Type-1 Interferons Impair The Immunoregulatory Activity Of IL-10: Understanding The Mechanisms Of Abrogation Of Transplant Tolerance
Marcos Iglesias, DVM, PhD, Maria Chicco, MD, Darrel Bibicheff, BS, Gerald Brandacher, MD, Giorgio Raimondi, PhD.
Johns Hopkins University, Baltimore, MD, USA.
Induction of long term transplant survival by "costimulation blockade" (CoB) regimens is impaired by inflammatory responses. In particular, multiple studies reported that engagement of toll like receptors (TLR) abrogate the tolerogenic effect of CoB. Despite the identification of type-1 interferons (TI-IFN) as mediators of this effect in multiple models, their target population and specific pathway used remain undefined. To better understand how TI-IFN could interfere with the induction of transplant tolerance, we studied their impact on the immunomodulatory properties of IL-10.
Full mismatch skin transplants were performed using dorsal skin from Balb/c into C57BL/6 mice and animals received a peri-transplant regimen based on DST (107 T-depleted splenocytes; on day 0) and anti-CD154 mAb (MR-1; on day 0, 7, 14) +/- anti-IL-10R (on day 0, 7, 14). Mouse T cells were isolated by negative-selection and Tmem and Treg subsets identified by flow cytometry. IL-10R expression and phospho-STAT3 induction after IL-10 or IL-6 stimulation in T cells were measured via flow cytometry. The gene expression profile of T cell subsets exposed to TI-IFN was assessed by microarray and quantitative PCR analysis. Protein levels were measured by Western Blot.
Our result showed that IL-10 has a fundamental role in the protective effect of CoB (transplant survival: MST 105d with CoB vs 47d with anti-IL-10R administration). We then studied the impact of T cell exposure to TI-IFN on IL-10 signaling. Following 48h of in vitro incubation with IFN-β, memory T cells (Tmem) and Tregs presented a dramatic defect in the production of phospho-STAT3 in response to IL-10. This effect was very selective, as IL-6 signaling (post IFN-β exposure) induced normal levels of phospho-STAT3. The reduced accumulation of phospho-STAT3 in conditioned cells resulted in the inhibition of the upregulation of mRNAs for LIGHT, Sphk1 and Tarm-1 - three genes we have discovered are induced by IL-10 in T cells. Encouragingly, this effect was slowly reversible with removal of TI-IFN, suggesting a "druggable" pathway. Microarray and flow cytometry data indicated that this IL-10-specific unresponsiveness was not associated with any reduction of IL-10 receptor expression or an increase in SOCS (Suppressor of Cytokine Signaling) 1 and 3, nor with reduced STAT3 cytoplasmic availability. Instead, this analysis suggested a novel role for the transcription factor STAT1 in dampening IL-10 signaling. Using T cells from STAT1-KO mice, we show that the absence of STAT1 prevented IL-10 inhibition induced by IFN-β in Treg and Tmem, supporting our new model.
Overall, these results highlight the importance of IL-10 in the therapeutic effect of CoB regimens and reveal a new molecular mechanism whereby TI-IFN interfere with IL-10 signaling, and ultimately with regulation of alloreactivity. Identifying a strategy to target this mechanism could provide a valuable aid in the clinical efficacy of CoB-based immunomodulatory strategies.
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