RESUMO
CD4+ and CD8+ effector T cell subpopulations can display regulatory potential characterized by expression of the prototypically anti-inflammatory cytokine IL-10. However, the underlying cellular mechanisms that regulate expression of IL-10 in different T cell subpopulations are not yet fully elucidated. We recently showed that TNF inhibitors (TNFi) promote IL-10 expression in human CD4+ T cells, including IL-17+ CD4+ T cells. Here, we further characterized the regulation of IL-10 expression via blockade of TNF signaling or other cytokine/co-stimulatory pathways, in human T cell subpopulations. Addition of the TNFi drug adalimumab to anti-CD3-stimulated human CD4+ T cell/monocyte cocultures led to increased percentages of IL-10+ cells in pro-inflammatory IL-17+, IFNγ+, TNFα+, GM-CSF+, and IL-4+ CD4+ T cell subpopulations. Conversely, exogenous TNFα strongly decreased IL-10+ cell frequencies. TNF blockade also regulated IL-10 expression in CD4+ T cells upon antigenic stimulation. Using time course experiments in whole peripheral blood mononuclear cell (PBMC) cultures, we show that TNF blockade maintained, rather than increased, IL-10+ cell frequencies in both CD4+ and CD8+ T cells following in vitro stimulation in a dose- and time-dependent manner. Blockade of IL-17, IFNγ, IL-6R, or CD80/CD86-mediated co-stimulation did not significantly regulate IL-10 expression within CD4+ or CD8+ T cell subpopulations. We show that TNF blockade acts directly on effector CD4+ T cells, in the absence of monocytes or CD4+ CD25highCD127low regulatory T cells and independently of IL-27, resulting in higher IL-10+ frequencies after 3 days in culture. IL-10/IL-10R blockade reduced the frequency of IL-10-expressing cells both in the presence and absence of TNF blockade. Addition of recombinant IL-10 alone was insufficient to drive an increase in IL-10+ CD4+ T cell frequencies in 3-day CD4+ T cell/monocyte cocultures, but resulted in increased IL-10 expression at later time points in whole PBMC cultures. Together, these data provide additional insights into the regulation of IL-10 expression in human T cells by TNF blockade. The maintenance of an IL-10+ phenotype across a broad range of effector T cell subsets may represent an underappreciated mechanism of action underlying this widely used therapeutic strategy.
RESUMO
Although the functions of teleost IL-10 have been preliminarily determined, functional evidence for its receptor signaling is lacking. Particularly, the identity of fish IL-10 receptor 2 (IL-10R2) is ambiguous. Cytokine receptor family member b4 (CRFB4) and CRFB5 are likely the ortholog of mammalian IL-10R2. In this study, grass carp CRFB4 (gcCRFB4) and gcCRFB5 cDNAs were isolated and characterized. The relatively high expression levels of grass carp IL10 receptor 1 (gcIL-10R1), gcCRFB4 and gcCRFB5 in immune tissues and cells implied their importance in fish immunity. Accordingly, gcIL-10R1, gcCRFB4 and gcCRFB5 were overexpressed in a grass carp kidney cell line to identify the IL-10 receptor subunits upon grass carp IL-10 (gcIL-10) treatment. Results showed that gcIL-10R1 was essential for gcIL-10 stimulation on STAT3 activation and grass carp suppressor of cytokine signaling 3 (gcSOCS3) promoter activity, and also indicated that gcCRFB4 but not gcCRFB5 might be the ortholog of mammalian IL-10R2. Furthermore, mutation of a putative STAT3-binding element in gcSOCS3 promoter attenuated the stimulation of gcIL-10 on gcSOCS3 promoter activity, indicating that gcIL-10 may modulate gcSOCS3 transcription at least partly via STAT3 activation. This notion was further supported by our observation that gcIL-10 was able to induce STAT3 phosphorylation and STAT3 inhibitor could abolish the upregulation of gcSOCS3 mRNA expression by gcIL-10 in grass carp head kidney leukocytes. Taken together, this study for the first time functionally characterized the teleost IL-10 receptor subunits and clarified the conservation of fish IL-10 signaling during evolution, thus laying the ground for further understanding the critical immune events led by IL-10 in teleost.