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Background: The host genetic factors play important role in determining the outcome of visceral leishmaniasis (VL). Macrophage migration inhibitory factor (MIF) is an important host cytokine, which is a key regulator of innate immune system. Genetic variants in MIF gene have been found to be associated with several inflammatory and infectious diseases. Role of MIF is well documented in leishmaniasis diseases, including Indian visceral leishmaniasis, where elevated level of serum MIF has been associated with VL phenotypes. However, there was no genetic study to correlate MIF variants in VL, therefore, we aimed to study the possible association of three reported MIF gene variants −794 CATT, −173G > C and non-coding RNA gene LOC284889 in Indian VL phenotype. Methods: Study subjects comprised of 214 VL patients along with ethnically and demographically matched 220 controls from VL endemic regions of Bihar state in India. Results: We found no significant difference between cases and controls in allelic, genotypic and haplotype frequency of the markers analysed [-794 CATT repeats (χ2 = 0.86; p = 0.35; OR = 0.85; 95% CI = 0.61–1.19); −173 G > C polymorphism (χ2 = 1.11; p = 0.29; OR = 0.83; 95% CI = 0.59–1.16); and LOC284889 (χ2 = 0.78; p = 0.37; OR = 0.86; 95% CI = 0.61–1.20)]. Conclusion: Since we did not find any significant differences between case and control groups, we conclude that sequencing of complete MIF gene and extensive study on innate and adaptive immunity genes may help in identifying genetic variations that are associated with VL susceptibility/resistance among Indians. Keywords: Visceral leishmaniasis (VL), Macrophage Migration Inhibitory Factor (MIF), Leishmania major, Leishmania donovani, Genetic polymorphism, Indian population

Visceral leishmaniasis (VL) is a multifactorial disease, where the host genetics play a significant role in determining the disease outcome. The immunological role of anti-inflammatory cytokine, Interleukin 10 (IL10), has been well-documented in parasite infections and considered as a key regulatory cytokine for VL. Although VL patients in India display high level of IL10 in blood serum, no genetic study has been conducted to assess the VL susceptibility / resistance. Therefore, the aim of this study is to investigate the role of IL10 variations in Indian VL; and to estimate the distribution of disease associated allele in diverse Indian populations.All the exons and exon-intron boundaries of IL10 were sequenced in 184 VL patients along with 172 ethnically matched controls from VL endemic region of India.Our analysis revealed four variations; rs1518111 (2195 A>G, intron), rs1554286 (2607 C>T, intron), rs3024496 (4976 T>C, 3' UTR) and rs3024498 (5311 A>G, 3' UTR). Of these, a variant g.5311A is significantly associated with VL (χ2=18.87; p =0.00001). In silico approaches have shown that a putative micro RNA binding site (miR-4321) is lost in rs3024498 mRNA. Further, analysis of the above four variations in 1138 individuals from 34 ethnic populations, representing different social and linguistic groups who are inhabited in different geographical regions of India, showed variable frequency. Interestingly, we have found, majority of the tribal populations have low frequency of VL ('A' of rs3024498); and high frequency of leprosy ('T' of rs1554286), and Behcet's ('A' of rs1518111) associated alleles, whereas these were vice versa in castes. Our findings suggest that majority of tribal populations of India carry the protected / less severe allele against VL, while risk / more severe allele for leprosy and Behcet's disease. This study has potential implications in counseling and management of VL and other infectious diseases.

Sepsis is a consequence of systemic bacterial infections leading to hyper activation of immune cells by bacterial products resulting in enhanced release of mediators of inflammation. Endotoxin (LPS) is a major component of the outer membrane of Gram negative bacteria and a critical factor in pathogenesis of sepsis. Development of antagonists that inhibit the storm of inflammatory molecules by blocking Toll like receptors (TLR) has been the main stay of research efforts. We report here that a filarial glycoprotein binds to murine macrophages and human monocytes through TLR4 and activates them through alternate pathway and in the process inhibits LPS mediated classical activation which leads to inflammation associated with endotoxemia. The active component of the nematode glycoprotein mediating alternate activation of macrophages was found to be a carbohydrate residue, Chitohexaose. Murine macrophages and human monocytes up regulated Arginase-1 and released high levels of IL-10 when incubated with chitohexaose. Macrophages of C3H/HeJ mice (non-responsive to LPS) failed to get activated by chitohexaose suggesting that a functional TLR4 is critical for alternate activation of macrophages also. Chitohexaose inhibited LPS induced production of inflammatory molecules TNF-α, IL-1β and IL-6 by macropahges in vitro and in vivo in mice. Intraperitoneal injection of chitohexaose completely protected mice against endotoxemia when challenged with a lethal dose of LPS. Furthermore, Chitohexaose was found to reverse LPS induced endotoxemia in mice even 6/24/48 hrs after its onset. Monocytes of subjects with active filarial infection displayed characteristic alternate activation markers and were refractory to LPS mediated inflammatory activation suggesting an interesting possibility of subjects with filarial infections being less prone to develop of endotoxemia. These observations that innate activation of alternate pathway of macrophages by chtx through TLR4 has offered novel opportunities to cell biologists to study two mutually exclusive activation pathways of macrophages being mediated through a single receptor.