Tissue resident iNKT17 cells facilitate cancer cell extravasation in liver metastasis via interleukin-22.

During metastasis, cancer cells invade, intravasate, enter the circulation, extravasate, and colonize target organs. Here, we examined the role of interleukin (IL)-22 in metastasis. Immune cell-derived IL-22 acts on epithelial tissues, promoting regeneration and healing upon tissue damage, but it is also associated with malignancy. Il22-deficient mice and mice treated with an IL-22 antibody were protected from colon-cancer-derived liver and lung metastasis formation, while overexpression of IL-22 promoted metastasis. Mechanistically, IL-22 acted on endothelial cells, promoting endothelial permeability and cancer cell transmigration via induction of endothelial aminopeptidase N. Multi-parameter flow cytometry and single-cell sequencing of immune cells isolated during cancer cell extravasation into the liver revealed iNKT17 cells as source of IL-22. iNKT-cell-deficient mice exhibited reduced metastases, which was reversed by injection of wild type, but not Il22-deficient, invariant natural killer T (iNKT) cells. IL-22-producing iNKT cells promoting metastasis were tissue resident, as demonstrated by parabiosis. Thus, IL-22 may present a therapeutic target for prevention of metastasis.

Th17 cells express interleukin-10 receptor and are controlled by Foxp3⁻ and Foxp3+ regulatory CD4+ T cells in an interleukin-10-dependent manner.

T helper 17 (Th17) cells are important for host defense against extracellular microorganisms. However, they are also implicated in autoimmune and chronic inflammatory diseases, and as such need to be tightly regulated. The mechanisms that directly control committed pathogenic Th17 cells in vivo remain unclear. We showed here that IL-17A-producing CD4+ T cells expressed interleukin-10 receptor α (IL-10Rα) in vivo. Importantly, T cell-specific blockade of IL-10 signaling led to a selective increase of IL-17A+IFN-γ⁻ (Th17) and IL-17A+IFN-γ+ (Th17+Th1) CD4+ T cells during intestinal inflammation in the small intestine. CD4+Foxp3⁻ IL-10-producing (Tr1) cells and CD4+Foxp3+ regulatory (Treg) cells were able to control Th17 and Th17+Th1 cells in an IL-10-dependent manner in vivo. Lastly, IL-10 treatment of mice with established colitis decreased Th17 and Th17+Th1 cell frequencies via direct signaling in T cells. Thus, IL-10 signaling directly suppresses Th17 and Th17+Th1 cells.

Colitis Promotes a Pathological Condition of the Liver in the Absence of Foxp3+ Regulatory T Cells.

Inflammatory bowel disease is associated with extraintestinal diseases such as primary sclerosing cholangitis in the liver. Interestingly, it is known that an imbalance between Foxp3+ regulatory T cells (Treg) and Th17 cells is involved in inflammatory bowel disease and also in primary sclerosing cholangitis. To explain these associations, one hypothesis is that intestinal inflammation and barrier defects promote liver disease because of the influx of bacteria and inflammatory cells to the liver. However, whether and how this is linked to the Treg and Th17 cell imbalance is unclear. To address this, we used dextran sodium sulfate (DSS) and T cell transfer colitis mouse models. We analyzed the pathological conditions of the intestine and liver on histological, cellular, and molecular levels. We observed bacterial translocation and an influx of inflammatory cells, in particular Th17 cells, to the liver during colitis. In the DSS colitis model, in which Treg were concomitantly increased in the liver, we did not observe an overt pathological condition of the liver. In contrast, the T cell-mediated colitis model, in which Treg are not abundant, was associated with marked liver inflammation and a pathological condition. Of note, upon depletion of Treg in DEREG mice, DSS colitis promotes accumulation of Th17 cells and a pathological condition of the liver. Finally, we studied immune cell migration using KAEDE mice and found that some of these cells had migrated directly from the inflamed intestine into the liver. Overall, these data indicate that colitis can promote a pathological condition of the liver and highlight an important role of Treg in controlling colitis-associated liver inflammation.

IL-10 Receptor Signaling Is Essential for TR1 Cell Function In Vivo.

IL-10 is essential to maintain intestinal homeostasis. CD4+ T regulatory type 1 (TR1) cells produce large amounts of this cytokine and are therefore currently being examined in clinical trials as T cell therapy in patients with inflammatory bowel disease. However, factors and molecular signals sustaining TR1 cell regulatory activity still need to be identified to optimize the efficiency and ensure the safety of these trials. We investigated the role of IL-10 signaling in mature TR1 cells in vivo. Double IL-10eGFP Foxp3mRFP reporter mice and transgenic mice with impairment in IL-10 receptor signaling were used to test the activity of TR1 cells in a murine inflammatory bowel disease model, a model that resembles the trials performed in humans. The molecular signaling was elucidated in vitro. Finally, we used human TR1 cells, currently employed for cell therapy, to confirm our results. We found that murine TR1 cells expressed functional IL-10Rα. TR1 cells with impaired IL-10 receptor signaling lost their regulatory activity in vivo. TR1 cells required IL-10 receptor signaling to activate p38 MAPK, thereby sustaining IL-10 production, which ultimately mediated their suppressive activity. Finally, we confirmed these data using human TR1 cells. In conclusion, TR1 cell regulatory activity is dependent on IL-10 receptor signaling. These data suggest that to optimize TR1 cell-based therapy, IL-10 receptor expression has to be taken into consideration.

A Protective Function of IL-22BP in Ischemia Reperfusion and Acetaminophen-Induced Liver Injury.

Acute liver injury can be secondary to a variety of causes, including infections, intoxication, and ischemia. All of these insults induce hepatocyte death and subsequent inflammation, which can make acute liver injury a life-threatening event. IL-22 is a dual natured cytokine which has context-dependent protective and pathogenic properties during tissue damage. Accordingly, IL-22 was shown to promote liver regeneration upon acute liver damage. However, other studies suggest pathogenic properties of IL-22 during chronic liver injury. IL-22 binding protein (IL-22BP, IL-22Ra2) is a soluble inhibitor of IL-22 that regulates IL-22 activity. However, the significance of endogenous IL-22BP in acute liver injury is unknown. We hypothesized that IL-22BP may play a role in acute liver injury. To test this hypothesis, we used Il22bp-deficient mice and murine models of acute liver damage induced by ischemia reperfusion and N-acetyl-p-aminophenol (acetaminophen) administration. We found that Il22bp-deficient mice were more susceptible to acute liver damage in both models. We used Il22 × Il22bp double-deficient mice to show that this effect is indeed due to uncontrolled IL-22 activity. We could demonstrate mechanistically increased expression of Cxcl10 by hepatocytes, and consequently increased infiltration of inflammatory CD11b+Ly6C+ monocytes into the liver in Il22bp-deficient mice upon liver damage. Accordingly, neutralization of CXCL10 reversed the increased disease susceptibility of Il22bp-deficient mice. In conclusion, our data indicate that IL-22BP plays a protective role in acute liver damage, via controlling IL-22-induced Cxcl10 expression.

IL-22BP is regulated by the inflammasome and modulates tumorigenesis in the intestine.

Chronic mucosal inflammation and tissue damage predisposes patients to the development of colorectal cancer. This association could be explained by the hypothesis that the same factors and pathways important for wound healing also promote tumorigenesis. A sensor of tissue damage should induce these factors to promote tissue repair and regulate their action to prevent development of cancer. Interleukin 22 (IL-22), a cytokine of the IL-10 superfamily, has an important role in colonic epithelial cell repair, and its levels are increased in the blood and intestine of inflammatory bowel disease patients. This cytokine can be neutralized by the soluble IL-22 receptor, known as the IL-22 binding protein (IL-22BP, also known as IL22RA2); however, the significance of endogenous IL-22BP in vivo and the pathways that regulate this receptor are unknown. Here we describe that IL-22BP has a crucial role in controlling tumorigenesis and epithelial cell proliferation in the colon. IL-22BP is highly expressed by dendritic cells in the colon in steady-state conditions. Sensing of intestinal tissue damage via the NLRP3 or NLRP6 inflammasomes led to an IL-18-dependent downregulation of IL-22BP, thereby increasing the ratio of IL-22/IL-22BP. IL-22, which is induced during intestinal tissue damage, exerted protective properties during the peak of damage, but promoted tumour development if uncontrolled during the recovery phase. Thus, the IL-22-IL-22BP axis critically regulates intestinal tissue repair and tumorigenesis in the colon.