Harmful role of IL-22 in intestinal homeostasis

IL-22 has several properties that make it suitable as a therapeutic agent due to its role in maintaining intestinal homeostasis. In the next section, however, the focus will be on the harmful effects of IL-22 in the intestine and why it could be appealing to therapeutically target, in other words inhibit, IL-22 instead.

IL-22 and intestinal stem cells

Contrastingly to the results presented about the beneficial effect of IL-22 on ISC proliferation, some studies report IL-22 reducing the numbers of ISC. Zha et al. (2019) found that treating murine jejunal enteroids with murine recombinant IL-22 reduces the number of Lgr5+ ISCs. These in vitro results were supported by in vivoexperiments. Intraperitoneal injections with IL-22 for seven days (1 µg/day) led to a decrease in Lgr5+ stem cell population in jejunum as well as ileum in the treated mice (Zha et al., 2019). Another study reported that IL-22 treatment reduced human small intestine organoid formation efficiency, organoid budding, and increased cell death (He et al., 2022). Since ISCs are the source of all types of intestinal epithelial cells (reviewed in Barker, 2014), their maintenance is critical to maintaining intestinal homeostasis. IL-22 is upregulated in CD and UC (Andoh et al., 2005), and the results from the abovementioned studies suggest that too much IL-22 could be detrimental for intestinal epithelial regeneration and could be one of the factors responsible for impaired mucosal regeneration and impaired healing observed in IBD (reviewed in Sommer et al., 2021).

IL-22 and epithelial barrier integrity

Although we have discussed the beneficial effects of IL-22 in intestinal barrier integrity, some studies indicate that IL-22 may decrease the barrier integrity. Experiments with epithelial cell monolayers have shown that IL-22 treatment increases the paracellular permeability and reduces transepithelial electrical resistance (TEER) (Patnaude et al., 2021; Wang et al., 2017). Reduction in TEER indicates reduced epithelial integrity. Experiments with human colorectal Caco-2 cells demonstrated that treating the monolayer with recombinant human IL-22 for 72h from the basolateral side significantly decreased TEER compared with the control treatment (Wang et al., 2017). To confirm that IL-22 is responsible for reducing TEER, the monolayer was treated with recombinant human IL-22BP, a known negative regulator of IL-22. IL-22BP treatment counteracted the TEER-reducing effect of IL-22 on Caco-2 cell monolayer (Wang et al., 2017). Similar results were obtained in experiments with the human colon-derived T-84 cell line, where a significant decrease in TEER was observed 48h post-treatment with human recombinant IL-22 (Patnaude et al., 2021). IL-22 signalling from the exclusively basolateral side of the epithelial cells is also confirmed in experiments with T-84 cells by Patnaude et al. (2021). IL-22 exhibited no effect on TEER, nor the expression of IL-22-inducible genes, such as REG1A and REG3G , when administered from the apical side of the abovementioned cell lines (Patnaude et al., 2021; Wang et al., 2017). Moreover, cell viability was also not affected by IL-22 treatment (Patnaude et al., 2021; Wang et al., 2017), indicating that IL-22 had the TEER-reducing effect via basolaterally expressed receptors in these cell lines. Interestingly, it was shown that butyrate is able to override the disruptive effects of IL-22 on TEER (Patnaude et al., 2021). This indicates once again that the presence of butyrate in the (large) intestine might influence the effects IL-22 has in the mammalian gut.
IL-22 is thought reduce epithelial integrity by altering tight junction structures, namely by claudin-2 upregulation (Patnaude et al., 2021; Wang et al., 2017). Claudin-2 is a junctional protein expressed in the gastrointestinal tract in humans (Wang et al., 2017). Claudin-2 forms paracellular channels to allow the passage of solutes, such as Na+ (Tanaka et al., 2017). However, overexpression of claudin-2 may lead to excessive transepithelial paracellular leakage in the intestine, which can be harmful to the host. Upregulation of claudin-2, as well as IL-22, is seen in intestinal diseases, such as CD and UC (Andoh et al., 2005; Wang et al., 2017). Treating Caco-2 cells with IL-22 for 24, 48, 72, and 96 hours showed increased expression ofCLDN2 , a gene coding for claudin-2 protein, but no other measured tight junction protein-coding genes (Wang et al., 2017). Claudin-2 expression was also upregulated upon IL-22 treatment in human primary intestinal epithelium cells (Wang et al., 2017), as well as in micein vivo and human organoids derived from healthy and UC donors (Patnaude et al., 2021). To demonstrate that claudin-2 upregulation is indeed induced by IL-22, the Caco-2 cell monolayer was subsequently treated with IL-22BP, and the results showed that IL-22BP completely abrogated the effects of IL-22 on claudin-2 expression (Wang et al., 2017). Additionally, knocking down the CLDN2 gene in Caco-2 cells confirmed that claudin-2 causes the reduction of TEER (Wang et al., 2017). Taken together, in the experimental setting of epithelial cell monolayers, it has been shown that IL-22 can be detrimental by upregulating junctional protein claudin-2 and thereby reducing TEER. The epithelial integrity in the intestine is essential for proper functioning and the evidence that IL-22 can harmfully act upon this barrier illustrates that IL-22 may also be detrimental. However, it is important to realize that these experiments were conducted on cell monolayers and may not reflect physiological conditions present in the mammalian intestine. Other models such as intestinal organoids or even whole organism may be better to evaluate the effect of IL-22 on intestinal epithelial barrier.

IL-22 in colitis models

In some murine colitis models, IL-22 has been shown to have a harmful role in the intestine. In the innate colitis model, mice were injected with anti-CD40 antibodies to induce colitis. In an experiment withIl-23-/WTRag-/- mice, it was shown that neutralization of endogenous IL-22 with anti-IL-22 antibody after anti-CD40 injections leads to a significant reduction in weight loss, colitis scores, and colon pathology (Eken et al., 2014). When IL-22 levels were restored with IL-22-expressing plasmid injections inIl-23-/-Rag-/- mice, they again developed severe colitis upon injections with anti-CD40 (Eken et al., 2014). Injection of empty plasmids alone did not lead to colitis development. These observations suggest that in mice, IL-22 has a pathological effect in anti-CD40-induced acute innate colitis. However, colitis developed inIl-23-/-Rag-/- mice only after anti-CD40 injection, indicating that IL-22 plasmid injections alone do not cause colitis (Eken et al., 2014). Additionally, it was reported that Il-23-/-Rag-/-mice who received IL-22 plasmid after anti-CD-40 injections had significantly more neutrophils in the colonic lamina propria compared with empty-vector recipients. The authors suggested that IL-22 may facilitate colitis pathology by recruiting neutrophils to the site of intestinal damage (Eken et al., 2014). Neutrophils produce neutrophil extracellular traps (NETs) to bind pathogens, but too many NETs may also be harmful to the host (reviewed in Castanheira & Kubes, 2019). Excessive neutrophil recruitment in the intestine may lead to enhanced NET production and induce inflammation in the colon. The experiments by Eken et al. (2014) indicate that IL-22 on its own is likely not disruptive, but rather it initiates signals which recruit other cell types or cytokines, which lead to harmful outcomes.
IL-22 was also shown to have a detrimental effect in a different experimental colitis model. CD4+CD45RBhi T cells (naïve) or Treg cell-depleted CD4+CD45RBlo T cells (memory/effector) derived from wild-type (WT) mice were transferred intoRag1-/- mice to induce colitis (Kamanaka et al., 2011). Specifically, Kamanaka et al. (2011) found increased IL-17 and IL-22 mRNA expression after disease development in the colons ofRag1-/- mice who received Tregcell-depleted CD4+CD45RBlo T cells in comparison to mice who received the naive CD4+CD45RBhi T cells. They additionally report that transfer of IL-22 knock-out (KO), but not IL-17 KO T cells (CD4+CD45RBlo T and Treg depleted) to Rag1-/- mice reduced weight loss and colitis scores compared with mice who received WT T cells. This indicates that memory/effector T cell-derived IL-22 might be involved in the pathogenicity of the colitis model used in this study. Possible mechanisms by which IL-22 can influence colitis is by inducing epithelial hyperplasia as there is evidence that IL-22 promotes epithelial cell proliferation in the colon (Kamanaka et al., 2011; Patnaude et al., 2021). Additionally, the levels of Reg3γ were significantly reduced in IL-22 KO memory-effector transfer mice, suggesting that IL-22 may drive colitis by inducing Reg3γ, which may alter the microbiota composition and lead to dysbiosis (Kamanaka et al., 2011). Interestingly, the results of the study by Kamanaka et al. (2011) again emphasize that the source of IL-22 might be determining the effects in its target tissues in view of the fact that IL-22 was protective in the CD4+CD45RBhi(naïve T cell transfer) colitis model (Zenewicz et al., 2008).
Another colitis model often used to investigate intestinal inflammation mechanisms is by inhibiting, knocking down, or knocking out IL-10 in mice. IL-10 inhibits the expression of pro-inflammatory cytokines, such as TNF-α, IL-6 and IFN-γ (Gasche et al., 2000), and therefore regulates inflammation. Gunasekera et al. (2020) found that IL-22 levels, as well as a number of antimicrobial IL-22-target gene mRNA levels, were significantly higher in Il-10-/- mice compared to the WT mice. IL-22 protein levels were also increased in the colon and small intestine of Il-10-/- colitic mice compared to the WT mice (Gunasekera et al., 2020). These observations suggest that IL-10 negatively regulates IL-22 expression in the intestine. Since it is known that IL-22 upregulates AMPs in the intestine, the diversity of microbiota was evaluated inIl-10-/- mice.Il-10-/- mice had less diverse microbiota compared with WT mice, as well asIl-10-/-Il-22-/ -, and Il-22-/- mice (Gunasekera et al., 2020). Reduced microbiota diversity is usually correlated with gut dysbiosis. It is possible that IL-22 driven AMP upregulation in the intestine leads to dysbiosis and consequently to intestinal disorders. Several Reg-family AMPs, such as Reg1α/β, Reg3, and Reg4 are known to be overexpressed in the intestines of humans with UC and CD (Granlund et al., 2011; Tsuchida et al., 2017). To determine a more specific role of IL-22 in this colitis model,Il-10-/-Il-22-/- mice were used. Il-10-/-Il-22-/- , norIl-22-/- mice develop chronic colitis, indicating that IL-22 is involved in chronic colitis development (Gunasekera et al., 2020). While Il-10-/-colitic mice exhibited rectal prolapse, as well as ulcerations, crypt abscesses, and mucosal hyperplasia in the colon,Il-10-/-Il-22-/- , andIl-22-/- mice did not develop any of the symptoms (Gunasekera et al., 2020). However, it is important to bear in mind that in this study exogenous IL-22 inIl-10-/-Il-22-/- mice was not used to show that indeed IL-22 is the aberrant factor. Taken together, these data indicate that IL-10 is an important negative regulator of IL-22 and its downstream genes in the intestine, and without appropriate regulation, IL-22 may become aberrant and cause pathology in the intestine.

IL-22 regulation and its role in tumorigenesis

While IL-22 is seen as beneficial in the intestine by promoting epithelial cell proliferation, it may also be detrimental for the same reason. IL-22 is found excessively expressed in human colon cancer tissues compared to healthy donor tissues, and in vitroexperiments have shown that IL-22 enhances tumour proliferation (Jiang et al., 2013). Tumours are formed by uncontrolled cell proliferation, and IL-22 has been shown to promote epithelial cell proliferation in human and mouse models (Lindemans et al., 2015; Patnaude et al., 2021; Zha et al., 2019). It is therefore hypothesized that when IL-22 lacks correct inhibiting signals in the intestine during a steady state, for instance by IL-22BP, it may initiate tumour formation by signalling epithelial cells to continuously proliferate. IL-22BP, a potent IL-22 inhibitor produced by dendritic cells (Martin et al., 2014), is upregulated during homeostatic conditions and is downregulated in the colon upon mechanical damage, whereas IL-22 levels exhibit the opposite (Huber et al., 2012). It has been shown in murine models that IL-22 and IL-22BP exhibit inverse expression patterns (Huber et al., 2012). Additionally, Il22bp-/- mice showed increased epithelial cell proliferation during the DSS-induced colitis recovery phase whereas in WT mice, the cell proliferation during the recovery phase had reduced to a rate similar to steady-state conditions (Huber et al., 2012). Moreover, they showed that lack of IL-22BP lead to accelerated development and higher number tumours in the colon in comparison with WT mice (Huber et al., 2012). The study by Huber et al. (2012) illustrates how important the tight regulation of IL-22 is in the intestine. Tumour formation is unquestionably a multifactorial process and IL-22 alone certainly is not responsible for this process, but without appropriate regulation it may enhance tumorigenesis by initiating excessive epithelial cell proliferation.
IL-22BP is not only important in preventing IL-22 signalling in tumour formation but also in continuous regulation of IL-22 signalling in the intestine of healthy individuals where it helps to maintain homeostasis. IL-22BP is constitutively produced by dendritic cells (Martin et al., 2014), and IL-22BP levels are usually upregulated during steady-state conditions and downregulated during inflammation. It allows to maintain the levels of IL-22 low in a healthy gut and elevated during intestinal damage. However, both IL-22 and IL-22BP have been observed to be increased in human CD and UC (reviewed in Zenewicz, 2021). These observations emphasize that IL-22 is a potent cytokine which is actively upregulated during inflammation, and that elevated IL-22BP levels may not always be enough to inhibit IL-22 signalling.
While the study by Huber et al. (2012) clearly shows that it is important to appropriately inhibit IL-22 to suppress tumour formation, they do not elaborate on the mechanisms by which IL-22 enhances tumour growth. Others have suggested to therapeutically target (and thus inhibit) IL-22 since there is evidence that it promotes tumour angiogenesis (Protopsaltis et al., 2019). Protopsaltis et al. (2019) showed that IL-22 enhances human endothelial cell proliferation, survival, and migration in a dose-dependent manner in vitro . Using the ex vivo murine choroid explant model, they also showed that IL-22 treatment promotes vessel outgrowth significantly more than the controls. Finally, they demonstrated that blocking IL-22 significantly reduces the volume of tumours induced by EL4 T cell lymphoma cell line in Rag-/- and C57BL/6 micein vivo compared to controls. Even though the experiments by Protopsaltis et al. (2019) did not show the harmful effect of IL-22 in the intestine, their findings could also be relevant to types of cancer found in the intestine since the induction of angiogenesis is one of the hallmarks of cancer (Hanahan & Weinberg, 2011). Human colon cancer patients have been observed to have significantly higher levels of IL-22 in their cancer tissue than healthy controls (Jiang et al., 2013). It is thus possible that IL-22 enhances intestinal, as well as other tissue, tumour growth and development by promoting angiogenesis.
Taken together, there are many examples of IL-22 having a negative impact on intestinal homeostasis in mammals, when dysregulated. In vivo and in vitro experiments have demonstrated that IL-22 leads to a reduction of intestinal stem cells and intestinal epithelial barrier integrity, but also enhances paracellular permeability via upregulation of junctional protein claudin-2. It has also been shown that IL-22 can exacerbate colitis in some murine models. Lastly, it was shown that IL-22 promotes tumour angiogenesis by enhancing endothelial cell proliferation, survival, and migration. All studies referring to harmful effects of IL-22 are summarized in Table 2 .