Beneficial role of IL-22 in intestinal homeostasis

The integrity of the intestinal barrier is essential for a healthy gut. There are examples of intestinal diseases in which the criteria for homeostatic barrier functioning are not met, such as inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn’s disease (CD), but also necrotizing enterocolitis (NEC). These multifactorial conditions are usually characterized by excessive or insufficient immune responses leading to unresolved inflammation and disruption in the intestinal epithelial barrier (reviewed in Sommer et al., 2021), alongside other pathological symptoms (Mihi et al., 2021; reviewed in Zhang & Li, 2014). The multifactorial nature makes these pathologies difficult to treat and there is, therefore, active ongoing research to find possible therapeutic agents, which could help restore the epithelial barrier in the affected individuals. Enhancing IL-22 signalling has been suggested as such a therapeutic agent since IL-22 appears to be a communication bridge between the immune and tissue-resident non-immune cells, such as intestinal epithelial cells (Wolk et al., 2004). Here, the examples of how IL-22 may positively influence intestinal homeostasis will be elaborated on.

IL-22 and pathologies

One disease where IL-22 may have a positive influence is necrotizing enterocolitis (NEC). NEC is an intestinal inflammatory disorder primarily affecting neonates and premature infants, and it is thought to occur because of an underdeveloped immune system (Neu, 2014). A mouse study found fewer IL-22-producing cells as well as low levels of IL-22 in the intestinal lamina propria of neonate pups compared with adult mice (Mihi et al., 2021). Mihi et al. (2021) also showed that daily intraperitoneal injections with recombinant murine IL-22 for three days led to a reduction in pro-inflammatory cytokine IL-1β as well as in reduction in disease severity in NEC-induced pups compared with phosphate-buffered saline (PBS)-treated littermates. Treatment with IL-22 was further shown to decrease the damage of ileal epithelium in mice (Mihi et al., 2021). Reduction in pro-inflammatory cytokine profile and intestinal damage during in vivo experiments indicates that IL-22 treatment may have restorative effects in the treatment of NEC by reducing excessive inflammation and enhancing epithelium healing.
IL-22 has been shown to be protective in a colitis model applied in mice. This colitis model uses a transfer of CD4+CD45RBhi T cells (naïve T cells) into Rag1-/- mice to induce intestinal disease. Since these Rag1-/- mice lack all T cells (including regulatory T cells), there is no regulation of the transferred T cells and these cells rapidly increase in numbers, causing inflammation in the intestine. This Rag1-/- model has been employed to study the role of IL22 in colitis. For example, Rag1-/- orIl22-/- Rag1-/- mice received IL-22-deficient or wild-type CD4+CD45RBhi T cells intraperitoneally, and while all mice developed colitis, the mice fully lacking IL-22 developed most severe symptoms (Zenewicz et al., 2008). This suggests that IL-22 has a protective role in this murine colitis model. Moreover, it indicates that the source of IL-22 may be important in defining whether IL-22 confers protective or harmful effect in mammalian intestine.

IL-22 and mucus

Another way IL-22 may be beneficial is by inducing mucus secretion. The mucus in the intestine provides a vital protective layer separating gut microbiota from the epithelial cells and the host’s immune system, and it helps maintain the integrity of the intestinal barrier (reviewed in Okumura & Takeda, 2017). Commensal and pathogenic bacteria get trapped in the mucus and are expelled from the intestine by peristalsis, preventing excessive bacterial colonization (reviewed in Kim & Ho, 2010). Evidence suggests that IL-22 might play a role in maintaining the mucus layer. In both murine in vivo and human in vitroexperiments treatment with IL-22 increased the expression of membrane-bound mucins (Patnaude et al., 2021). Membrane-bound mucins are expressed on the apical side of intestinal epithelial cells, where they form a glycocalyx (a layer consisting of glycoproteins and glycolipids) by extending 200-1500 nm above the cell surface (reviewed in Kim & Ho, 2010). Murine in vivo experiments showed that intravenous injections with IL-22-Fc lead to an increase in Mucin-1 in the colon (Patnaude et al., 2021). IL-22-Fc in this experiment is a murine recombinant IL-22 linked to the Fc (fragment crystallizable) region of mouse IgG1. Fc-fusion proteins are used to prolong the plasma half-life of proteins and therefore increase their therapeutic efficacy (reviewed in Czajkowsky et al., 2012). In vitro experiments with human colon-derived organoids showed that treatment with recombinant human IL-22 increases the gene expression of membrane bound mucinsMUC1 , MUC4 , and MUC13 (Patnaude et al., 2021). IL-22 treatment has also shown to increase MUC1 expression in human jejunum- and duodenum-derived organoids (He et al., 2022). However, in mice, the expression of Mucin-2 and the presence of goblet cells have been shown to moderately decrease (although not significantly) upon treatment with IL-22 in the colon (Patnaude et al., 2021). It is possible that the effect of IL-22 on the expression of mucins may differ between the different sections of intestine. Human and mouse membrane-bound mucins have been shown to stimulate cell migration and inhibit apoptosis (reviewed in Kim & Ho, 2010). The evidence that IL-22 upregulates the expression of some of the membrane-bound mucins in human and mouse colon epithelial cells suggests that treatment with IL-22 may have a beneficial effect in conditions where patients are challenged with increased bacterial burden or a disrupted epithelial barrier, such as IBD or NEC.

IL-22 and butyrate

Butyrate is one of the short-chain fatty acids (SCFAs) produced by the microbes in the gut and it is the main source of energy for intestinal colonocytes (reviewed in Martin-Gallausiaux et al., 2021). SFCAs are products of anaerobic fibre fermentation by the microbes. The presence of butyrate in the intestine has been suggested to play a role in regulating the effects of IL-22 in the intestine (Patnaude et al., 2021). Interestingly, like the addition of IL-22, the addition of butyrate to human colon-derived organoids also increased MUC13expression, and its effects were further enhanced when IL-22 was added together with butyrate (Patnaude et al., 2021). Interestingly, recently research performed in zebrafish uncovered a link between acetate produced by microbes and upregulation of IL22 (Liao et al., 2023). Specifically, this study showed that vitamin D induced anti-microbial peptide expression by activating IL22 signalling by increasing the abundance of acetate-producing bacteria (Liao et al., 2023).

IL-22 and intestinal barrier function

The intestinal epithelial barrier is protected by AMP production as well as rapid epithelial cell renewal. The gastrointestinal epithelium is renewed in 2-3 days in mice and in 3-5 days in humans (reviewed in Goyal et al., 2021). The rapid regenerative capacity of the intestinal epithelium allows for a symbiotic relationship between the host and the microbiota within the gut. Intestinal stem cells (ISCs) are the sole source of all other cell types found in the intestine (reviewed in Barker, 2014). It is therefore essential that the ISCs are well protected from the possibly harmful luminal contents, and that these cells receive correct signals to appropriately differentiate and proliferate. ISCs are offered protection by their location deep within the crypts, the AMPs produced by neighbouring Paneth cells, and the mucus layer (reviewed in Barker, 2014). IL-22 appears to initiate signals for ISC and epithelial cell proliferation, differentiation, and functioning. A number of in vivo and in vitro studies have shown that IL-22 has the capability to induce the proliferation of epithelial cells, but also stem cells in the intestine (Lindemans et al., 2015; Patnaude et al., 2021; Zha et al., 2019; Zhang et al., 2020). In case of colonic epithelial cells, it has been shown that treatment with recombinant human IL-22 leads to epithelial cell proliferation in human primary colon tissue organoids (Patnaude et al., 2021). In agreement with this, Patnaude et al. (2021) also reported that by injecting mice intravenously with IL-22-Fc, colon epithelial cells exhibit increased proliferation compared with the control treatment. Some studies have also shown that IL-22 positively affects the stem cell compartment in the intestine. For example, treating murine small intestine-derived organoids with exogenous recombinant IL-22 increases organoid size by inducing Lgr5+ stem cell proliferation (Lindemans et al., 2015). Likewise, another study demonstrated that by administering bacteria carrying IL-22 to irradiated mice increased Lgr5+ stem cells in the ileum (Zhang et al., 2020). However, others (He et al., 2022; Mihi et al., 2021; Zha et al., 2019) have not been able to replicate the reported intestinal stem cell expansion. Nevertheless, it has been shown that IL-22 treatment leads to proliferation of transit-amplifying (TA) cells in jejunal enteroids (Zha et al., 2019). TA cells are a cell type in a developmental stage between stem cells and fully differentiated epithelial cells (reviewed in Rangel-Huerta & Maldonado, 2017). Moreover, it has been revealed that IL-22 drives the differentiation of Paneth cells, the major producers of AMPs, in human small intestine organoids (He et al., 2022), and it has also been linked to AMP production in the intestine (He et al., 2022; Lindemans et al., 2015; Mihi et al., 2021; Patnaude et al., 2021). Specifically, in vitroexperiments with small intestine tissue from humans and mice have shown that treatment with IL-22 increases the mRNA levels of different AMPs, such as Reg3β and Reg3γ (Lindemans et al., 2015; Mihi et al., 2021). Additionally, Reg1α, Reg1β, Reg3α, β-defensin 3, and lipocalin-2 gene expression has been observed to be upregulated in human colonic organoids as well as small intestine-derived organoids when treated with IL-22 (He et al., 2022; Patnaude et al., 2021). The aforementionedin vitro studies are also supported by results from murinein vivo experiments where Reg3β and Reg3γ expression was upregulated in the colon after treatment with IL-22 (Gronke et al., 2019; Lindemans et al., 2015; Patnaude et al., 2021). The abovementioned studies illustrate that IL-22 can be beneficial in maintaining the intestinal homeostasis by protecting the stem cells with inducing AMP production and initiating cell proliferation and differentiation in the small and large mammalian intestine.

IL-22 as therapeutic agent

As mentioned above, IL-22 can lead to proliferation of intestinal epithelial cells and could thus aid in the regeneration of the intestinal epithelium upon damage. The effects of IL-22 as a therapeutic agent have been investigated in the context of irradiation damage in the gut. Irradiation causes damage to different intestinal cell types, such as γδ T-cells and helper T-cells, but also intestinal stem cells, Paneth cells, and goblet cells (Zhang et al., 2020). γδ T-cells and helper T-cells are known sources of IL-22 (reviewed in Dudakov et al., 2015). Zhang et al. (2015) presented a novel approach to deliver IL-22 to the site of intestinal damage. They transformed Lactobacillus reuteriand Escherichia coli to carry recombinant plasmids with murine IL-22, which would be expressed by the bacteria in the intestine. They showed that treatment with L. reuteri and E. coli carrying the IL-22 transgene can increase the survival of mice up to 85%, compared to the control group, if administered by gavage 24h after total body irradiation. It was shown in vitro that E. colisecretes IL-22, whereas L. reuteri needs to be lysed in order to release the intracellular IL-22 (Zhang et al., 2020). While this study does not demonstrate that the irradiation damage in the intestine is entirely restored by administering IL-22-producing bacteria, Zhang et al. (2020) do show increased numbers of intestinal stem cells in the ileum, as well as Paneth cells and goblet cells in mice in vivoafter the treatment. Using recombinant bacteria to deliver IL-22 to the gut to diminish intestinal damage after irradiation, or other type of intestinal injury, may be a safe treatment method since E. coliwas cleared from the colon by day five after gavage, indicating that the recombinant bacteria does not colonize the intestine (Zhang et al., 2020). However, L. reuteri clearance is not reported. The reason why L. reuteri has been proposed as a vehicle to deliver the biotherapeutic agents to the intestine is because this bacteria is considered safe for the host and can be transformed to additionally carry prophages which would lyse the bacteria during the gastrointestinal transit, after which the therapeutic agent L. reuteri is carrying would be released (Alexander et al., 2019). It is important to bear in mind that IL-22 delivery to the intestine by bacteria carrying the IL-22 transgene would likely be beneficial only in cases when the epithelial barrier is severely disrupted, since the receptors for IL-22 are found on the basolateral side of the epithelial cells and not on the apical side (Patnaude et al., 2021; Wang et al., 2017), and IL-22 could thus not elicit its effects from the gut lumen.

IL-22 to combat genotoxic stress

The intestine is challenged daily by potentially pathogenic bacteria and possibly genotoxic food items ingested by the host. Genotoxicity is the ability of a substance to damage DNA, and possibly lead to mutations and tumours. One such source of genotoxic stress is glucosinolate-containing cruciferous vegetables, such as broccoli and cabbage (Schumacher et al., 2014). While glucosinolates themselves are not genotoxic to mammalian cells, some of their breakdown products (by bacterial enzymes), such as 1-methoxy-3-indolylmethyl alcohol (1-MIM-OH), can be (Schumacher et al., 2014). A study demonstrated that stimulating murine intestinal cellsin vivo by gavage with 1-MIM-OH led to an increase in DNA adduct formation in the caecal tissue compared to the controls (Gronke et al., 2019). DNA adducts are formed when substances bind to DNA, leading to nucleotide mispairings and eventually mutations. Remarkably, it was shown that 1-MIM-OH treatment increased IL-22+ ILC3s and γδ T cells, as well as IL-22 production by these cell types (Gronke et al., 2019). 1-MIM-OH treatment also led to an increase in the expression of a known IL-22 response gene, Reg3γ , in primary colon epithelial cells in vivo (Gronke et al., 2019). This experiment shows that even though glucosinolate metabolites may have a genotoxic effect on intestinal cells, they significantly increase the amount of IL-22+ cells as well as IL-22 in the intestine. It is noteworthy that the concentrations of 1-MIM-OH used in the experiment are considerably higher than the average estimated daily intake of glucosinolates in men and women (Schumacher et al., 2014). Thus, humans would have to consume kilograms of cruciferous vegetables per day to attain harmful concentrations of glucosinolate metabolites in their intestines. Nevertheless, IL-22 may protect against genotoxic stress by inducing DNA damage response (DDR) pathways (Gronke et al., 2019). DDR is a safety mechanism containing a network of activated genes which protect the cells from accumulating mutations when challenged with genotoxic stress. Mice subjected to a glucosinolate-free diet exhibited a significant reduction in DDR gene expression as well as IL-22 production in their primary colon epithelial cells (Gronke et al., 2019). On the contrary, DDR effector gene expression and IL-22 production were upregulated in mice who received glucosinolate-containing diet (Gronke et al., 2019). These results indicate that IL-22 may be necessary for an appropriate initiation of DDR in the mammalian intestine.
Taken together, IL-22 appears to participate in many processes involved in maintaining intestinal homeostasis. It has been shown to protect against genotoxic stress, decrease the damage to ileal epithelium, and decrease the expression of proinflammatory cytokine during induced NEC. IL-22 has also been demonstrated to increase the production of membrane-bound mucins as well as AMPs by intestinal epithelial cells. Additionally, it has been reported that IL-22 can increase the proliferative capacity of intestinal stem and epithelial cells. All the above-mentioned processes are necessary for the maintenance and restoration of intestinal homeostasis, and it appears that IL-22 can shift the homeostasis in the mammalian gut towards a positive direction. However, the role of IL-22 in intestinal homeostasis is not as explicit. IL-22 has also been demonstrated to have detrimental effects in the intestine. All studies referring to the beneficial effects of IL-22 are summarized in Table 1 .