Discussion

Intestinal homeostasis depends on the intricate signalling network between gut microbiota, the host’s immune system, and the intestinal epithelium integrity. It is clear that even with the knowledge there is about IL-22 today, it’s role remains yet contradictory in the mammalian intestine. Some studies report highly beneficial effects of IL-22 in the intestine, while others show the opposite. Moreover, IL-22 appears to have an opposing role also in other tissues, such as the skin, lungs, and kidneys (reviewed in Dudakov et al., 2015). The fact that IL-22 is produced by plethora of immune cells and it has a systemic effect in the mammalian organism indicates that this cytokine and its production must be strictly regulated.
One of the beneficial effects designated to IL-22 in the intestine is the promotion of AMP secretion by intestinal epithelial cells. Several studies have shown that IL-22 induces epithelial cells to increase the production of AMPs, such as β-defensin 3, lipocalin-2, and a number of Reg-family peptides (Gronke et al., 2019; Gunasekera et al., 2020; He et al., 2022; Lindemans et al., 2015; Patnaude et al., 2021). AMPs, as the name indicates, target microbes and are thus an integral part of the protection at the mucosal surfaces against intestinal inhabitants. AMPs are produced mainly by Paneth cells that reside in the bottom of small intestinal crypts (reviewed in Goyal et al., 2021). Importantly, IL-22 has been revealed to drive the differentiation of human Paneth cells (He et al., 2022). The location of Paneth cells creates a concentration gradient of AMPs in the crypts, with the highest concentration of AMPs in the bottom of the crypt, protecting the most important cell types within the intestine – stem cells. IL-22, therefore, enhances the protection of the source of all cell types in the intestine and thus contributes to the maintenance of intestinal homeostasis. However, several of the Reg-family AMPs are found to be overexpressed during UC and CD in humans (Tsuchida et al., 2017). AMP overexpression may lead to a significant decrease, or even elimination, of some bacterial species resulting in an ecological disbalance of intestinal microbiota, allowing possibly harmful bacterial species to thrive and cause damage to the host. Additionally, elevated IL-22 in the intestine has been linked to reduced microbial diversity because of an IL-22-induced increase in AMP production (Gunasekera et al., 2020). Taken together, it appears that when appropriately regulated, IL-22 induces epithelial cells to secrete just the right amount of AMPs to ward off intestinal microbes at the mucosal surfaces without causing dysbiosis. However, excessive amounts of IL-22 may lead to overexpression of AMPs in the intestine which, as a consequence, trigger microbial dysbiosis possibly leading to intestinal disorders, such as UC and CD.
IL-22 has been reported to increase the proliferative capacity of the intestinal epithelial cells in a number of studies (Lindemans et al., 2015; Patnaude et al., 2021; Zha et al., 2019). This feature of IL-22 certainly makes it beneficial in the mammalian intestine since the rapid renewal of the epithelium is the foundation of the symbiotic relationship between gut microbiota and the host. Without the fast regeneration of the epithelium, the intestinal microbes would likely overwhelm the host and its immune system. Because IL-22 appears to induce epithelial regeneration in the intestine, it has been suggested to use this cytokine as a therapeutic agent in dysbiotic and intestinal inflammatory disorders, such as IBD or NEC. However, the effect IL-22 has on ISC proliferation remains controversial. There are studies reporting an increase in ISC numbers upon IL-22 treatment (Lindemans et al., 2015; Zhang et al., 2020), while one reports the opposite (Zha et al., 2019). Intriguingly, the positive and negative effects of IL-22 on the intestinal stem cell population was observed in vitro and in vivo (Lindemans et al., 2015; Zha et al., 2019; Zhang et al., 2020). The discrepancy in these results could be due to differences in tissues used to derive organoids, the concentration of IL-22 used in the experiments, the expression of IL-22 receptor, or the time period the of IL-22 treatment. It is noteworthy that while Zha et al. (2019) report a reduction in Lgr5+ active stem cell population upon IL-22 treatment, TA cell proliferation in the intestine was promoted by IL-22 in mice.
Furthermore, it has been demonstrated that IL-22 specifically induces Paneth cell differentiation and proliferation in human, but not murine, small intestine derived organoids (He et al., 2022). This is a major difference in the effects IL-22 has in human and murine intestinal models. It is, thus, possible that the differences reported in effects of IL-22 in the intestine could be due to differences in the experimental setups. Differences such as, the species of origin (human vs mouse) of tissues and cell lines used for in vitro studies, the specific intestinal sections examined, or the timing and the duration of IL-22 treatment. It is also important to recognize that while intestinal organoids are an excellent in vitro model to study molecular interactions under various physiological conditions, they have limitations. For instance, IL-22 may potentially be differently regulated in different parts of the intestine (small vs large intestine), or in structures not found in the organoids. Furthermore, the presence of IL-22 regulators, such as IL-22BP varies between in vivo and in vitro experiments. In vitromodels usually do not include such compounds, whereas they are likely present in in vivo systems. This can consequentially lead to different outcomes of the experiments. Therefore, a whole organism still remains to be the best model to study complex immunological interactions.
Another aspect that might lead to difference in IL-22 signalling outcome is a difference in the cell types that produce IL-22. This is well illustrated by the T cell transfer colitis model in mice. It has been shown that IL-22 originating from memory/effector T cells induces pathogenicity (Kamanaka et al., 2011), whereas IL-22 produced by naïve T cells had a protective role in the T cell transfer colitis model (Zenewicz et al., 2008). Although, it is also possible that the effects on the intestinal tissue result from the activity of naïve T cells themselves instead of IL-22. Next to this, different immune cell types arrive at the site of the infection at different times. Neutrophils are among the first immune cells to arrive at the site of infection and are known to produce IL-22 (reviewed in Dudakov et al., 2015). However, adaptive immune cells, such as T cells, arrive at the site of infection much later. It allows for a sustained production of IL-22 during different stages of intestinal infection or damage, but it may also define the outcome of IL-22 signalling in the intestine. This might also hold true for normal early life development. Mao and colleagues showed that during early life, the sequential development of innate and adaptive immunity influences IL22 signalling outcome. Innate cells (such as ILC3s) might be activated by the microbiota before an effective adaptive immune response develops. In their research, the authors show differences in IL-22-dependent IEC signalling between wild-type and Rag1−/− adult mice. They showed that the developing adaptive immune cells (CD4+ T cells), silenced the ILC3-induced pSTAT3 signalling (as a result of IL22R binding) in the epithelial cells of wildtype mice. They also showed that in Rag-/- mice, in the absence of a dominant adaptive immune response, the persistent activation of ILC3s resulted in impaired lipid metabolism. Together these findings indicate that a possible delay or alteration in adaptive immunity might influence IL22 effector functions in terms of lipid metabolism as well as host microbe interactions. A host-microbiome feedback loop also seems to exist in mice and humans in which the microbiome can influence IL-22 production via synthesis (of precursors) of AhR ligands and IL-22 in turn influences the microbiota composition through its actions on AMP and mucus (Mar et al, 2023).
Moreover, the presence of other cytokines can affect how adaptive immune cells are activated. For instance, IL-22 together with IL-10 may have a different effect on T cell activation compared to IL-22 together with IL-17. These are important details to keep in mind when finding answers to questions related to IL-22 signalling. Next to temporal differences in IL-22 production in the intestine, there is also spatial variability. For example, ILC3s – potent IL-22 producers – are more abundant in the small intestine than in the large intestine (reviewed in Kim et al., 2016). The location of IL-22-producing and IL-22 receptor-expressing cells may therefore affect the outcome of IL-22 signalling. Moreover, the presence of IL-22BP in the intestine is highly important in defining the outcome of IL-22 signalling. Additionally, the presence and the expression of IL-22 regulators differs between intestinal sections; for example butyrate is solely produced in the colon upon microbial fermentation. Furthermore, IL-22BP is highly expressed in the colon compared to small intestine in healthy mice (Huber et al., 2012). This can significantly change the results as well as interpretations ofin vivo experiments. Another important point to consider is how far-reaching the IL-22 signalling is. When IL-22 production is elicited at the location of damage in one specific segment of the intestine and the cytokine remains at that site, it is likely beneficial as it induces cell proliferation and healing where needed. However, if IL-22 moves to another site in the intestine, or other tissues, where there is no damage, it may induce needless epithelial cell proliferation, leading to possibly pathological conditions.
IL-22 has been linked to increased paracellular permeability and decreased epithelial integrity by upregulating claudin-2 protein (Wang et al., 2017; Zha et al., 2019). This may lead to the flux of water and solutes into the gut lumen, and this is an efficient way to remove parasites and other pathogens from the intestine. However, a persistent and uncontrolled flux of solutes and water into the gut may cause diarrhoea and dehydration. Furthermore, increased permeability allows the microbes to leave the intestinal environment and enter the tissue surrounding the intestine, potentially leading to (acute) inflammation. On top of that, IL-22-induced paracellular permeability, and not proliferation of cells, has been suggested to be the reason why organoids increase in size during in vitro experiments because of increased claudin-2 expression (He et al., 2022; Zha et al., 2019). Thus, the technique used to evaluate organoid growth – surface area or proliferative capacity of the cells – could lead to different interpretations of results.
An additional important factor in the intestine affecting IL-22 is butyrate, a SCFA produced by some types of intestinal bacteria, and a main energy source for intestinal epithelial cells (reviewed in Martin-Gallausiaux et al., 2021). Interestingly, butyrate has been shown to increase MUC13 expression in human colon organoids synergistically with IL-22 (Patnaude et al., 2021), suggesting that it may enhance the beneficial effects of IL-22 in mammalian intestine. Furthermore, butyrate was shown to over-ride the disruptive effects to the epithelial barrier elicited by IL-22 (Patnaude et al., 2021). The effects butyrate has on IL-22 may be seen as a therapeutic opportunity. Treating patients with intestinal pathologies with probiotics and/or prebiotics, which stimulate butyrate-producing bacteria, together with IL-22 may increase beneficial effects of IL-22 in the intestine. But also, by supplementing only pre- and/or probiotics it may be possible to diminish the pathological effects that IL-22 elicits in the intestine. The effects of butyrate on IL-22 illustrate the diversity of signals and interactions involved in regulating IL-22 and maintaining intestinal homeostasis.