https://orcid.org/0000-0002-1670-8332
Figures
Abstract
Visceral leishmaniasis (VL) is a neglected tropical disease caused by parasites from the Leishmania (L.) donovani complex. VL is characterised by uncontrolled parasite replication in spleen, liver and bone marrow, and by an impaired immune response and high systemic levels of inflammation. Monocytes have been poorly characterised in VL patients. The aim of this study was to evaluate the expression levels of markers involved in the regulation of T cell responses on different subsets of monocytes from the blood of VL patients and healthy non-endemic controls (HNEC). Monocytes can broadly be divided into three subsets: classical, intermediate and non-classical monocytes. Our results show that the percentages of all three subsets stayed similar at the time of VL diagnosis (ToD) and at the end of anti-leishmanial treatment (EoT). We first looked at co-stimulatory receptors: the expression levels of CD40 were significantly increased on classical and intermediate, but not non-classical monocytes, at ToD as compared to EoT and HNEC. CD80 expression levels were also increased on intermediate monocytes at ToD as compared to EoT and HNEC, and on classical monocytes only as compared to HNEC. The levels of CD86 were similar at EoT and ToD and in HNEC on classical and intermediate monocytes, but significantly higher at EoT on non-classical monocytes. We also looked at an inhibitory molecule, PD-L1. Our results show that the expression levels of PD-L1 were significantly higher on all three monocyte subsets at ToD as compared to HNEC, and to EoT on classical and intermediate monocytes. These results show that monocytes from the blood of VL patients upregulate both co-stimulatory and inhibitory receptors and that their expression levels are restored at EoT.
Author summary
Visceral leishmaniasis (VL) is a neglected tropical disease caused by Leishmania parasites. VL is characterised by uncontrolled parasite replication in internal organs and is often fatal if untreated.
Monocytes are part of a group of cells called white blood cells. These cells can become activated and play an important role against infections. They have been poorly characterised in VL patients. The aim of this study was to evaluate the expression levels of markers of activation on different subsets of monocytes from the blood of VL patients and healthy non-endemic controls (HNEC).
We assessed the expression levels of monocyte markers that are essential for activating and regulating the immune response. Our results how that these are increased on some monocyte subsets at the time of diagnosis as compared to the end of treatment and HNEC. We also looked at a marker that can inhibit the immune response and show that it is highly expressed at the time of diagnosis as compared to the end of treatment and HNEC.
These results show that monocytes from the blood of VL patients upregulate both activating and inhibitory receptors and that their expression levels are restored at the end of treatment.
Citation: Adem E, Yizengaw E, Mulaw T, Nibret E, Müller I, Takele Y, et al. (2024) Altered co-stimulatory and inhibitory receptors on monocyte subsets in patients with visceral leishmaniasis. PLoS Negl Trop Dis 18(8): e0012417. https://doi.org/10.1371/journal.pntd.0012417
Editor: Susan Madison-Antenucci, Wadsworth Center, UNITED STATES OF AMERICA
Received: March 18, 2024; Accepted: July 30, 2024; Published: August 19, 2024
Copyright: © 2024 Adem 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: Y.T. is funded by a Wellcome Trust Training Fellowship in Public Health and Tropical Medicine (204797/Z/16/Z). 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
Visceral leishmaniasis (VL) is caused by parasites from the Leishmania donovani complex, that are transmitted during the blood meal of sand fly vectors. It is endemic in 80 countries, with 12,773 cases reported in 2022 [1]. However, the real number of VL cases are likely to be significantly higher, as the surveillance systems in place are often inadequate and VL occurs in places that are often difficult to access [2,3]. 90% of VL cases occur in India, Bangladesh, Nepal, Sudan, South Sudan, Ethiopia and Brazil [2].
Whereas the majority of individuals infected with L. donovani remain asymptomatic, infection can also develop as a progressive disease. Common symptoms are fever, weight loss, hepatosplenomegaly and pancytopenia [4]. VL patients require hospitalisation and treatment. Existing treatments can cause severe side effects and have long durations. Drug resistance is also a growing problem [5]. One of the primary immunological features of VL patients is their profound immunosuppression: typically, whole blood cells and peripheral blood mononuclear cells (PBMCs) from VL patients display a reduced ability to produce IFNγ and proliferate when exposed to Leishmania antigen; and the leishmanin skin test does not induce a delayed-type hypersensitivity reaction. These impaired responses to antigen challenge improve after successful chemotherapy (reviewed in [6–8].
Monocytes in VL patients have been poorly characterised. Monocytes are heterogeneous and can be divided into at least three subsets, based on the expression levels of CD14 and CD16: CD14highCD16low (classical), CD14highCD16high (intermediate) and CD14lowCD16high (non-classical) [9,10]. It has been shown that classical monocytes emerge first in circulation and gradually change their expression levels of CD14 and CD16 into first intermediate monocytes and finally into non-classical monocytes [11]. These three subsets can also each display different effector functions: some of the main functions of classical monocytes are the ability to phagocytose, migrate and produce anti-microbial responses; intermediate monocytes specialise in antigen presentation, regulation of apoptosis and transendothelial migration; and non-classical monocytes into complement and FcR-mediated phagocytosis, transendothelial migration and adhesion (summarised in [12]).
Initial studies showed that monocytes can phagocytose and kill intracellular L. donovani by oxidative burst [13–15]. Hoover et al. showed that L. donovani can replicate within monocytes and that activation of these monocytes with IFNγ results in killing of the parasites [16]; this can be counteracted by IL-4 [17]. Monocytes infected with L. donovani did not produce TNFα and IL-1, unless primed with staphylococcus aureus or lipopolysaccharide [18]. Monocytes pre-treated with lipophosphoglycan (LPG), a glycolipid present on the surface of parasites, display a lower oxidative burst [19], showing that LPG plays a crucial role in the survival of Leishmania parasites in monocytes.
Monocytes from VL patients express lower levels of HLA-DR [20]; as well as CD54 and CD86 and display an impaired oxidative burst [21]. Singh et al. showed that monocytes isolated from VL patients exhibit an anti-inflammatory phenotype, characterised by decreased parasite phagocytosis and impaired oxidative burst [22]. We have recently shown that PD-L1 is increased on all three subsets of monocytes at the time of VL diagnosis [23]. In the current study, we extend these results to the analyses of other molecules also involved in the modulation of T cell responses, such as CD40, CD80 and CD86. We assessed their expression levels on the three different subsets of monocytes at the time of VL diagnosis (ToD) and the end of treatment (EoT). These parameters were compared with those of healthy non-endemic controls.
Materials and methods
Ethics statement
The study was approved by the Institutional Review Board of the University of Gondar (IRB, reference O/V/P/RCS/05/1572/2017).
Subjects and sample collection
For this cross-sectional study, 20 male VL patients were recruited at the time of diagnosis (ToD) from the Leishmaniasis Treatment and Research Centre of the Gondar University Hospital; a further 20 males were recruited at the end of successful treatment (EoT), as defined by patients looking improved, afebrile, and having smaller spleen and liver sizes than on admission [24]. 10 non-endemic male controls with no prior history of VL were recruited amongst the staff of the University of Gondar. The median ages of the three cohorts were similar: VL patients at ToD: 25 [20–30]; VL patients at EoT: 23.5 [18.5–26.8] and controls: 24 [20.5–27.3] years old (p = 0.7495). Informed written consent was obtained from each patient and control. The exclusion criteria were age (<18 years) and co-infection with HIV. The diagnosis of VL was based on positive serology (rK39) and the presence of amastigotes in spleen or bone marrow aspirates [25]. Patients were treated with a combination of sodium stibogluconate (SSG, 20mg/kg body weight/day) and paromomycin (PM, 15mg/kg body weight/day) injections, given intramuscularly for 17 days.
Ten ml of blood was collected in heparin tubes and was processed immediately after collection: following density gradient centrifugation on Histopaque-1077 (Sigma), the peripheral mononuclear cells (PBMCs) were isolated from the interphase and were used immediately for flowcytometry.
Flow cytometry
The different subsets of monocytes were defined based on the expression levels of CD14 and CD16 (S1A Fig for healthy non-endemic controls, S2A Fig for VL patients at time of diagnosis and S3A Fig for VL patients at the end of treatment). The following antibodies were used: anti-CD14APC (clone 61D3, eBioscience), anti-CD16PE (clone B73.1, eBioscience) and anti-CD16FITC (clone B73.1, Biolegend). The following antibodies were used to assess the levels of CD40: anti-CD40PE-Cyanine7 (clone 5C3, eBioscience); CD80: anti-CD80FITC (clone 2D10.4, eBioscience); CD86: anti-CD86pe-cyanine7(clone IT2.2, eBioscience); and PD-L1: anti-PD-L1PE (clone MIH1, eBioscience). The percentages for the isotype controls were <1%.
Acquisition was performed using a BD Accuri C6 flow cytometer, at least 5,000 monocytes were acquired, and data were analysed using BD Accuri C6 analysis software.
Results
Percentages of monocyte subsets
We first determined whether the percentages of the three subsets of monocytes: classical (CD14high CD16low), intermediate (CD14high CD16high) and non-classical (CD14low CD16high) differ between VL patients at the time of diagnosis (ToD) and at the end of treatment (EoT) and as compared to healthy non-endemic controls (HNEC). As expected, the percentages of classical monocytes were higher as compared to the intermediate and the non-classical subsets (Fig 1, Table 1). There were no differences between the percentages of the different subsets at ToD and EoT (Fig 1 and Table 1); as well as in the percentages of classical monocytes at ToD in VL patients and controls, however, there were significantly less classical and more intermediate monocytes in the PBMCs of VL patients at EoT as compared to HNEC (Fig 1, Table 1).
PBMCs were purified from VL patients at ToD (n = 20) and EoT (n = 20) and from HNEC (n = 10) as described in Materials and Methods. The percentages of each subset were determined by flow cytometry, as described in S1A, S2A and S3A Figs. Each symbol represents the value for one individual and the straight line represents the median. Statistical differences shown on this figure between VL patients at ToD and EoT and HNEC were determined by Dunn’s multiple comparisons test. ToD = Time of Diagnosis; EoT = End of Treatment; HNEC = healthy non-endemic controls.
Activation status of the different monocyte subsets
We first measured the expression levels of CD14 and CD16. Results presented in Table 2 show no significant differences between the different subsets in VL patients at ToD and EoT and in HNEC.
Next, we measured the expression levels of the co-stimulatory molecules CD40, CD80 and CD86 and the immune regulatory molecule PD-L1 on the different monocyte subsets (S1, S2 and S3 Figs).
Results presented in Fig 2 and Table 3 show that CD40 MFI was significantly higher at ToD as compared to EoT and as compared to HNEC on the classical and intermediate monocyte subsets isolated from VL patients. There were no significant differences for these 2 subsets between VL patients at EoT and HNEC; or between VL patients at ToD and EoT and HNEC on non-classical monocytes (Fig 2 and Table 3). Of note, the intermediate monocytes from VL patients expressed the highest levels of CD40 as compared to the classical and the non-classical monocytes at both time points. The MFI of CD40 was similar on all three subsets of monocytes from HNEC (S1 Table).
PBMCs were purified from VL patients at ToD (n = 20) and EoT (n = 20) and from HNEC (n = 10) as described in Materials and Methods. The expression levels (MFI = Median Fluorescence Intensity) of CD40 were measured on the different monocyte subsets by flow cytometry as described in Materials and Methods (S1B, S2B and S3B Figs). Each symbol represents the value for one individual and the straight line represents the median. Statistical differences shown on this figure between VL patients at ToD and EoT and HNEC were determined by Dunn’s multiple comparisons test. ToD = Time of Diagnosis; EoT = End of Treatment; HNEC = healthy non-endemic controls.
CD80 MFI was significantly higher on classical and intermediate monocytes from VL patients at ToD as compared to HNEC and between ToD and EoT on intermediate monocytes. There were no significant differences between VL patients at EoT and HNEC; or between VL patients at ToD and EoT and HNEC on non-classical monocytes (Fig 3 and Table 4). The classical monocytes from VL patients at EoT and ToD and from the HNEC expressed the lowest levels of CD80 (S2 Table).
PBMCs were purified from VL patients at ToD (n = 17) and EoT (n = 17) and from HNEC (n = 10) as described in Materials and Methods. The expression levels (MFI = Median Fluorescence Intensity) of CD80 were measured on the different monocyte subsets by flow cytometry as described in Materials and Methods (S1C, S2C and S3C Figs). Each symbol represents the value for one individual and the straight line represents the median. Statistical differences shown on this figure between VL patients at ToD and EoT and HNEC were determined by Dunn’s multiple comparisons test. ToD = Time of Diagnosis; EoT = End of Treatment; HNEC = healthy non-endemic controls.
CD86 was significantly lower on non-classical monocytes at ToD as compared to EoT. All the other comparisons were not significant (Fig 4 and Table 5). Whereas intermediate and non-classical monocytes from HNEC and VL patients at EoT expressed higher levels of CD86 as compared to the classical monocytes (Table S3), at ToD, the intermediate subsets expressed the highest levels of CD86 (S3 Table).
PBMCs were purified from VL patients at ToD (n = 20) and EoT (n = 20) and from HNEC (n = 10) as described in Materials and Methods. The expression levels (MFI = Median Fluorescence Intensity) of CD86 were measured on the different monocyte subsets by flow cytometry as described in Materials and Methods (S1D, S2D and S3D Figs). Each symbol represents the value for one individual and the straight line represents the median. Statistical differences shown on this figure between VL patients at ToD and EoT and HNEC were determined by Dunn’s multiple comparisons test. ToD = Time of Diagnosis; EoT = End of Treatment; HNEC = healthy non-endemic controls.
Results presented in Fig 5 and Table 6 show that at ToD, PD-L1 expression levels on all three different subsets were significantly higher as compared to those from HNEC. PDL-1 MFI decreased significantly at EoT on classical and intermediate, but not on non-classical monocytes. There were no significant differences in PD-L1 MFI between EoT and HNEC in all three monocyte subsets. Whereas intermediate and non-classical monocytes from HNEC and VL patients at EoT expressed higher levels of PD-L1 as compared to the classical monocytes (S4 Table), at ToD, the intermediate expressed the highest levels of PD-L1 (S4 Table).
PBMCs were purified from VL patients at ToD (n = 20) and EoT (n = 20) and from HNEC (n = 10) as described in Materials and Methods. The expression levels (MFI = Median Fluorescence Intensity) of PD-L1 were measured on the different monocyte subsets by flow cytometry as described in Materials and Methods (S1E, S2E and S3E Figs). Each symbol represents the value for one individual and the straight line represents the median. Statistical differences shown on this figure between VL patients at ToD and EoT and HNEC were determined by Dunn’s multiple comparisons test. ToD = Time of Diagnosis; EoT = End of Treatment; HNEC = healthy non-endemic controls.
Discussion
Our results show that the percentages of classical, intermediate and non-classical monocytes in the blood of VL patients are similar at ToD and EoT. However, the expression levels of CD40, CD80, CD86 and PD-L1 vary on the different monocyte subsets and some of these markers change their expression levels at the end of anti-leishmanial treatment. Our results also show that at EoT, all of these markers are back to similar levels as those from HNEC.
CD40 is a member of the tumor necrosis factor (TNF) receptor family and is expressed on B cells, monocytes, dendritic cells, macrophages as well as on non-hematopoietic cells such as fibroblast (summarised in [26]). Its ligand, CD40L, is predominantly expressed on activated T and B cells and on platelets and can be shed (soluble CD40L (sCD40L)). CD40/CD40L interaction stimulates cells to produce chemokines and cytokines, and upregulate co-stimulatory and adhesion molecules, as well as enzymes, such as matrix metalloproteinases [27,28].
Little is known about CD40 expression on monocytes in VL patients. The levels of the soluble form of its ligand, sCD40L, have been shown to be lower in plasma of HIV/VL patients as compared to patients with HIV asymptomatically co-infected with Leishmania parasites [29]. De Oliveira et al. showed that Leishmania infantum-infected macrophages exposed to sCD40L harbour less parasites [30]. High levels of sCD40L in serum are associated with successful anti-leishmanial treatment [31]. Here we show that CD40 MFI is increased on both classical and intermediate monocytes at ToD and decreased at EoT. CD40 is expressed at low levels on monocytes and its upregulation is associated to inflammatory conditions such as atherosclerosis [32], multiple sclerosis [33] and tuberculosis [34]. It is therefore possible that the increased CD40 MFI measured on monocytes isolated from VL patients at ToD is a result of the high level of inflammatory cytokines we measured in the plasma of these patients [35]. The levels of CD40 MFI could also be increased as a positive feedback loop to boost T cell responses, as these are inefficient during the active phase of VL [35]. At EoT, CD40 expression levels decreased, concomitantly with a partial restoration of T cell response and lower levels of inflammatory cytokines [35]. Since CD40 was upregulated on classical and intermediate, but not on non-classical monocytes, these results suggest that the latter subset might not be as important in T cell activation via CD40.
CD80 and CD86 bind to two receptors on the surface of T cells: CD28 and cytotoxic T-lymphocyte associated protein 4 (CTLA-4). The interaction with CD28 results in T cell activation and that with CTLA-4 in T cell suppression [36]. CD80 binds more efficiently to CTLA-4 as compared to CD28, and CD86 binds to both receptors less efficiently than CD80 [37]. It has also been shown that CD80 can interact with PD-L1 and thereby prevent the binding of PD-1 with PD-L1 [38]. Here we show that the expression levels of CD80 were higher at ToD on classical and intermediate monocytes. Since CD80 has a higher affinity to CTLA-4, this might contribute to the impaired antigen-specific responses observed at ToD [35]. CD86 was significantly higher on non-classical monocytes at EoT as compared to ToD. This suggests that non-classical monocyte might play a role in T cell activation via CD86 at ToD.
We have recently shown that PD-L1 was increased on all three subsets of monocytes at ToD [23]. Here we extended these results to EoT and show that PD-L1 was significantly decreased on classical and intermediate monocytes. This concurs with the more efficient immune response observed at EoT in VL patients, and indeed, our previous results show that PD1 was also lower on CD4+ T cells at EoT [35].
In summary our results show for the first time that that monocytes isolated from VL patients in Ethiopia upregulate both co-stimulatory and inhibitory molecules during VL and that at EoT, these levels are similar to those of HNEC. We haven’t measured the levels of CD28, CD40L and CTLA-4 on T cells at ToD and EoT. All these molecules exist as a soluble form and could interact with their receptors on monocytes: for example, sCTLA-4 could interact with CD80/86 and thereby prevent the binding with CD28 and T cell activation, as shown in autoimmune diseases [39]. The interaction of the inhibitory molecules PD-1 and PD-L1 is better characterised in VL: we have recently shown that IFNγ production is improved by inhibition of PD-1/PD-L1 ligation [23]. Therefore, the high expression of both molecules at ToD and their lower expression at EoT is in agreement with the efficiency of the immune response at these two time points [35].
Even though monocytes can be broadly divided into classical, intermediate and non-classical subsets based on the expression levels of CD14 and CD16, discrepancies, as well as redundancies in effector functions have been shown between the different subsets (summarised in [10]). Recently, more subsets have been identified with techniques including mass cytometry [40] and by using other markers such as CD62L, CD49d and CD43 and size [41]. Due to the heterogeneity of monocytes, conducting more detailed functional and phenotypic analyses of the different subsets will contribute to a better understanding of the role of these cells in the immunopathology of visceral leishmaniasis.
Supporting information
S1 Fig. PBMCs were purified from healthy non-endemic controls as described in Materials and Methods.
A. Identification of non-classical (R1), intermediate (R2) and classical (R3) monocytes based on the expression levels of CD14 and CD16. Histogram representing the expression level of CD40 (B), CD80 (C), CD86 (D) and PDL-1 (E) in non-classical (R1), intermediate (R2) and classical (R3) monocytes as measured by flow cytometry as described in Materials and Methods. One representative histogram is shown for each cell surface marker.
https://doi.org/10.1371/journal.pntd.0012417.s001
(TIFF)
S2 Fig. PBMCs were purified from VL patients at time of diagnosis as described in Materials and Methods.
A. Identification of non-classical (R1), intermediate (R2) and classical (R3) monocytes based on the expression levels of CD14 and CD16. Histogram representing the expression level of CD40 (B), CD80 (C), CD86 (D) and PDL-1 (E) in non-classical (R1), intermediate (R2) and classical (R3) monocytes as measured by flow cytometry as described in Materials and Methods. One representative histogram is shown for each cell surface marker.
https://doi.org/10.1371/journal.pntd.0012417.s002
(TIFF)
S3 Fig. PBMCs were purified from VL patients at the end of treatment as described in Materials and Methods.
A. Identification of non-classical (R1), intermediate (R2) and classical (R3) monocytes based on the expression levels of CD14 and CD16. Histogram representing the expression level of CD40 (B), CD80 (C), CD86 (D) and PD-L1 (E) in non-classical (R1), intermediate (R2) and classical (R3) monocytes as measured by flow cytometry as described in Materials and Methods. One representative histogram is shown for each cell surface marker.
https://doi.org/10.1371/journal.pntd.0012417.s003
(TIFF)
S1 Table. CD40 MFI on monocyte subsets from VL patients at ToD and EoT and on monocytes from HNEC.
PBMCs were purified from VL patients at ToD (n = 20) and EoT (n = 20) and from HNEC (n = 10) and the expression levels (MFI = Median Fluorescence Intensity) of CD40 were measured on the different monocyte subsets by flow cytometry. Results are presented as median with interquartile range. Statistical differences were determined by Kruskall-Wallis test (*) and Dunn’s multiple comparisons test (#). ToD = Time of Diagnosis; EoT = End of Treatment; HNEC = healthy non-endemic controls. C = classical monocytes. I = intermediate monocytes. NC = non-classical monocytes.
https://doi.org/10.1371/journal.pntd.0012417.s004
(DOCX)
S2 Table. CD80 MFI on monocyte subsets from VL patients at ToD and EoT and on monocytes from HNEC.
PBMCs were purified from VL patients at ToD (n = 17) and EoT (n = 17) and from HNEC (n = 10) and the expression levels (MFI = Median Fluorescence Intensity) of CD80 were measured on the different monocyte subsets by flow cytometry. Results are presented as median with interquartile range. Statistical differences were determined by Kruskall-Wallis test (*) and Dunn’s multiple comparisons test (#). ToD = Time of Diagnosis; EoT = End of Treatment; HNEC = healthy non-endemic controls. C = classical monocytes. I = intermediate monocytes. NC = non-classical monocytes.
https://doi.org/10.1371/journal.pntd.0012417.s005
(DOCX)
S3 Table. CD86MFI on monocyte subsets from VL patients at ToD and EoT and on monocytes from HNEC.
PBMCs were purified from VL patients at ToD (n = 20) and EoT (n = 20) and from HNEC (n = 10) and the expression levels (MFI = Median Fluorescence Intensity) of CD86 were measured on the different monocyte subsets by flow cytometry. Results are presented as median with interquartile range. Statistical differences were determined by Kruskall-Wallis test (*) and Dunn’s multiple comparisons test (#). ToD = Time of Diagnosis; EoT = End of Treatment; HNEC = healthy non-endemic controls. C = classical monocytes. I = intermediate monocytes. NC = non-classical monocytes.
https://doi.org/10.1371/journal.pntd.0012417.s006
(DOCX)
S4 Table. PD-L1 MFI on monocyte subsets from VL patients at ToD and EoT and on monocytes from HNEC.
PBMCs were purified from VL patients at ToD (n = 20) and EoT (n = 20) and from HNEC (n = 10) and the expression levels (MFI = Median Fluorescence Intensity) of PD-L1 were measured on the different monocyte subsets by flow cytometry. Results are presented as median with interquartile range. Statistical differences were determined by Kruskall-Wallis test (*) and Dunn’s multiple comparisons test (#). ToD = Time of Diagnosis; EoT = End of Treatment; HNEC = healthy non-endemic controls. C = classical monocytes. I = intermediate monocytes. NC = non-classical monocytes.
https://doi.org/10.1371/journal.pntd.0012417.s007
(DOCX)
Acknowledgments
We thank Arega Yeshanew, Roma Melkamu, Saba Atnafu, Aschalew Tamiru, Zemenay Mulugeta, Tigist Mekonen, Yohannes Sinku and Eden G/Hiwot from the University of Gondar for their invaluable help and enthusiastic cooperation in this study.
References
- 1. Ruiz-Postigo JA, Jain S, Madjou S, Virrey Agua JF, Maia-Elkhoury AN, Valadas S, et al. Global leishmaniasis surveillance, 2022: assessing trends over the past 10 years. Weekly epidemiological record. 2023;40(98): 471–87.
- 2. Bern C, Maguire JH, Alvar J. Complexities of assessing the disease burden attributable to leishmaniasis. PLoS Negl Trop Dis. 2008;2(10):e313. Epub 2008/10/30. pmid:18958165; PubMed Central PMCID: PMC2569207.
- 3. Alvar J, Velez ID, Bern C, Herrero M, Desjeux P, Cano J, et al. Leishmaniasis Worldwide and Global Estimates of Its Incidence. PLoS ONE. 2012;7(5):e35671. Epub 2012/06/14. PONE-D-11-24894 [pii]. pmid:22693548; PubMed Central PMCID: PMC3365071.
- 4. Davidson RN. Visceral leishmaniasis in clinical practice. J Infect. 1999;39(2):112–6. Epub 1999/12/28. pmid:10609527.
- 5. Hendrickx S, Guerin PJ, Caljon G, Croft SL, Maes L. Evaluating drug resistance in visceral leishmaniasis: the challenges. Parasitology. 2018;145(4):453–63. Epub 20161121. pmid:27866478; PubMed Central PMCID: PMC5989324.
- 6. Nylen S, Sacks D. Interleukin-10 and the pathogenesis of human visceral leishmaniasis. Trends Immunol. 2007;28(9):378–84. pmid:17689290.
- 7. Goto H, Prianti MG. Immunoactivation and immunopathogeny during active visceral leishmaniasis. Rev Inst Med Trop Sao Paulo. 2009;51(5):241–6. Epub 2009/11/07. [pii]. pmid:19893975.
- 8. Kumar R, Nylen S. Immunobiology of visceral leishmaniasis. Front Immunol. 2012;3:251. Epub 2012/08/23. pmid:22912637; PubMed Central PMCID: PMC3418610.
- 9. Ziegler-Heitbrock L, Ancuta P, Crowe S, Dalod M, Grau V, Hart DN, et al. Nomenclature of monocytes and dendritic cells in blood. Blood. 2010;116(16):e74–80. pmid:20628149.
- 10. Wong KL, Yeap WH, Tai JJ, Ong SM, Dang TM, Wong SC. The three human monocyte subsets: implications for health and disease. Immunol Res. 2012;53(1–3):41–57. pmid:22430559.
- 11. Patel AA, Zhang Y, Fullerton JN, Boelen L, Rongvaux A, Maini AA, et al. The fate and lifespan of human monocyte subsets in steady state and systemic inflammation. J Exp Med. 2017;214(7):1913–23. Epub 20170612. pmid:28606987; PubMed Central PMCID: PMC5502436.
- 12. Kapellos TS, Bonaguro L, Gemund I, Reusch N, Saglam A, Hinkley ER, et al. Human Monocyte Subsets and Phenotypes in Major Chronic Inflammatory Diseases. Front Immunol. 2019;10:2035. Epub 20190830. pmid:31543877; PubMed Central PMCID: PMC6728754.
- 13. Murray HW, Cartelli DM. Killing of intracellular Leishmania donovani by human mononuclear phagocytes. Evidence for oxygen-dependent and -independent leishmanicidal activity. J Clin Invest. 1983;72(1):32–44. pmid:6308049; PubMed Central PMCID: PMC1129158.
- 14. Pearson RD, Harcus JL, Roberts D, Donowitz GR. Differential survival of Leishmania donovani amastigotes in human monocytes. J Immunol. 1983;131(4):1994–9. pmid:6619546.
- 15. Passwell JH, Shor R, Smolen J, Jaffe CL. Infection of human monocytes by Leishmania results in a defective oxidative burst. Int J Exp Pathol. 1994;75(4):277–84. pmid:7947230; PubMed Central PMCID: PMC2002238.
- 16. Hoover DL, Nacy CA, Meltzer MS. Human monocyte activation for cytotoxicity against intracellular Leishmania donovani amastigotes: induction of microbicidal activity by interferon-gamma. Cell Immunol. 1985;94(2):500–11. pmid:3928173.
- 17. Lehn M, Weiser WY, Engelhorn S, Gillis S, Remold HG. IL-4 inhibits H2O2 production and antileishmanial capacity of human cultured monocytes mediated by IFN-gamma. J Immunol. 1989;143(9):3020–4. pmid:2509562.
- 18. Reiner NE, Ng W, Wilson CB, McMaster WR, Burchett SK. Modulation of in vitro monocyte cytokine responses to Leishmania donovani. Interferon-gamma prevents parasite-induced inhibition of interleukin 1 production and primes monocytes to respond to Leishmania by producing both tumor necrosis factor-alpha and interleukin 1. J Clin Invest. 1990;85(6):1914–24. pmid:2112157; PubMed Central PMCID: PMC296659.
- 19. McNeely TB, Turco SJ. Requirement of lipophosphoglycan for intracellular survival of Leishmania donovani within human macrophages. J Immunol. 1990;144:2745–50.
- 20. Cillari E, Vitale G, Arcoleo F, D’Agostino P, Mocciaro C, Gambino G, et al. In vivo and in vitro cytokine profiles and mononuclear cell subsets in Sicilian patients with active visceral leishmaniasis. Cytokine. 1995;7(7):740–5. Epub 1995/10/01. [pii]. pmid:8580385.
- 21. Roy S, Mukhopadhyay D, Mukherjee S, Ghosh S, Kumar S, Sarkar K, et al. A Defective Oxidative Burst and Impaired Antigen Presentation are Hallmarks of Human Visceral Leishmaniasis. J Clin Immunol. 2015;35(1):56–67. pmid:25479930.
- 22. Singh N, Kumar R, Chauhan SB, Engwerda C, Sundar S. Peripheral Blood Monocytes With an Antiinflammatory Phenotype Display Limited Phagocytosis and Oxidative Burst in Patients With Visceral Leishmaniasis. J Infect Dis. 2018;218(7):1130–41. Epub 2018/07/28. pmid:30053070; PubMed Central PMCID: PMC6107747.
- 23. Takele Y, Adem E, Franssen SU, Womersley R, Kaforou M, Levin M, et al. Impaired in vitro Interferon-gamma production in patients with visceral leishmaniasis is improved by inhibition of PD1/PDL-1 ligation. PLoS Negl Trop Dis. 2022;16(6):e0010544. Epub 20220624. pmid:35749568; PubMed Central PMCID: PMC9262188.
- 24. Guidelines for diagnosis, treatment and prevention of leishmaniasis in Ethiopia, (2013). https://www.afrikadia.org/wp-content/uploads/2018/08/VL_Guidelines_Ethiopia_2013.pdf
- 25. Chulay JD, Bryceson AD. Quantitation of amastigotes of Leishmania donovani in smears of splenic aspirates from patients with visceral leishmaniasis. Am J Trop Med Hyg. 1983;32(3):475–9. Epub 1983/05/01. pmid:6859397.
- 26. Elgueta R, Benson MJ, de Vries VC, Wasiuk A, Guo Y, Noelle RJ. Molecular mechanism and function of CD40/CD40L engagement in the immune system. Immunol Rev. 2009;229(1):152–72. pmid:19426221; PubMed Central PMCID: PMC3826168.
- 27. Chatzigeorgiou A, Lyberi M, Chatzilymperis G, Nezos A, Kamper E. CD40/CD40L signaling and its implication in health and disease. Biofactors. 2009;35(6):474–83. pmid:19904719.
- 28. Grewal IS, Flavell RA. A central role of CD40 ligand in the regulation of CD4+ T cell responses. ImmunolToday. 1996;17:410–4. pmid:8854557
- 29. Adriaensen W, Abdellati S, van Henten S, Gedamu Y, Diro E, Vogt F, et al. Serum Levels of Soluble CD40 Ligand and Neopterin in HIV Coinfected Asymptomatic and Symptomatic Visceral Leishmaniasis Patients. Front Cell Infect Microbiol. 2018;8:428. Epub 2019/01/09. pmid:30619775; PubMed Central PMCID: PMC6297181.
- 30. de Oliveira FA, Barreto AS, Bomfim LG, Leite TR, Dos Santos PL, de Almeida RP, et al. Soluble CD40 Ligand in Sera of Subjects Exposed to Leishmania infantum Infection Reduces the Parasite Load in Macrophages. PLoS One. 2015;10(10):e0141265. Epub 20151021. pmid:26488744; PubMed Central PMCID: PMC4619201.
- 31. de Oliveira FA, Vanessa Oliveira Silva C, Damascena NP, Passos RO, Duthie MS, Guderian JA, et al. High levels of soluble CD40 ligand and matrix metalloproteinase-9 in serum are associated with favorable clinical evolution in human visceral leishmaniasis. BMC Infect Dis. 2013;13:331. Epub 2013/07/23. pmid:23870715; PubMed Central PMCID: PMC3733913.
- 32. Bosmans LA, Bosch L, Kusters PJH, Lutgens E, Seijkens TTP. The CD40-CD40L Dyad as Immunotherapeutic Target in Cardiovascular Disease. J Cardiovasc Transl Res. 2021;14(1):13–22. Epub 20200328. pmid:32222950; PubMed Central PMCID: PMC7892683.
- 33. Aarts S, Seijkens TTP, van Dorst KJF, Dijkstra CD, Kooij G, Lutgens E. The CD40-CD40L Dyad in Experimental Autoimmune Encephalomyelitis and Multiple Sclerosis. Front Immunol. 2017;8:1791. Epub 20171212. pmid:29312317; PubMed Central PMCID: PMC5732943.
- 34. Rambaran S, Maseko TG, Lewis L, Hassan-Moosa R, Archary D, Ngcapu S, et al. Blood monocyte and dendritic cell profiles among people living with HIV with Mycobacterium tuberculosis co-infection. BMC Immunol. 2023;24(1):21. Epub 20230721. pmid:37480005; PubMed Central PMCID: PMC10362598.
- 35. Takele Y, Mulaw T, Adem E, Shaw CJ, Franssen SU, Womersley R, et al. Immunological factors, but not clinical features, predict visceral leishmaniasis relapse in patients co-infected with HIV. Cell Rep Med. 2022;3(1):100487. Epub 20211230. pmid:35106507; PubMed Central PMCID: PMC8784791.
- 36. Linsley PS, Ledbetter JA. The role of the CD28 receptor during T cell responses to antigen. Annu Rev Immunol. 1993;11:191–212. pmid:8386518.
- 37. Collins AV, Brodie DW, Gilbert RJ, Iaboni A, Manso-Sancho R, Walse B, et al. The interaction properties of costimulatory molecules revisited. Immunity. 2002;17(2):201–10. pmid:12196291.
- 38. Sugiura D, Maruhashi T, Okazaki IM, Shimizu K, Maeda TK, Takemoto T, et al. Restriction of PD-1 function by cis-PD-L1/CD80 interactions is required for optimal T cell responses. Science. 2019;364(6440):558–66. Epub 20190418. pmid:31000591.
- 39. Simone R, Pesce G, Antola P, Rumbullaku M, Bagnasco M, Bizzaro N, et al. The soluble form of CTLA-4 from serum of patients with autoimmune diseases regulates T-cell responses. Biomed Res Int. 2014;2014:215763. Epub 20140129. pmid:24605322; PubMed Central PMCID: PMC3925529.
- 40. Hamers AAJ, Dinh HQ, Thomas GD, Marcovecchio P, Blatchley A, Nakao CS, et al. Human Monocyte Heterogeneity as Revealed by High-Dimensional Mass Cytometry. Arterioscler Thromb Vasc Biol. 2019;39(1):25–36. pmid:30580568; PubMed Central PMCID: PMC6697379.
- 41. Merah-Mourah F, Cohen SO, Charron D, Mooney N, Haziot A. Identification of Novel Human Monocyte Subsets and Evidence for Phenotypic Groups Defined by Interindividual Variations of Expression of Adhesion Molecules. Sci Rep. 2020;10(1):4397. Epub 20200310. pmid:32157175; PubMed Central PMCID: PMC7064612.