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 .