Altered Chemokine Receptor Expression in CD4+ T Cells: A Signature of Visceral Leishmaniasis

Abstract

Robust T helper 1 (Th1) cell responses, which activate macrophages to kill intracellular parasites, are required to control Leishmania infection. Yet, visceral leishmaniasis (VL) patients do not control the infection despite the expansion of CD4⁺ T cells and increased IFN-γ expression in the spleen. Chemokines and/or chemokine receptors are involved in cellular migration and are critical in the inflammatory response. In a recent study that defined a transcriptional signature for CD4⁺ T cells from active VL patients, we found several differentially expressed chemokine receptor genes in CD4⁺ T cells compared to healthy endemic controls (EC). Since CD4⁺ T cells play crucial roles in parasite clearance, a better understanding of the role of altered chemokine receptor expression on CD4⁺ T cells and their different subsets during VL could inform future treatment strategies. In this study, we examined the gene expression and surface protein expression of differentially expressed chemokine receptors found in human VL subjects, relative to endemic controls (EC) by real-time qPCR and multicolor flow cytometry, respectively. We measured chemokine levels in plasma by enzyme-linked immunosorbent assay (ELISA) and performed transwell migration assays and flow cytometry to measure the migratory potential of CD4⁺ T cell subsets. We found elevated mRNA and surface protein expression of CCR5, while reduced CCR4 and CCR6 expression in CD4⁺ T cells from VL patients compared to EC. The frequency of CCR5 expressing Th1 cells was increased in peripheral blood, indicating the expansion of CCR5⁺ Th1 cells that may be responsible for Th1 cells trafficking towards infected tissues. Lower CCR4 expression was found on regulatory T (Treg) cells and central memory T (Tcm) cells, possibly explaining the reduced frequencies of these cells in peripheral blood during VL. The frequency of CCR6 expressing CD4⁺ T cells was also found to be lower in VL patients. Our results show that VL patients possess unique chemokine receptor expression patterns on the cell surface of CD4⁺ T cells, compared to EC. We also found increased levels of CCL3, CCL5 and CCL20 in VL plasma compared to EC. However, no changes were observed for CCL17 levels in VL plasma compared to EC, but their levels increased following treatment. We also observed reduced migration of VL CD4⁺ T cells, relative to other lymphocytes and mononuclear cells, irrespective of exogenous CCL5 presence. However, the frequency of CD4⁺ T cells migrating in response to CCL5 in EC individuals was similar. Additionally, we noted the frequency of CCR5⁺ Th1 cells was increased in VL patients compared to EC. However, no enhancement was seen in the migratory capacity of CCR5⁺ CD4⁺ T cells in the presence of recombinant CCL5. This suggests that the CCR5 receptor signalling pathway is less responsive towards exogenous CCL5, potentially due to high levels of CCL3 and CCL5 present in VL plasma, which may saturate surface CCR5. The upregulation of CCR5 expression and downregulation of CCR4 and CCR6 by CD4⁺ T cells distinguished VL patients from healthy individuals. These findings provide new insights into VL pathogenesis and could direct the development of new and improved disease diagnostics and therapeutics for treatment of VL.

Keywords: T‐lymphocytes; cell movement; chemokine receptors; chemokines; visceral leishmaniasis.