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 .