Leishmania donovani-Induced Immune Dysregulation among Sudanese Patients with Visceral and Post Kala-Azar Dermal Leishmaniases: Possible Roles in Pathogenesis

Elsamani Elmotwafig Mohamed Elsammani^1#, Brima Younis Musa¹, Mona Elfaki Eltahir Elfaki², Ahmed Mudawi Mudawi¹, Eltahir Awad Gasim Khalil^1*

¹Institute of Endemic Diseases, University of Khartoum, Khartoum, Sudan.
²Department of Microbiology and Clinical Parasitology, College of Medicine, King Khalid University, Abha, KSA.

DOI: 10.4236/aid.2020.105021                

Abstract

L. donovani infections (visceral and post kala-azar dermal leishmaniases) are characterized by infection-induced reversible immune suppression. Autoimmunity is a well-documented phenomenon among patients with primary immune deficiencies. This study aimed to study auto-immune phenomena accompanying L. donovani infections. In a prospective case-controlled study and following informed consent, 155 individuals with visceral leishmaniasis (VL; n = 62), post kala-azar dermal leishmaniasis (PKDL; n = 31) and apparently healthy volunteers (n = 62) were recruited. Sera antinuclear (ANA), anti-dsDNA, anti-thyroid peroxidase (TPO), anti-smooth muscles (ASMA) and F-actin auto-antibodies were measured using ELISA and indirect immune-fluorescence assay. The mean ages of VL, PDKL patients and apparently healthy volunteers were: 17.5 ± 12.5, 15.0 ± 7.0 and 17.5 ± 9.5 years with Male:Female ratios of 2:0, 1:2 and 1:5 respectively. Significantly high frequencies of F-actin (74.2%; 46/62) and ASMA (50%; 31/62) auto-antibodies were seen among VL patients (p = 0.003, p = 0.001) compared to apparently healthy volunteers. Likewise, significantly high frequencies of F-actin (64.5%; 20/31; p = 0.001), ASMA (42%; 13/31; p = 0.003), ANA (36%; 11/31; p = 0.001) and anti-dsDNA (16%; 5/31; p = 0.01) auto-antibodies were seen among PKDL patients. Development of tissue-based autoantibodies in L. donovani infections probably indicates loss of peripheral tolerance with activation of circulating auto-reactive T and B cells probably contributing to disease pathogenesis (increased bilirubin/liver enzymes, prolonged QT interval/arrythmias and blood cytopenias). In conclusion, L. donovani infection-induced immune suppression with development of tissue-based auto-antibodies is prevalent among Sudanese patients with VL and PKDL leishmaniases and contributes to some aspects of the disease pathogenesis.

Keywords

L. donovani-Induced Immune Suppression, Tissue-Based Autoimmunity, Pathogenesis

1. Introduction

The leishmaniases are increasingly recognized major public health problems that constitute wide spectra of human and animal diseases. It is caused by obligate intra uni-cellular protozoan parasites of the genus Leishmania. Clinical manifestations depend on complex interactions between the Leishmania parasite and human host immune system. Visceral leishmaniasis (VL, Kala-azar) is the most severe form with high mortality rates if untreated. Control of VL focuses on case detection, drug treatment, control of animal reservoir/vectors and bed nets. Research on anti-leishmania candidate vaccines is on-going [1] - [6]. More than fifty per cent of successfully treated VL patients develop an immune-mediated dermatosis called post kala-azar dermatitis (PKDL). Early activation of the innate immune system takes place during L. donovani infection enabling the host immune system to determine the type of immune response (Th1/Th2). The majority of VL patients become immune-compromised with depressed cell-mediated immunity and anergy to some antigens (leishmanial and purified protein derivative). VL-induced immune suppression is transient with reversion to immune competence after six months of successful treatment. VL-immune deficiency is probably due to on-site generation of adenosine, a potent immune-suppressant that leads to immune impairment and disease progression. L. donovani infection-induced auto-immune phenomena probably happen when auto-reactive B and T cells escape tolerance check points with resultant hyper-gamma-globulinemia and immune-related peripheral blood cytopenias. Alternatively, the infection leads to T-cells suppression with enhanced leishmanial antigen-specific IL-10 production and increased CD8+ CD28-mediated T CD4+ cells anergy. The mechanisms of hyper-gamma-globulinemia that accompanies VL and PKDL are probably similar to what happens in Hyper Ig M syndrome (a primary immune deficiency syndrome [2] [4] [7] - [22] ). This study aimed to characterize auto-immunity (organ-specific and tissue-specific auto-antibodies) that accompanies L. donovani infection-induced immune suppression. Autoimmunity is generally induced by genetics and environmental factors (microbial infections etc.). A number of theories have been proposed to explain autoimmune phenomena in PID: failure of tolerance mechanisms with failure of deletion of autoreactive B cells), signaling pathway defects with failure of anergy, loss of development of T_reg cells, deranged T cell activation by innate system and defects in self-antigen clearance that follows tissue destruction. Autoantibodies (anti-ANA, anti-ds DNA, anti-RNP, anti-SS/A, anti-SS/B, anti-cardiolipin, anti-beta 2-glycoprotein I, anti-actin, anti-tubulin, anti-smooth muscle) have been detected in the sera of animals and humans with cutaneous and visceral leishmaniases [23] - [34]. Although a pathogenic role for autoimmunity in leishmaniasis was reported (immune pancytopenia), it has to be clearly understood that reactivity to one or more autoantibodies should not be mis-interpreted as autoimmune disorders. In addition, a number of autoimmune diseases (SLE, rheumatoid arthritis, polyarteritis nodosa and autoimmune hemolytic anemia, autoimmune hepatitis) could be mimicked or exacerbated by leishmaniasis [29] [35] - [40]. This study aimed to determine the emergence of auto-immunity following secondary immune suppression caused by L. donovani infections.

2. Materials and Methods

2.1. Study Type and Site

This was a prospective and case-controlled study with a case: control ratio of 1:1 - 1:2. The study proposal was reviewed and passed by the Scientific and Ethics Committees of the Institute of Endemic Diseases; University of Khartoum. Written informed consents were obtained from participating individuals. Visceral and PKDL patients were recruited at Professor Elhassan Centre, Dooka, Eastern Sudan (VL endemic area). Apparently Healthy controls were recruited in Greater Khartoum, Khartoum (VL non-endemic area).

2.2. Study Patients and Apparently Healthy Controls

One hundred and fifty five VL (n = 62), PKDL (n = 31) parasitologically-confirmed patients and apparently healthy volunteers (n = 62) were enrolled. The sample size was calculated using EpiInfo version 3.04.04 software. Taking into consideration that the proportion of VL patients who will develop PKDL is 50% (0.5), two-sided significant level as 0.05 and a power of 80% and the ratio of the sample size of exposed/non-exposed = 1. The total sample size was calculated at 155 [Odds ratio = 3.5 and prevalence ratio = 1.75]. VL/PKDL patients and volunteers were recruited sequentially from patients/individuals of all ages from both sexes who report to with HBV, HCV, HIV and chronic diseases (Diabetes mellitus, chronic renal failure etc.), pregnant and lactating females were excluded.

2.3. Methods

Anti-ANA (anti-nuclear antibody), anti-ds-DNA and anti-TPO auto-antibodies were measured in sera by indirect ELISA using commercial ELISA kits as described by the manufacture (EUROIMMUN Medizinische Labor Diagnostika AG, Germany). ASMA and F-actin auto-antibodies were measured in serum by indirect immune-fluorescence technique using commercial Immune-fluorescence kits as described by the manufacture (EUROIMMUN, Medizinische Labor Diagnostika AG, Germany).

2.4. Statistical Analysis

The data were analysed using Epidemiological Information Software (Epi Info 7). The frequencies of auto-antibodies (ANA, ds-DNA, TPO, ASMA and F-actin) were calculated. The frequencies of auto-antibodies detected were compared between the VL group and apparently healthy volunteers group; PKDL group and apparently healthy volunteers group and between PKDL and VL groups. P value < 0.05 was considered statistically significant.

3. Results

The means ages and the male; female ratios of the study groups were: VL patients (n = 62, mean ± SD age = 17.5 ± 12.5 and a Male: Female ratio of 1.2:1), PKDL patients (n = 31, mean ± SD age = 15 ± 7 years and a Male: Female ratio of 2:1) and apparently healthy volunteers from VL non-endemic area (n = 62, mean ± SD age, 27.5 ± 9.5 years; and a Male: Female ratio of 1.5:1). Significantly high frequencies of F-actin (74.2%, 46/62; p = 0.003) and ASMA (50%, 31/62; p = 0.001) auto-antibodies were reported among VL patients compared to apparently healthy volunteers. Likewise, significantly high frequencies of F-actin (64.5%; 20/31; p = 0.001), ASMA (42%; 13/31; p = 0.003), ANA (36%, 11/31; p = 0.001) and anti-dsDNA (16%, 5/31; p = 0.01) auto-antibodies were reported among PKDL patients. Eleven apparently healthy volunteers (11/62, 17.7%) in total had one or more auto-antibodies (Table 1).

4. Discussion

Autoimmunity in different forms has been described intensively in patients with primary immune deficiency (PID) several explanations have been put forward to explain this ironic discrepancy. The mechanisms of autoimmunity during L. donovani infection and the clinical significance of this autoimmunity among Sudanese VL/PKDL patients are not clear and remain to be determined, but

Table 1. Frequencies of auto-antibodies among different study groups.

*ANA = antinuclear antibody.

appear to be similar to that accompany PID. Development of tissue-based auto-antibodies in L. donovani infections as was commonly seen among our VL/PKDL patients probably indicates absence of peripheral tolerance mechanisms contributing to activation of autoreactive T and B cells. The clinical significance of these differences remains unclear, but the development of autoimmunity can explain some pathogenetic features of L. donovani infection, especially pre-treatment prolonged QT interval, cardiac arrhythmias, increased serum bilirubin and liver enzymes. These abnormalities could indicate infection-induced heart and liver involvement and might explain sudden deaths encountered during antimony treatment. It is our assumption that the presence of high titers of smooth-muscle auto antibodies (ASMA) with significant reactivity against antinuclear and filamentous actin (F-actin) in VL/PKDL patients, could explain cardiac and liver involvement in VL/PKDL patients. The presence of F-actin has been recognized as specific to auto immune hepatitis (AIH) [37].

5. Conclusion

In conclusion, tissue-specific auto-antibodies (ASMA, F-actin) are prevalent among Sudanese visceral and post kala-azar dermal leishmaniasis patients and could explain aspects of the disease pathogenesis.

Declaration

This work is a result original work conducted as part of research study that was conducted at treatments firm of the Institute of Endemic Diseases, University of Khartoum, Sudan. The authors declare that this work is not under consideration for publication by any publishing firm.

Funding

This study received no financial support from any source.

Authors’ Contribution

Conceptualization, EEEM and EAGK; data curation, EEEM, MEEE; methodology, BYM, AMM and MEEE; data analysis, EEEM, MEEE, EAGK; writing—original draft, EEEM, MEEE, EAGK; writing—review & editing, EEEM, MEEE, EAGK. All authors approved the final version of the manuscript.

NOTES

^#Deceased.

Conflicts of Interest

The authors declare no conflicts of interest regarding the publication of this paper.

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