Shashi Bhushan Chauhan

Visceral leishmaniasis (VL), a parasitic disease caused by Leishmania donovani , requires a robust CD4 ⁺ T‐cells response to control parasite replication by interferon‐gamma (IFN‐γ) production and activation of macrophages. However, in VL patients, the anti‐parasitic CD4 ⁺ T‐cell responses are ineffective for reasons that are still unclear. Our recent study reporting a transcriptional signature of CD4 ⁺ T‐cell isolated from the peripheral blood of active VL patients showed enhanced expression of genes related to cytotoxicity. This study investigates the cytotoxic potential of CD4 ⁺ T‐cells in VL patients, focusing on the expression of the key cytotoxic molecules granzyme B (GZMB), granulysin (GNLY), perforin (PRF), natural killer cell granule protein 7 (NKG7) and the degranulation capacity of CD4 ⁺ T‐cells during infection. We observed significant upregulation of these cytotoxic markers in CD4⁺ T‐cells from VL patients, particularly prior to anti‐parasitic drug treatment (D0), suggesting activation of these cells. However, the degranulation capacity, as indicated by CD107a expression, was comparable between VL patients and endemic controls (ECs), suggesting potential functional impairment in the cytotoxic response. Yet, antigen‐specific GZMB secretion in whole blood cultures and intracellular GZMB production in CD4 ⁺ T‐cell subsets (notably Th1, Th9 and Th17/22 cells) were enhanced in VL patients, indicating a robust antigen‐specific response, though this did not translate into effective parasite control. These findings highlight a paradox whereby CD4 ⁺ T‐cells from VL patients have heightened production of cytotoxic molecules, but lack translocation of CD107a to the cell surface, which is associated with an inability to eliminate parasites. This study provides new insights into the immune dysfunction in VL, highlighting a potential role for the cytotoxic phenotype that develops in parasite‐specific CD4 ⁺ T‐cells. Targeting these pathways may offer novel therapeutic strategies to enhance immune responses and improve clinical outcomes in VL.

Apolipoprotein E (ApoE) has been associated with several diseases including Parkinson's disease, Alzheimer's and multiple sclerosis. ApoE also has documented immunomodulatory functions. We investigated gene expression in circulating monocytes and in bone marrows of patients with visceral leishmaniasis (VL) living in an endemic area in Bihar, India, and contrasted these with control healthy subjects or other diagnostic bone marrows from individuals in the same region. Samples from VL patients were obtained prior to initiating treatment. Our study revealed significant upregulated expression of the apoE transcript in patients with VL. Furthermore, the levels of ApoE protein were elevated in serum samples of subjects with VL compared with healthy endemic controls. These observations may provide clues regarding the complex interactions between lipid metabolism and immunoregulation of infectious and inflammatory diseases.

Control of visceral leishmaniasis (VL) depends on proinflammatory Th1 cells that activate infected tissue macrophages to kill resident intracellular parasites. However, proinflammatory cytokines produced by Th1 cells can damage tissues and require tight regulation. Th1 cell IL-10 production is an important cell–autologous mechanism to prevent such damage. However, IL-10–producing Th1 (type 1 regulatory; Tr1) cells can also delay control of parasites and the generation of immunity following drug treatment or vaccination. To identify molecules to target in order to alter the balance between Th1 and Tr1 cells for improved antiparasitic immunity, we compared the molecular and phenotypic profiles of Th1 and Tr1 cells in experimental VL caused by Leishmania donovani infection of C57BL/6J mice. We also identified a shared Tr1 cell protozoan signature by comparing the transcriptional profiles of Tr1 cells from mice with experimental VL and malaria. We identified LAG3 as an important coinhibitory receptor in patients with VL and experimental VL, and we reveal tissue-specific heterogeneity of coinhibitory receptor expression by Tr1 cells. We also discovered a role for the transcription factor Pbx1 in suppressing CD4 ⁺ T cell cytokine production. This work provides insights into the development and function of CD4 ⁺ T cells during protozoan parasitic infections and identifies key immunoregulatory molecules.

Immune-modulating therapies have revolutionized the treatment of chronic diseases, particularly cancer. However, their success is restricted and there is a need to identify new therapeutic targets. Here, we show that natural killer cell granule protein 7 (NKG7) is a regulator of lymphocyte granule exocytosis and downstream inflammation in a broad range of diseases. NKG7 expressed by CD4⁺ and CD8⁺ T cells played key roles in promoting inflammation during visceral leishmaniasis and malaria—two important parasitic diseases. Additionally, NKG7 expressed by natural killer cells was critical for controlling cancer initiation, growth and metastasis. NKG7 function in natural killer and CD8⁺ T cells was linked with their ability to regulate the translocation of CD107a to the cell surface and kill cellular targets, while NKG7 also had a major impact on CD4⁺ T cell activation following infection. Thus, we report a novel therapeutic target expressed on a range of immune cells with functions in different immune responses.

CD8⁺ T‐cell function is compromised in chronic diseases such as visceral leishmaniasis (VL). However, little is known about the changes in gene expression that cause CD8+ T‐cell dysfunction during VL. We used targeted transcriptional profiling of peripheral blood CD8⁺ T cells from VL patients pre‐ and post‐anti‐parasitic drug treatment, and compared them with the same cell population from healthy endemic controls to assess their activation, differentiation and functional status during disease. We found a predominance of downregulated immune genes in CD8⁺ T cells from VL patients. However, genes encoding several notable immune checkpoint molecules, including LAG‐3, TIM‐3 and CTLA‐4, cytolytic molecules, such as granzymes A, B and H and perforin, as well as SOCS3, STAT1, JAK2 and JAK3 cytokine signalling genes were found to be increasingly expressed by VL patient CD8⁺ T cells. Additional studies confirmed increased expression of the inhibitory receptors LAG3 and TIM3 on VL patient CD8⁺ T cells, thereby identifying these molecules as potential targets to improve antigen‐specific CD8⁺ T‐cell responses during disease.

Oncogenic Human papillomavirus (HPV) infections are closely associated with anal cancer which is high among human immunodeficiency virus (HIV) infected males. There are no data regarding anal HPV infection and cytological abnormalities in HIV positive males receiving free therapy in the national program. Thus, this cross-sectional study was performed to assess the prevalence and risk factors of anal HPV infection and cytological abnormalities in HIV positive males. We screened 126 HIV-positive male patients attending the antiretroviral treatment center (ART) between 2014 and 2015 with anal papanicolaou smear cytology and HPV-DNA testing. HPV-DNA was detected by using polymerase chain reaction (PCR) method with two consensus primer sets E6 and MY09/11 and further analyzed for the presence of various HPV genotype by Sanger sequencing. Risk factors associated with anal cytological abnormalities and HPV infection was analyzed by using univariate and multivariate logistic regression models. Out of 126, 52 were on antiretroviral therapy. 91% were married to female partners but during the study 48 (38%) gave positive history of anal intercourse with other men. Anal cytology was done in 95 patients, out of which 60 (63.15%) had cytological abnormalities. LSIL (low-grade squamous intraepithelial lesions) was present in 27 (45%), ASCUS (atypical squamous cells of undetermined significance) in 31 (52%) and ASC-H (atypical squamous cells cannot exclude a high-grade squamous intraepithelial lesion) in 2 (3.33%). In multivariate analysis, the risk factors for cytological abnormality were presence of history of anal intercourse (OR, 6.1; 95% CI, 2.0–18.7) and WHO stage III & IV (OR, 2.7; 95% CI, 1.1–7.5). HPV-DNA was detected in 33/119 (27.73%) patients. The most prevalent HPV type in the study was HPV-16 (10.08%), other HPV types detected were 18,31,35,17,66,72,52,68 and 107 (17.65%). High prevalence of anal cytological abnormalities in our study suggests that regular anal Pap smear screening should be done in HIV positive males in the ART center.

Monocytes are important effector cells during Leishmania infection, and changes in their functions may impact development of immunity. However, functional characteristics of monocytes in patients with visceral leishmaniasis (VL) remains poorly understood. Peripheral blood monocytes from patients with VL and healthy endemic controls from Muzaffarpur, India, were isolated and compared in an ex vivo setting, using cell-culture techniques, flow cytometry, and reverse transcription quantitative polymerase chain reaction analysis. A blood monocyte population with a gene signature comprising upregulated expression of TGM2, CTLRs, VDR, PKM, SOCS1, and CAMP1 and downregulated expression of NOS2 and HIF1A was observed in patients with VL but not in controls. Monocytes from patients with VL also had impaired expression of chemokine receptors and adhesion molecules and decreased frequencies of interleukin 1β– and interleukin 6–producing cells. Importantly, monocytes from patients with VL had a markedly reduced capacity for phagocytosis of amastigotes, p47phox and p67phox expression, and reactive oxygen species production. Monocytes from patients with VL express antiinflammatory molecules and lack a classically activated phenotype. They have reduced expression of molecules related to activation and antiparasitic effector functions, indicating that monocytes are skewed toward an antiinflammatory phenotype. These findings provide insights into the functional status of monocytes during VL and advise that therapeutic manipulation of this important cell population may result in favorable patient outcomes.