Bone Marrow Mesenchymal Stromal Cells Induce Proliferative, Cytokinic and Molecular Changes During the T Cell Response: The Importance of the IL-10/CD210 Axis.

BACKGROUND: Bone marrow mesenchymal stromal cells (BM-MSCs) display immunomodulatory features, representing a promising tool for cell-based therapies. However, the mechanisms used by MSCs to regulate T cell fate remain unclear. AIMS: We investigated the potential of BM-MSCs to modulate T cell activation, proliferation, cytokine secretion and immunophenotype. MATERIALS AND METHODS: T cells were co-cultured with BM-MSCs to assess their immunomodulatory impact. T cell characterization was performed using cell tracing, ELISA, intracellular and surface staining, flow cytometry analysis and qPCR. RESULTS: The activation and proliferation of T cells were downregulated during coculture with BM-MSCs. We also observed that BM-MSCs upregulated IL-10 secretion as well as the expression of its receptor CD210 on T cells, thus creating a loop favoring the expansion of IL-10-producing T cells. IL-10 neutralization restored T cell proliferation, demonstrating that IL-10 is functionally relevant during immunomodulation. Moreover, BM-MSCs differently modulated CD4 and CD8 T-cell immunophenotype by inducing broad changes in their molecular pattern. CONCLUSIONS: We provide a comprehensive functional and molecular characterization of T cells that are immunomodulated by BM-MSCs. Indeed, a better understanding of the immunological interplay between T cells and MSCs will facilitate the development of new efficient approaches to improve cell-based immune therapies.

IFN-γ-mediated induction of an apical IL-10 receptor on polarized intestinal epithelia.

Cytokines secreted at sites of inflammation impact the onset, progression, and resolution of inflammation. In this article, we investigated potential proresolving mechanisms of IFN-γ in models of inflammatory bowel disease. Guided by initial microarray analysis, in vitro studies revealed that IFN-γ selectively induced the expression of IL-10R1 on intestinal epithelia. Further analysis revealed that IL-10R1 was expressed predominantly on the apical membrane of polarized epithelial cells. Receptor activation functionally induced canonical IL-10 target gene expression in epithelia, concomitant with enhanced barrier restitution. Furthermore, knockdown of IL-10R1 in intestinal epithelial cells results in impaired barrier function in vitro. Colonic tissue isolated from murine colitis revealed that levels of IL-10R1 and suppressor of cytokine signaling 3 were increased in the epithelium and coincided with increased tissue IFN-γ and IL-10 cytokines. In parallel, studies showed that treatment of mice with rIFN-γ was sufficient to drive expression of IL-10R1 in the colonic epithelium. Studies of dextran sodium sulfate colitis in intestinal epithelial-specific IL-10R1-null mice revealed a remarkable increase in disease susceptibility associated with increased intestinal permeability. Together, these results provide novel insight into the crucial and underappreciated role of epithelial IL-10 signaling in the maintenance and restitution of epithelial barrier and of the temporal regulation of these pathways by IFN-γ.

TLR4, IL10RA, and NOD2 mutation in paediatric Crohn's disease patients: an association with Mycobacterium avium subspecies paratuberculosis and TLR4 and IL10RA expression.

Mycobacterium avium subspecies paratuberculosis (MAP) has been implicated in the pathogenesis of Crohn's disease (CD). The role of CD susceptibility genes in association with these microbes is not known. Sixty-two early onset paediatric CD patients and 46 controls with known MAP status were analysed for an association with 34 single nucleotide polymorphisms (SNPs) from 18 CD susceptibility genes. Functional studies on peripheral blood mononuclear cells (PBMCs) were conducted on 17 CD patients with known CD mutations to assess IL-6, IL-10, and TNF-α expression upon stimulation with MAP precipitated protein derivative (PPD) and lipopolysaccharide (LPS). In addition, surface expression of IL10R and TLR4 on resting B cells, NK cells, T cells, and monocytes was assessed. A mutation in TLR4 (rs4986790) and IL10RA (rs22291130) was significantly associated with MAP-positive CD patients compared to MAP-negative CD patients (27.6 vs. 6.1 %, p = 0.021, and 62.1 vs. 33.3 %, p = 0.024, respectively). PPD and LPS significantly increased IL-6, IL-10, and TNF-α production in PBMCs. IL-10 and TNF-α production were significantly lower in a subgroup of CD patients (5/12) with a known NOD2 mutation. Receptor for IL-10 was significantly higher expressed on NK cells (CD56low) and on NK T cells harbouring a NOD2 mutations compared to wildtype cells (p = 0.031 and 0.005, respectively). TLR4 was significantly higher expressed on NK cells (CD56high) harbouring a NOD2 mutations compared to wildtype cells (p = 0.038).

Type I and III interferons enhance IL-10R expression on human monocytes and macrophages, resulting in IL-10-mediated suppression of TLR-induced IL-12.

Currently, only about 30-50% of chronic hepatitis C virus (HCV) and hepatitis B virus (HBV) patients respond to IFN-based therapy. It has been suggested that IL-10 is involved in suppressing the activity of type I IFNs on antigen-presenting cells (APCs). However, the interaction between type I IFNs and IL-10 is still not clear. Here we report that IFN-α priming upregulated the expression of IL-10R1 on monocytes, and subsequently IL-10 induced a higher level of STAT3 phosphorylation in IFN-primed cells. This indicates that IFN-α increased the sensitivity of monocytes to IL-10, and as a result, TLR-induced IL-12p70 by IFN-pretreated cells was suppressed. Interestingly, both IFN-ß and IL-29, a member of the type III IFN family, comparably sensitized monocytes and macrophages to IL-10 stimulation, indicating a general effect of IFN on the activity of IL-10 in APCs. In summary, we demonstrate that one of the consequences of priming human APCs with IFN is to promote the cells' sensitivity to IL-10, which leads to the inhibition of TLR-induced IL-12p70 production. Therefore, type I and III IFNs induce a suboptimal activation of immune cells. These findings are relevant for the development of strategies to further improve IFN-based therapy for patients with multiple sclerosis or viral hepatitis.

Lipopolysaccharide-induced interleukin (IL)-4 receptor-α expression and corresponding sensitivity to the M2 promoting effects of IL-4 are impaired in microglia of aged mice.

In several models of aging, microglia become more inflammatory and reactive to immune challenges. For example, peripheral LPS injection causes exaggerated microglial activation associated with prolonged sickness and depressive-like behavior in aged BALB/c mice. Therefore, the purpose of this study was to determine the extent to which age-related amplified microglial activation was associated with reduced sensitivity to the anti-inflammatory and M2 promoting cytokines interleukin (IL)-10 and IL-4. In initial studies with adult mice, LPS induced a time-dependent increase in M1 and M2 mRNA profiles in microglia. Furthermore, peripheral LPS injection markedly increased surface expression of IL-4 receptor-alpha (IL-4Rα), but not IL-10 receptor-1 (IL-10R1) on microglia. In BV-2 cells, IL-4, but not IL-10, re-directed LPS-activated microglia towards an M2 phenotype. Based on these findings, comparisons of M1 and M2 activation profiles, induction of IL-4Rα, and sensitivity to IL-4 were determined in microglia from adult (3-4 mo) and aged (18-22 mo) mice. In aged microglia, LPS promoted an exaggerated and prolonged M1 and M2 profile compared to adults. Moreover, IL-4Rα protein was not increased on aged microglia following LPS injection. To determine the consequence of impaired IL-4Rα upregulation, adult and aged mice were injected with LPS and activated microglia were then isolated and treated ex vivo with IL-4. While ex vivo IL-4 induced an M2 profile in activated microglia from adult mice, activated microglia from aged mice retained a prominent M1 profile. These data indicate that activated microglia from aged mice are less sensitive to the anti-inflammatory and M2-promoting effects of IL-4.

Circulating neutrophils of septic patients constitutively express IL-10R1 and are promptly responsive to IL-10.

Previous studies have demonstrated that neutrophils isolated from the blood of healthy donors do not respond to IL-10 in terms of either activation of signal transducer and activator of transcription-3 (STAT3) tyrosine phosphorylation or induction of suppressor of cytokine signalling (SOCS)-3 protein, unlike autologous mononuclear cells. This was explained by the fact that circulating neutrophils of healthy donors express only IL-10R2, but not IL-10R1, the latter IL-10R chain being essential for mediating IL-10 responsiveness. In this study, we report that peripheral blood neutrophils of septic patients constitutively display, besides IL-10R2, also abundant levels of surface IL-10R1. Consequently, septic neutrophils are promptly responsive to IL-10 in vitro, as revealed by a direct IL-10-mediated induction of STAT3 tyrosine phosphorylation and SOCS-3 gene transcription, mRNA and protein expression. Consistent with the presence of a fully functional IL-10R, modulation of LPS-induced CXCL8, CCL4, tumour necrosis factor-alpha and IL-1ra gene expression was also rapidly induced by IL-10 in septic, but not normal, neutrophils. Collectively, these data uncover that neutrophils of septic patients are predisposed to be promptly responsive to IL-10, presumably to help limiting their pro-inflammatory state. They also fully validate our previous observations, herein in the context of a human disease, that responsiveness of human neutrophils to IL-10 is strictly dependent upon the modulation of IL-10R1 expression.