Induction of autophagy in Cx3cr1+ mononuclear cells limits IL-23/IL-22 axis-mediated intestinal fibrosis

Abstract

Intestinal fibrosis is an excessive proliferation of myofibroblasts and deposition of collagen, a condition frequently seen in Crohn’s disease (CD). The mechanism underlying myofibroblast hyper-proliferation in CD needs to be better understood. In this report, we found that mTOR inhibitor rapamycin or mTOR deletion in CX3Cr1+ mononuclear phagocytes inhibits expression of interleukin (IL) −23, accompanied by reduced intestinal production of IL-22 and ameliorated fibrosis in the TNBS-induced fibrosis mouse model. This inhibition of IL-23 expression is associated with elevated autophagy activity. Ablating the autophagy gene Atg7 increases the expression of IL-23, leading to increased expression of IL-22 and increased fibrosis. Both induction of IL-22 and intestinal fibrosis occurred in RAG−/− mice and depletion of innate lymphoid cells (ILCs) attenuates the fibrotic reaction, suggesting that the pro-fibrotic process is independent of T and B cells. Moreover, IL-22 facilitates the transformation of fibroblasts into myofibroblasts. Finally, the fibrotic reaction was attenuated upon neutralization of either IL-23 or IL-22. Altogether, this study elucidated a signaling cascade underlying intestinal fibrosis in which altered mTOR/autophagy in CX3Cr1+ mononuclear phagocytes up-regulates the IL-23/IL-22 axis, leading to an excessive fibrotic response. Thus, our findings suggest that this cascade could be a therapeutic target for alleviation of CD fibrosis.

The role of IL-22 in intestinal health and disease

“…IL-22 may also play different roles based on the location of the inflammation; for example, IL-22 neutralization prevented only colonic, but not cecal, inflammation in Helicobacter hepaticus –infected, anti–IL-10R–treated mice (Morrison et al, 2015). Finally, neutralization of IL-22 prevented up-regulation of profibrotic genes in trinitrobenzene sulfonic acid–induced colitis, thus implicating IL-22 as a potential mediator of fibrotic complications commonly observed in Crohn’s disease (Mathur et al, 2019). Collectively, preclinical models of intestinal inflammation suggest that IL-22 effects are strongly dependent on context, complicating predictions of its role in human disease.…”

Mechanistic Insight into the Development of TNBS-Mediated Intestinal Fibrosis and Evaluating the Inhibitory Effects of Rapamycin

“…To further validate and quantify fibrotic responses, we determined the αSMA, collagen and TGFβ expressions by qPCR, and αSMA expression by flow cytometry in TNBS-treated colon (Figure 3B, 3C). We have also shown Cx3Cr1 + mononuclear phagocytes induce an inflammatory immune response to injury 9 . Thus, we wanted to see if administration of rapamycin in TNBS-treated mice could reverse the inflammatory and fibrotic effects.…”

“…During tissue injury with a chemical, mechanical and infection condition, an inflammatory response triggers the tissue repair process. However, a dysregulated and pathological inflammatory response leads to developing scarring or a fibrotic reaction, which could impair the tissue repair function 9,18,19 . Here, we show the procedure for the TNBS-induced fibrosis animal model, which significantly shares pathophysiology with human Crohn's disease.…”

“…mTOR/autophagy role is broadly implicated on intestinal homeostasis and in IBD pathogenesis 20,21 . We identified that mTOR/autophagy signaling is critical to modulate proinflammatory responses from IL-23 and IL1β cytokines from Cx3Cr1 + mononuclear phagocytes that affect pro-fibrotic IL-23/IL-22 axis ( Figure 3A) 9 . Thereby, purifying single Cx3Cr1 + mononuclear cells for immunoprofiling and gene expression is a critical step of the model.…”

“…This model is technically simple; disease onset is rapid, inexpensive, and could widely be used in different animals (e.g., mouse, rat and guinea pig 7 ). Co-administration of ethanol and TNBS (2,4,6-trinitrobenzene sulfonic acid) abruptly damages the intestinal barrier and exposes colon tissue protein to TNBS and elicit substantial immunologic responses 8,9 . Repeated exposure of TNBS leads to an overreactive repair process responding to inflammation and injury, and develops a fibrotic reaction in the gut.…”