The blots were incubated at 4 overnight?C with primary antibody followed by incubation with anti-rabbit-HRP or anti-mouse-HRP secondary antibody in 5% BSA for 2?h
The blots were incubated at 4 overnight?C with primary antibody followed by incubation with anti-rabbit-HRP or anti-mouse-HRP secondary antibody in 5% BSA for 2?h. T cells (Tregs) are one of the subsets of CD4+ T helper cells that are necessary for immune homeostasis. Apart from their roles in maintaining U-69593 peripheral tolerance, Tregs U-69593 have been heavily implicated for regulating immune responses against the invading pathogens1,2,3. Exhibiting contrast functions, Tregs can not only suppress protective immune responses and the collateral damage caused due the excessive inflammation during infections, but also provide suitable niche for facilitating the persistence of chronic infections such as tuberculosis4,5,6. Thus, induction and expansion of Tregs mark as one of the immune evasion strategies of mycobacteria for their survival in the host. During mycobacterial infections, accumulation and proliferation of Tregs at the site of infection contributes to inhibition of bacterial clearance5,7 as well as inhibition of antigen-specific protective responses exhibited by T cells8. Further, patients with active tuberculosis have increased population of circulating Tregs that suppress IFN- production by Th1 cells9. Correspondingly, depletion of Tregs from the PBMCs alleviates the IFN- production in response to mycobacterial antigens10. While depletion of CD25+ T cells in mice enhanced the IFN- production, adoptive transfer of CD25+ T cells to mice infected with facilitates bacterial survival11. Thus, understanding the mechanisms that define such Treg-mediated survival strategies of mycobacteria is important. Investigations in both murine and human models of tuberculosis have identified several mechanisms of Treg generation and expansion12,13. Of current interest, studies have highlighted the roles for PD-L1 (B7-H1/CD274) and COX-2-catalyzed PGE2 during mycobacteria-induced Treg induction and expansion. While PD-L1 deficient mice were increasingly sensitive to tuberculosis infection14, studies on human DCs showed that infection-induced PD-L1 was essential for expansion of Tregs15,16. Though PD-L1 KO mice exhibited elevated CD4+ T and CD8+ T cell responses, PD-1 expression was higher in CD4+ T cells in the PD-L1 KO mice, suggesting a possible suppression of PD-1 by PD-L1. Further, possibly due to chronic activation of immune cells and inflammation, PD-L1 KO mice exhibited increased mycobacterial CFUs in lung and death of mice14. Likewise, PGE2-responsive human Treg expansion was found during mycobacterial infection17. However, the mechanisms that mediate the expression of the molecules like PD-L1 and COX-2 in DCs are not established. In this context, it is well constituted that mycobacterial infection of the cells instigates a plethora of signaling pathways that ultimately regulate the immune mediators to determine cell-fate decisions and outcome/s of the infection. Previous investigations from our laboratory implicates the roles for SHH, WNT, NOTCH1 and PI3K signaling pathways in modulating macrophage18,19,20,21 and DC22,23,24 responses. Further, NOTCH25,26, WNT27,28, and PI3K29,30 pathways were entailed to regulate the DC functions. Thus, we explored the roles for these signaling pathways in modulating the mycobacteria-induced Treg expansion and functions. Here we demonstrate that infection-responsive activation of SHH-PI3K-mTOR-NF-B signaling in human DCs was necessary for BCG-induced Treg KIT expansion. On the other hand, NOTCH signaling hindered the ability of the infected DCs to expand Tregs while the contribution of WNT signaling was not evident. Although no apparent influence of SHH and NOTCH1 signaling on DC phenotype in terms of the maturation markers HLA-DR, CD40, CD83, CD80 and CD86 was observed, pro-inflammatory cytokines such as TNF-, IL-2, IL-1 and IL-6 were moderately NOTCH1-responsive and suppressed by SHH signaling. Further, experiments utilizing pharmacological inhibitors and conventional siRNAs indicated that both PD-L1 U-69593 and COX-2/PGE2 were induced in DCs upon stimulation with BCG and and were regulated by SHH signaling. While SHH-responsive transcription factor, GLI1 arbitrated COX-2 expression, mycobacteria-stimulated SHH signaling was found to suppress miR-324-5p and miR-338-5p, bonafide miRNAs that target PD-L1, to aid increased expression of PD-L1 and Treg expansion. Interestingly, inhibition of NOTCH1 signaling resulted in elevated expression of infection-induced PD-L1 whereas inhibition of SHH signaling showed increased transcripts of and NICD, markers for NOTCH activation. These results establish the mechanism of Treg expansion during mycobacterial infections, a testimony of its survival capabilities in the host. Results SHH and NOTCH signaling regulate BCG-induced Treg expansion To investigate the molecular circuitry regulating mycobacteria-mediated Treg expansion, U-69593 role for signaling pathways like NOTCH, WNT and SHH were assessed. Twenty-four hours post-infection with BCG, DCs were washed and co-cultured with autologous CD4+ T cells for 5 days to analyze the expansion of.