsters infected with L. interrogans serovar Pyrogenes has been reported earlier [64]. In conclusion, severe leptospirosis due to the L. interrogans strain HP358 could be characterized as a sudden and increased pro-inflammatory response with delayed and significantly low expression of anti-inflammatory IL-10. The severe leptospirosis characterized in the hamster model in the present study is in accordance with the severe form of leptospirosis in humans where patients showed mild symptoms during the early course of the disease and developed a rapidly worsening condition leading to fatality within 72 hours [70,103]. As observed in this study, the rapid evolution to severe illness and fatality in hamsters occurred when most inflammatory mediators were expressed and all organs (lungs, liver and kidneys) were affected. Likewise, the severe form of human leptospirosis involves haemorrhage and multiple organ damages.
Identification of biomarkers in leptospirosis is important not only for diagnosis but also to predict the progression to severity. The main characteristic of ideal biomarkers is their early detection [104] for timely intervention in patients management. A panel of biomarkers will increase the specificity and sensitivity of the diagnosis compared to a single biomarker [101,105]. Serum biochemistry may not be a good predictor for severe leptospirosis as these markers are detected only after the occurrence of serious damage to the liver and kidneys. Cytokines play a major role in host-pathogen interaction and prognosis [18]. Several earlier studies have identified significant expression of IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10 and TNF-α in severe leptospirosis [6,102,106]. However, these studies were conducted in one-time sampling. Progressive monitoring is important to elucidate the progression of cytokines levels and to determine the most appropriate biomarker for disease severity. As the damage in leptospirosis surge rapidly, we recommend performing blood and cytokines profiling at 24 hours interval to monitor the biomarkers for the severe illness that could prevent substantial damage to the organs. From the present investigation, we found the expression of IL-1β, CXCL10/IP-10 and CCL3/MIP-α increased in the blood and most organs day by day as the infection progressed. On the other hand, neutrophils increased progressively from day 1 p.i. while lymphocytes and platelets showed a declining trend. Taken all these data together, we suggest that high levels of IL-1β, CXCL10/IP-10, CCL3/MIP-α, neutrophils and low levels of lymphocytes and platelets could serve as a cumulative panel of potential biomarkers in the disease progression from mild to severe in leptospirosis. As this study was conducted in an animal model, a progressive validation study in human leptospirosis is recommended.
Conclusion
Severe leptospirosis is characterized by a sudden over-expression of pro-inflammatory cytokines after infection of L. interrogans strain HP358 and without prominent expression of regulatory cytokines. The massive expression of cytokines and chemokines led to sudden and rapid damage to the liver and kidneys. Damages in the lungs, liver and kidneys were preceded by the early occurrence of haemorrhage in the lungs. High levels of IL-1β, CXCL10/IP-10, CCL3/MIP-α, neutrophils and low levels of lymphocytes and platelets might serve as a cumulative panel of biomarkers in severe leptospirosis.
Supporting information
S1 Table. Leptospiral copies number in blood and organs.
https://doi.org/10.1371/journal.pntd.0010409.s001
(DOC)
S2 Table. Fold gene expression value of pro-inflammatory cytokines.
https://doi.org/10.1371/journal.pntd.0010409.s002
(DOC)
S3 Table. Fold gene expression value of pro-inflammatory chemokines.
https://doi.org/10.1371/journal.pntd.0010409.s003
(DOC)
S4 Table. Fold gene expression value of anti-inflammatory cytokines.
https://doi.org/10.1371/journal.pntd.0010409.s004
(DOC)
Acknowledgments
We thank the staff in the Animal Research Facility—Low-level infection at the Faculty of Veterinary, UPM for their help in providing the equipment needed for this study.
References
- 1. Vijayachari P, Sugunan AP, Singh SS, Mathur PP. Leptospirosis among the self-supporting convicts of Andaman Island during the 1920s—the first report on pulmonary haemorrhage in leptospirosis? Indian J Med Res. 2015; 142(1):11–22. pmid:26261162
- 2.
Scoot GM, Coleman TJ. Leptospirosis. In: Cook C, Zumla AI, editors. Manson’s tropical diseases. British: Saunders Elsevier; 2009. pp. 1161–7. https://doi.org/10.1002/cphc.200800403 pmid:19142925
- 3.
Day NPJ, Edward CN. 2010 Leptospirosis. In: Cohen J, Powderly WG, Opal SM, editors. Infectious diseases. 3rd ed. Britsih: Mosby Elsevier; 2010. pp. 1241–6.
- 4.
Watt G. Leptospirosis. In: Ryan E, Hill D, Solomon T, Magill A. Hunter’s tropical medicine and emerging infectious diseases. 9th ed. British: Saunders Elsevier; 2013. pp 597–601.
- 5.
Hartskeerl RA, Wagenaar JF. Leptospirosis. In: Kasper DL, Fauci A, Hauser S, Longo D, Jameson J, editors. Harrison’s principles of internal medicine. 19th ed. New York: McGraw-Hill Education; 2015. pp. 1140–5.
- 6. Reis EA, Hagan JE, Ribeiro GS, Teixeira-Carvalho A, Martins-Filho OA, Montgomery RR, et al. Cytokine response signatures in disease progression and development of severe clinical outcomes for leptospirosis. PLoS Negl Trop Dis. 2013; 7(9):e2457. pmid:24069500
- 7. Chirathaworn C, Kongpan S. Immune responses to Leptospira infection: roles as biomarkers for disease severity. Braz J Infect Dis. 2014; 18(1):77–81. pmid:24275371
- 8. Haake DA, Levett PN. Leptospirosis in humans. Curr Top Microbiol Immunol. 2015; 387:65–97. pmid:25388133
- 9. Cagliero J, Villanueva SYAM, Matsui M. Leptospirosis pathophysiology: Into the storm of cytokines. Front Cell Infect Microbiol. 2018; 8:204. pmid:29974037
- 10. Dupont H, Dupont–Perdrizet D, Perie JL. Leptospirosis: prognostic factors associated with mortality. Clin Infect Dis. 1997; 25(3):720–4. pmid:9314467
- 11. Panaphut T, Domrongkitchaiporn S, Thinkamrop B. Prognostic factors of death in leptospirosis: a prospective cohort study in Khon Kaen, Thailand. Int J Infect Dis. 2002; 6(1):52–9. pmid:12044303
- 12. Spichler AS, Vilaça PJ, Athanazio DA, Albuquerque JOM, Buzzar M, Castro B, et al. Predictors of lethality in severe leptospirosis in urban Brazil. Am J Trop Med Hyg. 2008; 79(6):911–4. pmid:19052303
- 13. Fann RJ, Vidya RR, Chong HE, Indralingam V, Chan WSC. Clinical presentations and predictors of mortality for leptospirosis- a study from suburban area in Malaysia. Med J Malaysia. 2020;75(1):52–6. pmid:32008021
- 14. Wang HK, Lee MH, Chen YC, Hsueh PR, Chang SC. Factors associated with severity and mortality in patients with confirmed leptospirosis at a regional hospital in northern Taiwan. J Microbiol Immunol Infect. 2020; 53(2):307–14. pmid:29934034
- 15. Azhari NN, Ramli SNA, Joseph N, Philip N, Mustapha NF, Ishak SN, et al. Molecular characterization of pathogenic Leptospira sp. in small mammals captured from the human leptospirosis suspected areas of Selangor state, Malaysia. Acta Trop. 2018; 188:68–77. pmid:30145261
- 16. Philip N, Jani J, Azhari NN, Sekawi Z, Neela V.K. In vivo and in silico virulence analysis Leptospira species isolated from environments and rodents in leptospirosis outbreak areas in Malaysia. Front Microbiol. 2021; 12:753328. pmid:34803975
- 17. Matsui M, Rouleau V, Bruyere-Ostells L, Goarant C. Gene expression profiles of immune mediators and histopathological findings in animal models of leptospirosis: comparison between susceptible hamsters and resistant mice. Infect Immun. 2011; 79(11):4480–92. pmid:21844232
- 18. Vernel-Pauillac F, Goarant C. Differential cytokine gene expression according to outcome in a hamster model of leptospirosis. PLoS Negl Trop Dis. 2010; 4(1):e582. pmid:20076757
- 19. Villanueva SY, Saito M, Tsutsumi Y, Segawa T, Baterna RA, Chakraborty A, et al. High virulence in hamsters of four dominant Leptospira serovars isolated from rats in the Philippines. Microbiology. 2014;160(2):418–28. pmid:24257815
- 20. Marinho M, Oliveira-Júnior IS, Monteiro CM, Perri SH, Salomão R. 2009. Pulmonary disease in hamsters infected with Leptospira interrogans: histopathologic findings and cytokine mRNA expressions. Am J Trop Med Hyg. 2009; 80(5):832–6. pmid:19407133
- 21. Stoddard RA, Gee JE, Wilkins PP, McCaustland K, Hoffmaster AR. 2009. Detection of pathogenic Leptospira spp. through TaqMan polymerase chain reaction targeting the lipL32 gene. Diagn Microbiol Infect Dis. 2009; 64(3):247–55. pmid:19395218
- 22. Vernel-Pauillac F, Merien F. Proinflammatory and immunomodulatory cytokine mRNA time course profiles in hamsters infected with a virulent variant of Leptospira interrogans. Infect Immun. 2006; 74(7):4172–9. pmid:16790792
- 23. Matsui M, Roche L, Geroult S, Soupé-Gilbert ME, Monchy D, Huerre M, et al. Cytokine and chemokine expression in kidneys during chronic leptospirosis in reservoir and susceptible animal models. PLoS One. 2016; 11(5):e0156084. pmid:27219334
- 24. Doudier B, Garcia S, Quennee V, Jarno P, Brouqui P. Prognostic factors associated with severe leptospirosis. Clin Microbiol Infect. 2006; 12(4):299–300. pmid:16524404
- 25. Hinjoy S, Kongyu S, Doung-Ngern P, Doungchawee G, Colombe SD, Tsukayama R, et al. Environmental and behavioral risk factors for severe leptospirosis in Thailand. Trop Med Infect Dis. 2019; 4(2):79. pmid:31100812
- 26. Spichler A, Moock M, Chapola EG, Vinetz J. Weil’s disease: an unusually fulminant presentation characterized by pulmonary hemorrhage and shock. Braz J Infect Dis. 2005; 9(4):336–40. pmid:16270127
- 27. Gulati S, Gulati A. Pulmonary manifestations of leptospirosis. Lung India. 2012; 29(4):347–53. pmid:23243349
- 28. Predescu A, Diaconu S, Tiuca N, Purcareanu A, Tomescu A, Cuciureanu D, et al. Leptospirosis-a case report. Internal Medicine. 2018; 15(4):45–53.
- 29. Haake DA. Hamster model of leptospirosis. Curr Protoc Microbiol. 2006; 2(1):12E–2. pmid:18770576
- 30. Coutinho ML, Matsunaga J, Wang LC, de la Peña Moctezuma A, Lewis MS, Babbitt JT, et al. Kinetics of Leptospira interrogans infection in hamsters after intradermal and subcutaneous challenge. PLoS Negl Trop Dis. 2014; 8(11):e3307. pmid:25411782
- 31. Nikaido Y, Ogawa M, Fukuda K, Yokoyama M, Kanemaru T, Nakayama T, et al. Transbronchial invasion and proliferationof Leptospira interrogans in lung without inflammatory cell infiltration in a hamster model. Infect Immun. 2019; 87(12):e00727–19. pmid:31548321
- 32. Barnacle J, Gurney S, Ledot S, Singh S. Leptospirosis as an important differential of pulmonary haemorrhage on the intensive care unit: a case managed with VV-ECMO. J Intensive Care. 2020; 8(1):1–7. pmid:32351698
- 33. Gouveia EL, Metcalfe J, de Carvalho ALF, Aires TSF, Villasboas-Bisneto JC, Queirroz A, et al. Leptospirosis-associated severe pulmonary hemorrhagic syndrome, Salvador, Brazil. Emerg Infect Dis. 2008; 14(3):505–8. pmid:18325275
- 34. Allen P, Raftery S, Phelan D. Massive pulmonary haemorrhage due to leptospirosis. Intensive Care Med. 1989; 15:322–4. pmid:2768647
- 35. Dong WH, Chen Z. Leptospirosis with pulmonary haemorrhage and multiple organ failure: a case report and literature review. J Int Med Res. 2021; 49(5):1–9. pmid:34044641
- 36. Wunder EA, Figueira CP, Benaroudj N, Hu B, Tong BA, Trajtenberg F, et al. A novel flagellar sheath protein, FcpA, determines filament coiling, translational motility and virulence for the Leptospira spirochete. Mol Microbiol. 2016;101(3):457–470. pmid:27113476
- 37. Wang B, Sullivan JA, Sullivan GW, Mandell GL. Role of specific antibody in interaction of leptospires with human monocytes and monocyte-derived macrophages. Infect Immun. 1984; 46(3):809–13. pmid:6500713
- 38. Isogai E, Isogai H, Wakizaka H, Miura H, Kurebayashi Y. Chemiluminescence and phagocytic responses of rat polymorphonuclear neutrophils to leptospires. Zentralbl Bakteriol. 1989; 272(1):36–46. pmid:2610812
- 39. Scharrig E, Carestia A, Ferrer MF, Cedola M, Pretre G, Drut R, et al. Neutrophil extracellular traps are involved in the innate immune response to infection with Leptospira. PloS Negl Trop Dis. 2015; 9(7):e0003927. pmid:26161745
- 40. Vieira ML, Teixeira AF, Pidde G, Ching ATC, Tambourgi DV, Nascimento A, et al. Leptospira interrogans outer membrane protein LipL21 is a potent inhibitor of neutrophil myeloperoxidase. Virulence. 2018; 9(1):414–25. pmid:29235397
- 41. Charo N, Scharrig E, Ferrer MF, Sanjuan N, Carrera Silva EA, Schattner M, et al. Leptospira species promote a pro-inflammatory phenotype in human neutrophils. Cell Microbiol. 2019; 21(2):e12990. pmid:30537301
- 42. Raffray L, Giry C, Vandroux D, Fayeulle S, Moiton MP, Gerber A, et al. The monocytosis during human leptospirosis is associated with modest immune cell activation states. Med Microbiol Immunol. 2019; 208(5):667–78. pmid:30542761
- 43. Isogai E, Kitagawa H, Isogai H, Matsuzawa T, Shimizu T, Yanagihara Y, et al. Effects of leptospiral lipopolysaccharide on rabbit platelets. Zentralbl Bakteriol. 1989; 271(2):186–96. pmid:2775427
- 44. Hoffmeister KM, Felbinger TW, Falet H, Denis CV, Bergmeier W, Mayadas TN, et al. The clearance mechanism of chilled blood platelets. Cell. 2003; 112(1):87–97. pmid:12526796
- 45. Tunjungputri RN, Gasem MH, van der Does W, Sasongko PH, Isbandrio B, Urbanus RT, et al. Platelet dysfunction contributes to bleeding complications in patients with probable leptospirosis. PLoS Negl Trop Dis. 2017; 11(9):e0005915. pmid:28934202
- 46. Fang JQ, Imran M, Hu WL, Ojcius DM, Li Y, Ge YM, et al. vWA proteins of Leptospira interrogans induce hemorrhage in leptospirosis by competitive inhibition of vWF/GPIb-mediated platelet aggregation. EBioMedicine. 2018; 37:428–41. pmid:30337247
- 47. Vieira ML, Nascimento ALTO. Virulent Leptospira interrogans induce cytotoxic effects in human platelets in vitro through direct interactions. Front Microbiol. 2020; 11:572972. pmid:33117318
- 48. Edwards CN, Nicholson GD, Everard CO. Thrombocytopenia in leptospirosis. Am J Trop Med Hyg. 1982; 31(4):827–9. pmid:7102918
- 49. Casiple LC. Thrombocytopenia and bleeding in leptospirosis. Phil J Microbiol Infect Dis. 1998; 27(1):18–22.
- 50. Nicodemo AC, Duarte MI, Alves VA, Takakura CF, Santos RT, Nicodemo EL. Lung lesions in human leptospirosis: microscopic, immunohistochemical, and ultrastructural features related to thrombocytopenia. Am J Trop Med Hyg. 1997; 56(2):181–7. pmid:9080878
- 51. Jaureguiberry S, Roussel M, Brinchault-Rabin G, Gacouin A, Le Meur A, Arvieux C, et al. Clinical presentation of leptospirosis: a retrospective study of 34 patients admitted to a single institution in metropolitan France. Clin microbiol Infect. 2005; 11(5):391–4. pmid:15819866
- 52. Craig SB, Graham GC, Burns MA, Dohnt MF, Smythe LD, McKay DB. Haematological and clinical-chemistry markers in patients presenting with leptospirosis: a comparison of the findings from uncomplicated cases with those seen in the severe disease. Ann Trop Med Parasitol. 2009; 103(4):333–41. pmid:19508751
- 53. Craig SB, Collet TA, Wynwood SJ, Smythe LD, Weier SL, McKay DB. Neutrophil counts in leptospirosis patients infected with different serovars. Trop Biomed. 2013; 30(4):579–83. pmid:24522125
- 54. De Silva NL, Niloofa MJR, Fernando N, Karunanayake L, Rodrigo C, De Silva HJ, et al. Changes in full blood count parameters in leptospirosis: a prospective study. Int Arch Med. 2014; 7(1):1–4. pmid:24387244
- 55. George T, Pais MLJ, Adnan M, Pereira R, Jakribettu RP, Baliga MS. Clinicolaboratory profile of leptospirosis: Observations from a tertiary care hospital. J Appl Hematol. 2020; 11(3):102–7. https://doi.org/10.4103/joah.joah_11_20.
- 56. Daher EF, Lima RS, Junior GBS, Silva EC, Karbage NN, Kataoka RS, et al.Clinical presentation of leptospirosis: a retrospective study of 201 patients in a metropolitan city of Brazil. Braz J Infect Dis. 2010; 14(1):3–10. pmid:20428646.
- 57. Goswami RP, Goswami RP, Basu A, Tripathi SK, Chakrabarti S, Chattopadhyay I. Predictors of mortality in leptospirosis: an observational study from two hospitals in Kolkata, eastern India. Trans R Soc Trop Med Hyg. 2014;108(12):791–6. pmid:25359320
- 58. Divakar A, Pillai MGK, Thomas E. Clinical profile of mortality predictors in leptospirosis: a prospective study in a tertiary care center. Int J Med Res Rev 2017; 5(9):857–64. https://doi.org/10.17511/ijmrr.2017.i09.05.
- 59. Sandhu RS, Ismail HB, Ja’afar MH, Rampal S. The predictive factors for severe leptospirosis cases in Kedah. Trop Med Infect Dis. 2020;5(2):79. pmid:32422911
- 60. De Fost M, Chierakul W, Limpaiboon R, Dondorp A, White NJ, van Der Poll T. Release of granzymes and chemokines in Thai patients with leptospirosis. Clin Microbiol Infect. 2007; 13(4):433–6. pmid:17359329
- 61. Kyriakidis I, Samara P, Papa A. Serum TNF-α, sTNFR1, IL-6, IL-8 and IL-10 levels in Weil’s syndrome. Cytokine. 2011; 54(2):117–20. pmid:21316985
- 62. Senavirathna I, Rathish D, Agampodi A. Cytokine response in human leptospirosis with different clinical outcomes: a systematic review. BMC Infect Dis. 2020; 20(1):268. pmid:32264832
- 63. Tomizawa R, Sugiyama H, Sato R, Ohnishi M, Koizumi N. Male-specific pulmonary hemorrhage and cytokine gene expression in golden hamster in early-phase Leptospira interrogans serovar Hebdomadis infection. Microb Pathog. 2017; 111:33–40. pmid:28811249
- 64. Lowanitchapat A, Payungporn S, Sereemaspun A, Ekpo P, Phulsuksombati D, Poovorawan Y, et al. Expression of TNF-alpha, TGF-beta, IP-10 and IL-10 mRNA in kidneys of hamsters infected with pathogenic Leptospira. Comp Immunol Microbiol Infect Dis. 2010; 33(5):423–34. pmid:19559480
- 65. Medeiros FR, Spichler A, Athanazio DA. Leptospirosis associated disturbances of blood vessels, lungs and hemostasis. Acta Trop. 2010; 115(1–2):155–62. pmid:20206112
- 66. Luks AM, Lakshminarayanan S, Hirschmann JV. Leptospirosis presenting as diffuse alveolar hemorrhage–case report and literature review. Chest. 2003; 123(2):639–43. pmid:12576395
- 67. Dolhnikoff M, Mauad T, Bethlem EP, Carvalho CR. Pathology and pathophysiology of pulmonary manifestations in leptospirosis. Braz J Infect Dis. 2007; 11(1): 142–8. pmid:17625743
- 68. Croda J, Neto AN, Brasil RA, Pagliari C, Nicodemo AC, Duarte MIS. Leptospirosis pulmonary haemorrhage syndrome is associated with linear deposition of immunoglobulin and complement on the alveolar surface. Clin Microbiol Infect. 2010; 16(6):593–9. pmid:19778300
- 69. De Brito T, Aiello VD, da Silva LFF, da Silva AMG, da Silva WLF, Castelli JB, et al. Human hemorrhagic pulmonary leptospirosis: pathological findings and pathophysiological correlations. PloS one. 2013; 8(8):e71743. pmid:23951234
- 70. Silva JJ, Dalston MO, Carvalho JE, Setubal S, Oliveira JM, Pereira MM. Clinicopathological and immunohistochemical features of the severe pulmonary form of leptospirosis. Rev Soc Bras Med Trop. 2002; 35(4):395–9. pmid:12170336
- 71. Evangelista K, Franco R, Schwab A, Coburn J. Leptospira interrogans binds to cadherins. PLoS Negl Trop Dis. 2014; 8(1):e2672. pmid:24498454
- 72. Evangelista KV, Hahn B, Wunder EA Jr, Ko AI, Haake DA, Coburn J. Identification of cell-binding adhesins of Leptospira interrogans. PLoS Negl Trop Dis. 2014; 8(10):e3215. pmid:25275630
- 73. Martinez-Lopez DG, Fahey M, Coburn J. Responses of human endothelial cells to pathogenic and non-pathogenic Leptospira species. PLoS Negl Trop Dis. 2010; 4(12):e918. pmid:21179504
- 74. Chen Q, Wang Q, Zhu J, Xiao Q, Zhang L. Reactive oxygen species: key regulators in vascular health and diseases. Br J Pharmacol. 2018; 175(8):1279–92. pmid:28430357
- 75. Mantovani A, Cassatella MA, Costantini C, Jaillon S. Neutrophils in the activation and regulation of innate and adaptive immunity. Nat Rev Immunol. 2011; 11(8):519–31. pmid:21785456
- 76. Finder J, Stark WW Jr, Nakayama DK, Geller D, Wasserloos K, Pitt BR, et al. TGF-beta regulates production of NO in pulmonary artery smooth muscle cells by inhibiting expression of NOS. Am J Physiol. 1995; 268(5):L862–7. pmid:7539224
- 77. Vodovotz Y. Control of nitric oxide production by transforming growth factor-β1: mechanistic insights and potential relevance to human disease. Nitric oxide. 1997; 1(1):3–17. pmid:9701040
- 78. Levine AB, Punihaole D, Levine TB. Characterization of the role of nitric oxide and its clinical applications. Cardiology. 2012; 122(1):55–68. pmid:22722323
- 79. Bruckdorfer KR, Dee G, Jacobs M, Rice-Evans CA. The protective action of nitric oxide against membrane damage induced by myoglobin radicals. Bio-chem Soc Trans. 1990; 18(2):285–6. pmid:2379721
- 80. Von Knethen A, Brüne B. Activation of peroxisome proliferator-activated receptor γ by nitric oxide in monocytes/macrophages down-regulates p47phox and attenuates the respiratory burst. J Immunol. 2002; 169(5):2619–26. pmid:12193733
- 81. Pautz A, Art S, Hahn S, Nowag S, Voss C, Kleinert H. Regulation of the expression of inducible nitric oxide synthase. Nitric Oxide. 2010; 23(2):211–67. pmid:20438856
- 82. Lowenstein CJ, Padalko E. iNOS (NOS2) at a glance. J Cell Sci. 2004; 117(14):2865–7. pmid:15197240
- 83. d’Andon MF, Quellard N, Fernandez B, Ratet G, Lacroix-Lamandé S, Vandewalle A, et al. Leptospira interrogans induces fibrosis in the mouse kidney through Inos-dependent, TLR-and NLR-independent signaling pathways. Plos Negl Trop Dis. 2014; 8(1):e2664. pmid:24498450
- 84. Pretre G, Olivera N, Cedola M, Haase S, Alberdi L, Brihuega B, et al. Role of inducible nitric oxide synthase in the pathogenesis of experimental leptospirosis. Microb Pathog. 2011; 51(3):203–8. pmid:21497651
- 85. Avdeeva MG, Bondarenko IN, Peredirii IR. Clinical significance of the activity of nitric oxide synthase in patients with leptospirosis. Klin Lab Diagn. 2008; 1:40–3. pmid:18314776
- 86. Cordonin C, Turpin M, Bascands JL, Dellagi K, Mavingui P, Tortosa P, et al. Three Leptospira strains from western Indian ocean wildlife show highly distinct virulence phenotypes through hamster experimental infection. Front Microbiol. 2019; 10:382. pmid:30915044
- 87. MacNee W. ABC of chronic obstructive pulmonary disease: Pathology, pathogenesis, and pathophysiology. BMJ. 2006; 332(7551):1202–1204.
- 88. Lappalainen U, Whitsett JA, Wert SE, Tichelaar JW, Bry K. Interleukin-1beta causes pulmonary inflammation, emphysema, and airway remodeling in the adult murine lung. Am J Respir Cell Mol Biol. 2005; 32(4):311–8. pmid:15668323
- 89. Churg A, Zhou S, Wang X, Wang R, Wright JL. The role of interleukin-1beta in murine cigarette smoke-induced emphysema and small airway remodeling. Am J Respir Cell Mol Biol. 2009; 40(4):482–90. pmid:18931327
- 90. Couillin I, Vasseur V, Charron S, Gasse P, Tavernier M, Guillet J, et al. IL-1R1/MyD88 signaling is critical for elastase-induced lung inflammation and emphysema. J Immunol. 2009; 183(12):8195–202. pmid:20007584
- 91. Pauwels NS, Bracke KR, Dupont LL, Pottelberge GRV, Provoost S, Berghe TV, et al. Role of IL-1alpha and the Nlrp3/caspase-1/IL-1beta axis in cigarette smoke-induced pulmonary inflammation and COPD. Eur Respir J. 2011; 38(5):1019–28. pmid:21622588
- 92. Carpio C, Villasante C, Galera R, Romero D, de Cos A, Hernanz A, et al. Systemic inflammation and higher perception of dyspnea mimicking asthma in obese subjects. J Allergy Clin Immunol. 2016; 137(3):718–26. pmid:26768410
- 93. Zou Y, Chen X, Liu J, Zhou DB, Kuang X, Xiao J, et al. Serum IL-1β and IL-17 levels in patients with COPD: associations with clinical parameters. Int J Chron Obstruct Pulmon Dis. 2017; 12:1247–54. pmid:28490868
- 94. Neville LF, Mathiak G, Bagasra O. The immunobiology of interferon-gamma inducible protein 10 kD (IP-10): a novel, pleiotropic member of the C-X-C chemokine superfamily. Cytokine Growth Factor Rev. 1997; 8(3):207–19. pmid:9462486
- 95. Maurer M, von Stebut E. Macrophage inflammatory protein-1. Int J Biochem Cell Biol. 2004; 36(10):1882–6. pmid:15203102
- 96. Vazirinejad R, Ahmadi Z, Arababadi MK, Hassanshahi G, Kennedy D. The biological functions, structure and sources of CXCL10 and its outstanding part in the pathophysiology of multiple sclerosis. Neuroimmunomodulation. 2014; 21(6):322–30. pmid:24642726
- 97.
Bhavsar I, Miller CS, Al-Sabbagh M. Macrophage inflammatory protein-1 alpha (MIP-1 alpha)/CCL3: as a biomarker. In: Preedy VR, Patel VB, editors. General methods in biomarker research and their applications. Switzerland: Springer Nature; 2015.pp. 223–49.
- 98. Tisoncik JR, Korth MJ, Simmons CP, Farrar J, Martin TR, Katze MG. Into the eye of the cytokine storm. Microbiol Mol Biol Rev. 2012; 76(1):16–32. pmid:22390970
- 99. Zhao HQ, Li WM, Lu ZQ, Sheng ZY, Yao YM. The growing spectrum of anti-inflammatory interleukins and their potential roles in the development of sepsis. J. Interferon Cytokine Res. 2015; 35(4):242–51. pmid:25517926
- 100. Cohen J. The immunopathogenesis of sepsis. Nature. 2002; 420(6917):885–91. pmid:12490963
- 101. Tajiki MH, Nakama ASY, Salomão R. The ratio of plasma levels Of Il-10/Tnf-alpha and its relationship to disease severity and survival in patients with leptospirosis. Braz J Infect Dis. 1997; 1(3):138–41. pmid:11105129
- 102. Mikulski M, Boisier P, Lacassin F, Soupe-Gilbert ME, Mauron C, Bruyere-Ostells L, et al. Severity markers in severe leptospirosis: a cohort study. Eur J clin Microbiol Infect Dis. 2015; 34(4):687–95. pmid:25413923
- 103. Ittyachen AM. Covid-19 and leptospirosis: Cytokine storm and the use of steroids. Trop Doct. 2021; 51(1):128–30. pmid:33236692
- 104. Bennett MR, Devarajan P. Proteomic analysis of acute kidney injury: biomarkers to mechanisms. Proteomics Clin Appl. 2011; 5(1–2):67–77. pmid:21280238
- 105. Johann DJ Jr, Veenstra TD. Multiple biomarkers in molecular oncology. Expert Rev Mol Diagn. 2007; 7:223–5. pmid:17489728
- 106. Chirathaworn C, Supputtamongkol Y, Lertmaharit S, Poovorawan Y. Cytokine levels as biomarkers for leptospirosis patients. Cytokine. 2016; 85:80–2. pmid:27295614