Abstract
The function of interleukin-22 (IL-22) in intestinal barrier
homeostasis remains controversial. Here, we map the transcriptional
landscape regulated by IL-22 in human colonic epithelial organoids and
evaluate the biological, functional and clinical significance of the
IL-22 mediated pathways in ulcerative colitis (UC). We show that IL-22
regulated pro-inflammatory pathways are involved in microbial
recognition, cancer and immune cell chemotaxis; most prominently those
involving CXCR2^+ neutrophils. IL-22-mediated transcriptional
regulation of CXC-family neutrophil-active chemokine expression is
highly conserved across species, is dependent on STAT3 signaling, and
is functionally and pathologically important in the recruitment of
CXCR2^+ neutrophils into colonic tissue. In UC patients, the magnitude
of enrichment of the IL-22 regulated transcripts in colonic biopsies
correlates with colonic neutrophil infiltration and is enriched in
non-responders to ustekinumab therapy. Our data provide further
insights into the biology of IL-22 in human disease and highlight its
function in the regulation of pathogenic immune pathways, including
neutrophil chemotaxis. The transcriptional networks regulated by IL-22
are functionally and clinically important in UC, impacting patient
trajectories and responsiveness to biological intervention.
Subject terms: Mucosal immunology, Inflammatory bowel disease,
Neutrophils, Interleukins, miRNA in immune cells
__________________________________________________________________
Mechanisms of non-response to ustekinumab, a biologic targeting IL-23,
are currently unclear. Here, the authors show that the transcriptional
program regulated by IL-22, an IL-23 responsive cytokine, is enriched
in patients with ulcerative colitis unresponsive to ustekinumab and
associated with higher colon neutrophil recruitment and activation of
upstream IL-22 regulators.
Introduction
Ulcerative colitis (UC) is the most common form of inflammatory bowel
disease (IBD). It is an incurable, chronic, immune-mediated
inflammatory disease (IMID) that selectively affects the colon and is
associated with significant complications, including
cancer^[72]1,[73]2. Dysregulated mucosal immune responses are at the
heart of UC pathogenesis characterized by increased production of
cytokines and the local accumulation of immune cells, most notably
mononuclear cells and neutrophils which are associated with
architectural distortion of tissue, crypt destruction and crypt abscess
formation^[74]3. Accordingly, targeting individual cytokines or the
cells that produce them are the most effective therapeutic strategies
in UC.
Ustekinumab is a monoclonal antibody (mAb) targeting the p40 subunit
common to both interleukin (IL)−12 and IL-23^[75]4. Selective targeting
of IL-23 is a conceptually attractive approach, since IL-23 is strongly
implicated in IMIDs affecting the skin^[76]5, brain^[77]6, joints^[78]7
and intestine^[79]8,[80]9. IL-23 overexpressing transgenic mice develop
multi-system inflammatory disease, including severe neutrophilic
inflammation in the gut^[81]10. In preclinical models of UC, genetic
deletion, or therapeutic neutralization of the specific p19 subunit of
IL-23 significantly attenuates colitis^[82]11,[83]12. IL-23 stimulates
effector function of innate and adaptive lymphocytes, triggering
production of IL-17A, IL-17F, interferon-γ (IFNγ) and GM-CSF^[84]13.
Although clinical trials evaluating the efficacy of IL-23 blockade are
now underway, important theoretical concerns about targeting IL-23
exist, since the downstream pathways regulated by IL-23 are also
implicated in tissue restitution. For instance, IL-22 is one of the key
cytokines regulated by IL-23, and several lines of evidence point to
IL-22 having an important protective function in the gut. IL-22 induces
production of anti-microbial peptides and is involved in intestinal
epithelial barrier recovery after acute injury by promoting LGR5^+
intestinal epithelial stem cell proliferation^[85]14. Clinical trials
evaluating recombinant IL-22 to promote recovery of epithelial injury
are currently underway ([86]NCT02749630). Confusingly, in several
chronic models of IBD, IL-22 has been shown to be pathogenic^[87]15.
Accordingly, new insights into the function of IL22 in mucosal immunity
are now needed, especially in human disease, to help reconcile these
discrepancies.
In this study, we probe the clinical and functional significance of the
IL-22 responsive transcriptional program in diseased tissue of UC
patients treated with ustekinumab and in multiple models of colitis. We
provide further insights into cytokine-mediated regulation of the
intestinal epithelium and how this influences pathogenic pathways and
patient outcomes in UC. We show that IL-22 is a functionally important
regulator of neutrophil recruitment to the colon by controlling the
expression of neutrophil-active CXC-family chemokines. Augmented
expression of IL-22 responsive transcripts and increased recruitment of
colonic neutrophils is associated with treatment resistance to
ustekinumab.
Results
Enrichment of IL-22 responsive transcriptional networks is associated with
poor response to ustekinumab therapy in ulcerative colitis
As IL-22 selectively targets the intestinal epithelium we generated a
human mini-gut colonic epithelial organoid system in order to
investigate the IL-22 regulated molecular pathways. Colonic organoids
were treated with or without human recombinant IL-22, and the
IL-22-responsive transcriptome was mapped by RNA-seq (Fig. [88]1a).
IL-22 induced differential expression of 1251 transcripts (upregulated:
579, downregulated: 672, FDR < 0.01) (Fig. [89]1b). Significantly
upregulated transcripts encoded anti-microbial peptides (REG1A, REG1B),
mucins (MUC1, MUC4, MUC12), chemokines (CXCL1, CXCL2, CXCL5, CXCL8),
cytokines (TNF, IL-1, IL18, IL33), caspase family members (CASP1,
CASP4, CASP5, CASP10), matrix metalloproteinases (MMP1, MMP7, MMP10),
enzymes involved in the generation of reactive oxygen species (DUOXA2,
NOS2, SOD2), and immunoregulatory molecules, such as SOCS1, SOCS2,
SOCS3 and IDO. Pathway analysis identified tumor necrosis factor-alpha
(TNF) and IFNγ responses among other pro-inflammatory pathways to be
enriched in the IL-22 regulated DEG list (Fig. [90]1c).
Fig. 1. Clinical significance of the IL-22 responsive transcriptional network
in ulcerative colitis.
[91]Fig. 1
[92]Open in a new tab
a Experimental schema of IL-22 stimulation of colonic organoids (n = 4
biological replicates, IL-22 concentration: 10 ng/ml, duration: 24 h).
b Volcano plot demonstrating fold change and false discovery rate (FDR)
of differentially expressed genes in human colonoids treated with
recombinant IL-22. c Pathway analysis of DEG regulated by IL-22 (top
10, hallmark gene sets as defined in MSigDB, NES: normalized enrichment
score). d Expression of the top 50 upregulated transcripts regulated by
IL-22 in colonic mucosa separates healthy controls (blue, n = 11),
patients with endoscopically inactive UC (green, n = 23) and active UC
(red, n = 74) (principal component analysis, reposited dataset:
[93]GSE50971). e Enrichment of the IL-22 regulated transcriptional
program (gene set variation analysis, gene set: top 50 upregulated
genes) in the reposited dataset [94]GSE50971 (healthy control: control,
iUC-inactive UC, aUC-active UC, Kruskal–Wallis test, ****p < 0.0001). f
Validation of the enrichment for the IL-22 regulated transcriptional
program in biopsies taken from healthy controls (control, n = 18) and
patients with UC (n = 550) participating in UNIFI trial (Mann–Whitney
test, two-sided test, ***p < 0.001). g, h, i Association of the IL-22
enrichment score with clinical outcomes in UNIFI. Clinical remission
(defined as a total Mayo score of ≤2 and no subscore >1) and deep
remission [which required both clinical remission and mucosal healing
defined as histologic improvement (neutrophil infiltration in <5% of
crypts, no crypt destruction, and no erosions, ulcerations, or
granulation tissue) and endoscopic improvement] at week 8 in UC
patients enrolled in the UNIFI clinical trial program stratified
according to IL-22 enrichment score in baseline biopsies sampled
immediately prior to initiation of ustekinumab (n = 358) or placebo
(n = 184). Blue bar shows response rate in placebo-treated UC patients.
Gray bar shows response for all patients treated with ustekinumab. Red
bars show response for all patients treated with ustekinumab stratified
based on the extent of the IL-22 transcriptional program’s activation
(ES). Patient numbers and statistical analysis shown in Supplementary
Fig. [95]1. Source data for b, c, d, e, f, h and i are provided as a
Source Data file.
Next, we asked whether core IL-22 regulated transcripts were
differentially expressed in diseased tissue of UC patients. Principal
component analysis based on the expression of the top 50 most highly
upregulated genes induced by IL-22 in colonic biopsies showed
separation of patients with active UC from those with quiescent
(inactive) disease and healthy controls (previously reposited dataset:
[96]GSE59071, Fig. [97]1d). A similar picture emerged by testing for
the activation of this gene set (top 50 upregulated genes by IL-22)
using Gene Set Variation Analysis (GSVA) (Fig. [98]1e). This is an
algorithm which tests whether a group (set) of genes is enriched in
complex and heterogeneous samples. The enrichment score varies between
+1 (upregulated) to −1 (downregulated) and depends on the distribution
of gene expression across the samples tested. We validated these
findings in endoscopically acquired colonic biopsies sampled from the
sigmoid colon of patients with moderate-to-severe UC (n = 550,
Supplementary Table [99]1) enrolled to the UNIFI phase III clinical
trial, a randomized, placebo-controlled trial evaluating the efficacy
of ustekinumab, a monoclonal antibody (mAb) that blocks the p40 subunit
shared by the human IL-12 and IL-23 cytokines. Our analysis
demonstrated that the transcriptional program regulated by IL-22
(Mann–Whitney, two-tailed test, P < 0.0001, Fig. [100]1f) was
significantly enriched in UC in comparison with non-IBD control
subjects.
Although the IL22 responsive transcriptional program was enriched at
population level in UC patients, there was considerable variation in
magnitude of enrichment. Since it was unlikely that this variation was
being driven by differences in disease severity (all patients in the
UNIFI trial program had moderate-to-severe UC, with Mayo endoscopy
subscores of either 2 or 3), we considered the possibility that this
molecular heterogeneity might represent important differences in
underlying disease immunobiology. If molecular stratification of UC
patients according to the magnitude of IL-22 responsive transcript
enrichment was biologically and/or clinically meaningful, we reasoned
that UC patients with different degrees of enrichment would experience
different outcomes and follow different trajectories. To test this
hypothesis, we stratified patients from the UNIFI trial program
according to their IL-22 enrichment score (in colonic biopsies sampled
immediately prior initiation with ustekinumab) and evaluated whether
these differences in molecular phenotype impacted treatment response.
Enrichment scores in colonic tissue could differentiate responders and
non-responders to ustekinumab induction therapy (including patients on
130 mg and 6 mg/kg dose), (Fig. [101]1g–i, Supplementary Fig. [102]1).
Remarkably, in comparison with unstratified patients, remission rates
in patients with low IL22 enrichment scores (ES < 0) were approximately
doubled, including clinical remission (13% vs 25%), mucosal healing
(16% vs 26%) and deep remission (a combination of clinical, endoscopic
and histologic remission, 12% vs 22%). Conversely, outcomes in UC
patients with high IL-22 enrichment scores were broadly comparable to
placebo treated patients. In other words, stratification of UC patients
according to the magnitude of enrichment of IL-22 responsive
transcriptional modules in baseline biopsies sampled at baseline prior
to treatment, associates with response to ustekinumab induction
therapy.
Interestingly, IL22 enrichment scores were slightly higher in UNIFI
participants previously treated with a biologic (anti-TNF), suggesting
that high IL-22 enrichment scores may be associated with poor responses
to other treatments (Supplementary Fig. [103]2A). To investigate this
possibility, we analyzed four previously published datasets of UC
patients treated with other biologics where outcome data and baseline
tissue transcriptomic data was available (anti-TNF: [104]GSE23597,
[105]GSE16879, [106]GSE92415, vedolizumab: [107]GSE73661). Although
there was no significant association of IL-22 enrichment scores with
drug response in 3 of these studies, in one study of infliximab
treatment, we observed a significant association of IL-22 enrichment
scores with primary non-response (Supplementary Fig. [108]2B). These
data suggest that enrichment of IL-22 responsive transcripts in colonic
tissue can predict outcomes to ustekinumab and might also portent poor
outcomes more generally in UC, including poor responses to other
therapies.
Patient stratification according to the magnitude of enrichment of the
IL-22-regulated transcriptional program identifies immunological mechanisms
of treatment resistance
Since patients with the greater enrichment of the IL-22-regulated
transcriptome were more likely to experience lack of response to
ustekinumab, we reasoned that immunological pathways differentiating
these patients from those with low IL-22 enrichment might provide
insights into mechanisms of treatment resistance. To probe differences
in the molecular profile of patients stratified by IL-22 responsive
transcripts, we analyzed genome-wide transcript expression changes in
biopsies sampled from UC patients from the UNIFI program with high
IL-22 enrichment scores (IL22 ES ≥ 0.25) in comparison with patients
with low IL-22 enrichment scores (IL-22 ES < 0.25). Canonical Pathway
Analysis (IPA, Ingenuity) demonstrated that the most significantly
associated biological pathway in patients with high IL-22 enrichment
scores was Granulocyte Adhesion and Diapedesis (right-tailed Fisher’s
exact test, P = 1.8 × 10^−29, Fig. [109]2a). Other notable associations
included multiple Th17 associated pathways, autoimmune diseases, and
other pathways that have also previously been linked to treatment
resistance including oncostatin M and TREM1 signaling (Fig. [110]2a and
Supplementary Fig. [111]3). We also performed a Downstream Effects
Analysis^[112]16 (IPA, Ingenuity), to predict causal effects and
biological processes that were significantly activated in patients with
high IL-22 enrichment scores. Overall, there were 245 disease or
functional annotations significantly activated in patients with high
IL-22 enrichment scores, encompassing different biological and
inflammatory processes. Pathways involving immune cell trafficking were
especially activated and comprised the greatest number of functional
annotations recorded (Supplementary Fig. [113]4A, B). In patients with
high IL-22 enrichment scores, the highest-ranking causal network
associated with cell migration connected 84 nodes, encoding transcripts
involved in neutrophil chemotaxis, matrix metalloproteinases,
anti-microbial peptides, immunoglobulin Fc receptors and innate-immune
response proteins, including IL-1, IL-6, and oncostatin M
(Fig. [114]2b).
Fig. 2. Biological pathways and upstream regulators associated with
enrichment of the IL-22 regulated transcriptional program.
[115]Fig. 2
[116]Open in a new tab
a Pathway enrichment analysis (IPA) of the differentially expressed
genes in patients enrolled in UNIFI with high enrichment for the IL-22
transcriptional program (right-tailed Fisher’s exact test), b genes
belonging to “Migration of cells” pathway that are upregulated in
patients with high enrichment of the IL-22 transcriptional program, as
identified using IPA downstream analysis. Genes have been further
subdivided into functional categories. Symbols denote type of protein,
color denotes regulation and color of line denotes relationship, c
regulator effects analysis performed on the top 3 predicted upstream
activators by IPA in the patients with high enrichment for the IL22
transcriptional program, d normalized expression intensity (to healthy
controls, n = 18) of known IL-22 regulators stratified by the IL-22
transcriptional program enrichment (UNIFI, UC n = 550, Kruskal–Wallis
test with Dunn multiple comparisons test, *p < 0.05, **p < 0.01,
***p < 0.001). Source data for a and d are provided as a Source Data
file.
To probe which mediators were potentially driving the transcriptional
changes observed in patients with ustekinumab resistance, we performed
an Upstream Regulator Analysis (IPA, Ingenuity). This algorithm
identifies upstream mediators predicted to modulate the expression of
transcripts in a user-defined dataset using large-scale causal
networks. The top 3 predicted upstream regulators of the gene
expression changes observed in colonic biopsies of patients with high
IL-22 enrichment scores were lipopolysaccharide (z-score=5.1,
right-tailed Fisher’s exact test, P value = 2.76×10^−22), TNF (z-score
= 4.6, right-tailed Fisher’s exact test, P value = 2.97 × 10^−18), and
IL-1β (z-score = 5.7, right-tailed Fisher’s exact test, P value =
5.72 × 10^−16) (Supplementary Fig. [117]4C). To gauge the biological
impact of these predicted mediators we performed Regulator Effects
analysis (Ingenuity IPA), an algorithm which connects activated
regulators with downstream differentially expressed genes in the
dataset. All three of the top predicted upstream regulators had closely
related, overlapping mechanistic networks, converging around activation
of IL1β, and induction of the transcription factors NFKB1, JUN and RELA
(Fig. [118]2c).
These data also offer insights into unexpected observations in our
dataset. We initially anticipated that patients with the highest
enrichment scores for IL-22 responsive transcripts would respond
favorably to ustekinumab, based on the notion that these patients have
augmented IL-23/IL-22 axis activity, and hence are more likely to be
amenable to IL-23 blockade, since IL-23 is an important driver of IL-22
production^[119]13,[120]17–[121]21. However, IL-23 is not the only
driver of IL-22 production; other cytokines, such IL-1^[122]22,
IL-6^[123]23,[124]24 and TL1A^[125]25 can also trigger IL22 production.
Crucially, although there was only a small difference in the expression
of transcripts encoding the two subunits of IL-23 (IL-23A and IL-12B)
in patients with high IL-22 enrichment scores, there was a substantial
increase in the expression of other drivers of IL-22, including IL1B
and IL6 (Fig. [126]2d, Supplementary Fig. [127]5). One possible
explanation for these observations is that IL-23 blockade with
ustekinumab is likely to be ineffective in patients with augmented
expression of alternative drivers of IL-22 production, such as IL-1β,
and are consistent with the possibility of IL-1β being an important
driver of ustekinumab resistance, by triggering activation of IL-22
regulated pathways in an IL-23 independent manner.
IL-22 regulates expression of neutrophil-active chemokines and other
pro-inflammatory transcriptional modules in colonic epithelial cells
Our data imply that IL-22 is potentially involved in mediating a
harmful transcriptional program in colonic epithelial cells, and that
patients with the greatest magnitude of expression of IL-22 responsive
transcripts are likely to be resistant to ustekinumab therapy. To
further understand potential pathogenic mechanisms mediated by IL-22,
we conducted a more in-depth analysis of IL-22-induced transcriptional
changes in colonic organoids at both transcript and pathway level,
including a comparison of IL-22 mediated epithelial regulation with
changes induced by other cytokines elevated in UC mucosa, including
IFNγ, IL-17A, IL-13 and TNF. Canonical Pathway analysis of the IL-22
regulated transcriptional program in colonic epithelial cells confirmed
activation of IL-22 signaling and activation of other TREM1 signaling,
acute phase response, inflammasome activation, toll-like receptor
signaling, Th17 pathway activation and notch signaling (Fig. [128]3a
and Supplementary Fig. [129]6).
Fig. 3. Causal network analysis identifies induction of neutrophil-active
chemokines as a key biological activity of IL-22 in the colonic epithelium.
[130]Fig. 3
[131]Open in a new tab
a Top 10 pathways enriched in transcriptional changes regulated by
IL-22 in human colonoids (n = 4) as identified by IPA, b Circos plot
showcasing the shared differentially expressed transcripts regulated by
the different cytokines in human colonic organoids (purple lines
connecting same genes across DEG lists), c Venn diagrams of shared
canonical pathways identified in IPA between IL-22 and other
pro-inflammatory cytokines, d clique of neutrophil attracting
chemokines regulated by IL-22 identified by protein–protein interaction
(PPI) network analysis (STRING), colors depict fold changes [log[e]]. e
Rulation of transcripts coding for chemokines by IL-22 and other
cytokines in human colonoids, f cumulative effect of IL-22 and IL-17A
co-treatment in the expression of neutrophil-attracting chemokines
(FDR: false discovery rate). Source data for a, e and f are provided as
a Source Data file.
Of the 1251 transcripts regulated by IL-22, 322 (26%) were uniquely
regulated by IL-22, whereas 1573 (74%) were additionally regulated by
other cytokines. At transcript level, the greatest degree of overlap
was observed between IL-22 and IFNγ (Fig. [132]3b, Supplementary
Table [133]2). There was also substantial overlap between
IL-22-regulated transcripts and transcripts regulated by TNF and
IL-17A. Conversely, there was relatively low co-regulation of
transcripts induced by IL-22 and IL-13. A similar pattern was observed
at biological pathway level. The majority of canonical pathways
regulated by IL-22 were also regulated by IFNγ and TNF, whereas there
was little overlap with IL-13-regulated biological pathways
(Fig. [134]3c).
We also examined whether enrichment scores for these other
pro-inflammatory cytokines were associated with response to
ustekinumab. In the case of IL-13 and IL-17A, there was no association
between their enrichment scores and clinical response, mucosal healing
or deep remission (Supplementary Fig. [135]7). Interestingly,
enrichment scores for IFNγ, the cytokine which most strongly overlapped
with IL-22, was also associated with response to ustekinumab, albeit
slightly less than IL-22. TNF enrichment scores were only weakly
associated with some, but not all outcomes in response to ustekinumab.
Biological pathway and causal network analysis identified prominent
activation of cell trafficking pathways in IL-22 treated organoids,
most notably around neutrophil recruitment (Supplementary Fig. [136]8).
Analysis of the top molecular and cellular functions of the gene
expression changes induced by IL22 confirmed a highly significant
association with cell movement, which was the most significantly
associated annotation. Based on DEGs induced by IL-22, a
protein–protein interaction (PPI) network analysis identified cliques
of networks regulated by IL-22 in colonoids, and among the most highly
complex is a clique of neutrophil attracting chemokines, including
CXCL1, CXCL2, CXCL3, CXCL5, CXCL6 and CXCL8 (Fig. [137]3d) (Analysis in
Cytoscape utilizing the STRING database to generate a PPI network of
the IL-22 regulated DEG, complexity assessed by the M-Code algorithm.
Colors depict fold changes [log[e]]).
To further probe how IL-22, and other pro-inflammatory cytokines might
impact epithelial regulation of immune cell trafficking we investigated
the profile and pattern of chemokine expression in colonoids treated
with these different immune cues. Each cytokine studied regulated a
unique pattern of chemokine expression (Fig. [138]3e). IL-22
preferentially upregulated the neutrophil-active CXC-family chemokines
CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6 and CXCL8, a function shared
with IL-17A. IFNγ strongly upregulated CXCL9 and CXCL10, whereas IL-13
was only cytokine to strongly upregulate both eosinophil-active
chemokines CCL24 and CCL26. In view of the shared regulation of
neutrophil-active chemokines by IL-22 and IL-17A, we further
investigated how these 2 cytokines might interact by evaluating gene
expression changes occurring in colonic organoids treated with a
combination of IL-17A and IL-22. Together IL-17A and IL-22 created
synergistic effects for induction of CXC-family neutrophil-active
chemokines (Fig. [139]3f).
The IL-22 regulated transcriptome correlates with colonic neutrophil
accumulation which is associated with resistance to ustekinumab therapy in
ulcerative colitis
In keeping with the hypothesis that epithelial-derived IL-22 regulated
neutrophil-active chemokines were functionally important in UC
pathogenesis, we observed significant upregulation of CXC-family
chemokines in the colon of UC patients in comparison with healthy
control subjects in 2 independent, large datasets (UNIFI and
[140]GSE50971, Fig. [141]4a, b). Moreover, there was a significant
positive correlation observed between the IL-22 enrichment score and
the enrichment of CXC-family neutrophil-active chemokines
(Fig. [142]4c).
Fig. 4. The IL-22 regulated transcriptome correlates with colonic neutrophil
accumulation which is associated with resistance to ustekinumab therapy in
ulcerative colitis.
[143]Fig. 4
[144]Open in a new tab
a Relative expression of neutrophil attracting chemokines in sigmoid
biopsies of UC patients participating to the UNIFI study (n = 550) and
non-IBD controls (n = 18) (Mann–Whitney test, two-tailed, no multiple
testing correction applied), b relative expression of the neutrophil
attracting chemokines in the colonic mucosa of healthy controls (HC),
UC patients with inactive and active disease ([145]GSE50971), c
non-parametric correlation (Spearman two-tailed) between the enrichment
score for the IL-22 transcriptional program and the chemokine gene set
(CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8), r = 0.54, 95% CI (0.48,
0.60), d IL-22 enrichment scores stratified by the neutrophil subscore
of the Geboes histology score in the epithelium and lamina propria in
colonic biopsies sampled from the UNITI cohort prior to starting
ustekinumab. A higher subscore reflects an increase in neutrophil
infiltration (for lamina propria 0: no increase, 1: mild but
unequivocal increase, 2: moderate increase, 3: marked increase; for
epithelium 0: none, 1: <5% crypts involved, 2: <50% crypts involved, 3:
>50% involved) (Kruskal–Wallis test with Dunn’s multiple comparisons
test, ****p < 0.0001, ***p < 0.001, line denotes median, n = 550) e
bioinformatically computed proportion of neutrophils (Cibersort) in
mucosal biopsies of UC patients participating in the UNIFI trial
correlates positively (Spearman correlation, two-tailed) with the IL-22
enrichment score (single sample gene set enrichment analysis-ssGSEA), f
bioinformatically computed proportion of neutrophils (Cibersort) in
mucosal biopsies of UC patients participating in the UNIFI trial
stratified by response (mucosal healing, week 8) to ustekinumab and
healthy controls (HC, n = 18). UC: UC patients n = 358, R responders,
NR non-responders, *p < 0.0001 for NR vs. R and for UC vs. HC,
Mann–Whitney test, two-tailed, box & whiskers representing
interquartile range and overall range, median value depicted with
horizontal line, g non-parametric correlation (Spearman, two-tailed)
between the enrichment score for the IL-22 transcriptional program and
the enrichment score for the gene set defining UC1 and UC2 populations
by Czarnewski et al. (r = 0.79), 95% CI (0.77,0.83), (FDR false
discovery rate). Source data for a, c, d–g are provided as a Source
Data file.
We further investigated this possibility by exploring the relationship
between the IL-22 enrichment score and neutrophil recruitment in the
colonic mucosa in UC patients in the UNIFI cohort. The histological
severity of UC can be scored using haematoxylin and eosin-stained
colonic sections using the Geboes score, and severity grading includes
an assessment of neutrophil infiltration in both the lamina propria
(LP) and epithelium^[146]26. Strikingly, the magnitude of neutrophil
infiltration in the epithelium but not the lamina propria correlated
with the magnitude of the IL-22 ES (Fig. [147]4d).
It is possible to estimate the proportion of different immune cell
types in tissue sample based on gene expression profiles and cellular
deconvolution tools, such as Cibersort^[148]27. The estimated
proportion of neutrophils in colonic tissue in colonic tissue of UC
patients from the UNIFI cohort strongly correlated with IL-22
enrichment scores (Fig. [149]4e). Moreover, the proportion of
neutrophils in colonic tissue also differentiated responders and
non-responders to ustekinumab, consistent with the possibility that
IL-22-driven colonic neutrophil recruitment has an important function
in treatment resistance (Fig. [150]4f).
A gene set of 57 homolog genes, conserved between mouse and human
colitis, has been previously been shown to stratify patients with UC in
two groups (UC1, UC2) based on response to biological therapy^[151]28.
In that study, the most highly enriched pathways in patients with
refractory disease included neutrophil-related pathways and neutrophil
degranulation. We found that 19/57 (33%) of these genes were regulated
by IL-22 (21.95 fold enrichment, P = 1.26e^−27, hypergeometric test),
and there was a positive correlation in the enrichment of those 57
genes with the IL-22 ES in the UNIFI cohort (Fig. [152]4g), providing
further supportive evidence of the association between the IL22
transcriptional program, neutrophil chemoattraction and refractory
disease.
IL-22 mediated remodeling of the colonic epithelial transcriptome is
conserved across species at gene and pathway level
Next, we investigated whether IL-22-mediated regulation of
neutrophil-active chemokine expression was functionally important in
colitis. The first step was to evaluate whether IL-22-mediated
regulation of human colonic epithelial function was conserved across
species. A comparison of differentially expressed genes and biological
pathways induced by IL-22 in human and mouse colonoids, demonstrated
significant correlation at both transcript (Spearman, two-tailed,
[MATH:
r2=0.65 :MATH]
, P < 0.0001) and pathway (Spearman, two-tailed,
[MATH:
r2=0.79 :MATH]
and P < 0.0001) level (Fig. [153]5a, b). In mouse colonic organoids,
IL-22 selectively induced expression of the neutrophil-active
chemokines Cxcl1, Cxcl3 and Cxcl5, with minimal impact on the
expression of other CXC-family chemokines (Supplementary Fig. [154]9A).
In the CC family of chemokines, we observed induction of Ccl7 and
weaker induction of Ccl2 by IL-22, with little or no impact on other
chemokine genes (Supplementary Fig. [155]9B). We validated these
findings using real time PCR, which confirmed time and dose-dependent
induction of Cxcl5 (Supplementary Fig. [156]9C). As observed in human
colonoids, IL-22 induced expression of Cxcl1 and Cxcl5, and was
synergistically augmented by IL-17A (Fig. [157]5c). We confirmed these
observations at protein level by measuring chemokine production in
supernatants of mouse colonic organoids cultured with recombinant mouse
cytokines (Fig. [158]5d).
Fig. 5. IL-22 mediated remodeling of the colonic epithelial transcriptome is
conserved across species at gene and pathway level.
[159]Fig. 5
[160]Open in a new tab
a Non-parametric (Spearman, two-tailed) correlation of transcripts
regulated by IL-22 in human and mouse colonoids (FC fold change)
[r = 0.65, (0.60, 0.71), p < 0.0001], b non-parametric (Spearman, two
tailed) correlation of pathways regulated by IL-22 in human and mouse
colonoids (z-scores derived from Ingenuity Pathway Analysis- IPA)
[r = 0.79, (0.64, 0.88), p < 0.0001], c effects of IL-22, IL-17A and
their combination to the regulation of the neutrophil attracting
chemokines Cxcl1 and Cxcl5 expression in mouse colonoids, (n = 12,
Kruskal–Wallis with Dunn’s multiple comparisons test, **p < 0.01,
****p < 0.0001, median denoted with black line), d effects of IL-22,
IL-17A and their combination to the production of the neutrophil
attracting chemokines Cxcl1 and Cxcl5 in mouse colonoids (n = 6, paired
Wilcoxon, two tailed test, all comparisons against control, *p = 0.31,
median denoted with black line), e enrichment of the IL-22
transcriptional program (top 50 upregulated genes by IL-22 in mouse
colonoids) in preclinical models of colitis (single sample GSEA),
including the T-cell transfer model (induced by adoptive transfer of
naive CD4^+ T cells to Rag2^−/− mice), the spontaneous,
microbiota-dependent models of colitis developing in Il10^−/−, and the
Tbx21^−/− Rag2^−/− Ulcerative Colitis (TRUC) mice, colitis occurring
following administration of DSS administration in drinking water, or
rectal administration of DNBS, and innate-immune-mediated colitis
occurring in Rag1^−/− mice following administration of agonistic
anti-CD40 antibodies (n = 3 biological replicates per group,
Mann–Whitney test, one-tailed, for all comparisons *p = 0.05, besides
TRUC vs Rag2^−/− *p = 0.03, line representing median), f relative
expression of neutrophil attracting chemokines in colonic tissue of
different mouse models of colitis. (FDR: false discovery rate). Source
data for a–d are provided as a Source Data file.
We investigated whether IL-22 responsive transcripts were enriched in
the colon in mouse models of colitis. GSVA demonstrated significant
enrichment of the mouse IL-22 responsive transcriptional module across
6 different colitis models (Fig. [161]5e). Similar to our observations
in human UC, there was significant upregulation of the
neutrophil-active chemokines Cxcl1, Cxcl2, Cxcl3 and Cxcl5 across all
models of colitis tested, indicating that this core chemokine module is
conserved in colitis development across species (Fig. [162]5f).
IL-22 is a functionally important regulator of neutrophil recruitment in
chronic colitis
Next, we tested the functional significance of IL-22-induced regulation
of neutrophil-active chemokines in vivo. Tbx21^−/− Rag2^−/− Ulcerative
Colitis (TRUC) mice develop chronic, microbiota-dependent colitis with
important parallels with human UC. TRUC mice develop chronic, distal
colitis which is dependent on IL-23 and TNF^[163]11,[164]29.
Neutrophil-active chemokines were among the most elevated transcripts
in the colon of TRUC mice (Cxcl5 was 2^nd and Cxcl1 the 11^th most
highly expressed transcripts across the entire genome, Fig. [165]6a).
To test the functional activity of IL-22 in regulating
neutrophil-active chemokines we neutralized, genetically disrupted, or
administered recombinant IL22 to TRUC mice. In keeping with IL-22 being
an important regulator of neutrophil chemotaxis in the colon,
administration of neutralizing anti-IL-22 monoclonal antibody (mAb), or
genetic deletion of IL-22 resulted in significant loss of Cxcl1 and
Cxcl5 expression and a significant reduction in the numbers of
neutrophils accumulating in the colon (Fig. [166]6b–d). Moreover,
administration of recombinant (r)IL-22 reinstated Cxcl1 and Cxcl5
expression and restored excessive neutrophil recruitment in the colon
of TRUC Il22^−/− mice (Fig. [167]6b, d). The functional impact of this
axis was also examined by assessing disease activity. IL-22
neutralization or germline deletion of Il22 was associated with a
significant reduction in disease features, including reduced colitis
scores and reduced colon mass, whereas administration of rIL-22
restored colitis in otherwise disease free TRUC Il22^−/− mice
(Fig. [168]6e and Supplementary Fig. [169]10).
Fig. 6. IL-22 is a functionally important regulator of neutrophil recruitment
in chronic colitis.
[170]Fig. 6
[171]Open in a new tab
a Volcano plot demonstrating fold change and P value of differentially
expressed genes in TRUC mice vs controls (Rag2^−/−)(n = 6, biological
replicates), b effects of IL-22 neutralization to the expression of
neutrophil attracting chemokines Cxcl1 and Cxcl5 (n = 36 biological
replicates, Kruskal–Wallis with Dunn’s multiple comparison test for
TRUC + anti-IL22 and TRUC IL22^−/− vs TRUC, *p < 0.05, ****p < 0.0001
and Mann–Whitney two tailed test for TRUC IL22^−/− + IL-22 vs. TRUC
IL22^−/−, ***p < 0.001, line denotes median), c flow cytometry plot,
showing the relative frequency of neutrophils in colonic tissue of TRUC
mice treated with a monoclonal antibody blocking IL-22, d absolute
number of neutrophils in mouse colonic tissue of TRUC mice and TRUC
mice with IL-22 neutralization ((n = 36, biological replicates,
Kruskal–Wallis with Dunn’s multiple comparison test for
TRUC + anti-IL22 and TRUC IL22^−/− vs TRUC, *p < 0.05, ****p < 0.0001
and Mann–Whitney two tailed test for TRUC IL22^−/− + IL-22 vs. TRUC
IL22^−/−, ***p < 0.001, lines denote median)), e effects of IL-22
neutralization on the severity of TRUC colitis (n = 36, biological
replicates, Kruskal–Wallis with Dunn’s multiple comparison test for
TRUC + anti-IL22 and TRUC IL22^−/− vs TRUC, ****p < 0.0001 and
Mann–Whitney two tailed test for TRUC IL22^−/− + IL-22 vs. TRUC
IL22^−/−, ***p < 0.001, lines denote median), f schematic
representation of the co-culturing experiment using ILC3 isolated from
Il22^−/− and control mice and cultured with colonoids, g relative
expression of Cxcl1 and Cxcl5 in colonoids co-cultured with ILC3
derived from Il22^+/+ and Il22^−/− mice (n = 3, biological replicates
per group, Mann–Whitney, one-tailed test, line denotes median). h Colon
mass and i colitis score in TRUC mice treated with anti-CXCR2 (n = 4)
or control antibody (n = 14, biological replicates, Mann–Whitney,
two-tailed test). Neutralizing anti-IL-22 mAb (clone IL22-01, 200 μg
per mouse) were administered intraperitoneally (ip) every 3–4 days.
Recombinant IL-22 (rIL-22, 100 μg per mouse) were administered ip at
days 0, 4, 8 and 12. Source data for a, b, d, e, g–i are provided as a
Source Data file.
Group 3 innate lymphoid cells are the dominant producers of IL-17A and
IL-22 in TRUC disease^[172]11. Therefore, we purified group 3 ILCs from
the colon of TRUC and TRUC Il22^−/− mice and co-cultured them with
mouse colonic organoids (Fig. [173]6f). Unlike IL-22 sufficient ILC3,
which induced Cxcl1 and Cxcl5 expression in colonic organoids,
induction of these chemokine transcripts was significantly diminished
in colonic organoids co-cultured with IL-22 deficient ILC3
(Fig. [174]6g). This is based on the assumption that organoids were the
chief source of chemokines in this experimental system. Although we did
not formally test whether ILCs were a potential source of chemokines,
previously published work has failed to detect the expression of these
chemokines in colonic ILC3s^[175]30.
The functional importance of neutrophil recruitment was further probed
by blocking CXCR2, the common receptor expressed by neutrophils for
CXC-family neutrophil-active chemokines, including CXCL1 and CXCL5. In
vivo administration of anti-CXCR2 mAbs to TRUC mice significantly
attenuated TRUC disease (Fig. [176]6h, i). Taken together, these data
support the notion that IL-22-mediated induction of neutrophil-active
chemokines, including CXCL1 and CXCL5 is functionally important in the
recruitment of CXCR2^+ neutrophils, and that this pathway has an
important pathogenic function in colitis.
IL-22-mediated induction of neutrophil-active chemokines in colonic
epithelial cells is dependent on STAT3 signaling
Next, we sought to define the signaling requirements of IL-22-mediated
induction of neutrophil-active chemokine expression. In the intestinal
epithelium ligation of IL-22 with its specific receptor triggers
activation of different signaling pathways, including STAT3 and MAP
kinases, such as MAP3K8^[177]31–[178]34. Immunostaining confirmed
immunoreactivity for IL22RA1 only in the colonic epithelium of healthy
controls and patients with UC (Supplementary Fig. [179]11). Consistent
with STAT3 and MAP3K8 having an important function in epithelial
signaling in UC, we also observed increased immunostaining for pSTAT3
and MAP3K8 in the epithelial compartment in patients with active UC in
comparison to healthy controls (6% ± SEM0.2% vs. 19% ± SEM7% and
52% ± SEM19% vs. 91% ± SEM6% cells, respectively)(Fig. [180]7a and Fig
Supplementary Fig. [181]11).
Fig. 7. IL-22 mediated induction of neutrophil-active chemokines in colonic
epithelial cells is abrogated by JAK inhibition.
[182]Fig. 7
[183]Open in a new tab
a Representative images of immunostaining for IL22RA1, pSTAT3 and
MAP3K8 in the colon of a healthy control, UC patient without active
inflammation and a UC patients with active inflammation, b, c relative
expression of Cxcl1 and Cxcl5 in IL-22 treated colonoids generated from
Villin-Cre x Stat3^fl/fl mice (Stat3^ΔIEL), Map3k8^−/^− mice and
controls, (n = 12 biological replicates, Mann–Whitney test, two-tailed
*p < 0.05, **p < 0.01). d Effects of STAT3 genetic disruption in key
gene transcripts regulated by IL-22 ([184]GSE15955) e effects of a JAK
inhibitor (tofacitinib) in CXCL1 and CXCL5 expression in human colonic
organoids (n = 3, biological replicates, ANOVA with Tukey’s multiple
comparison test, *p < 0.05, ***p < 0.0001). Source data for b–d and are
provided as a Source Data file.
To examine the requirements of STAT3 and MAP3K8 signaling pathways for
the IL-22 regulated induction of CXC-family chemokines in colonic
organoids, we generated colonoids from mice with epithelial-specific
deletion of Stat3 (Villin-Cre x Stat3^fl/fl mice – subsequently termed
Stat3^ΔIEL), and from mice with germline deletion in MAP3K8. Unlike
colonic organoids from control mice (Stat3^fl/fl), in which IL22
induction of Cxcl5 was maintained, there was no induction of Cxcl5 in
Stat3^ΔIEL organoids (Fig. [185]7b). In contrast, IL-22 induction of
Cxcl5 was maintained in Map3k8^−/− colonoids (Fig. [186]7c).
To further investigate the dependence of colonic epithelial STAT3
activation for CXC-family chemokine induction in the context of
colitis, we analyzed genome-wide changes in the epithelial compartment
in DSS colitis, taking advantage of microarray data available from a
previously published study^[187]31. In this study, gene expression
profiling was performed on purified colonic epithelial cells from
Stat3^ΔIEL and control and mice following induction of colitis. STAT3
responsive genes were defined as transcripts upregulated in epithelial
cells from control mice that failed to upregulate in the colonic
epithelium of mice with epithelial-specific genetic disruption of
STAT3. In Stat3^ΔIEL mice there was no expression of several canonical
IL-22-regulated genes, such as Reg3b, Reg3g, Fut2 and Socs3, consistent
with STAT3 being required for the induction of these transcripts in the
epithelium. Moreover, in agreement with our in vitro observations,
there was also lower expression of Cxcl1 and Cxcl5 colonic epithelium
from Stat3^ΔIEL mice (Fig. [188]7d). These observations have important
clinical implications, as small inhibitors of JAK/STAT signaling are
now emerging into clinical practice. Tofacitinib, a selective JAK1/JAK3
inhibitor, which prevents phosphorylation and activation of STAT3, has
recently been approved for UC^[189]35. Importantly, tofacitinib
significantly inhibited IL-22 induced expression of CXCL1 and CXCL5 in
human colonoids, indicating that the IL-22/CXC chemokine axis can be
therapeutically targeted by JAK/STAT inhibition (Fig. [190]7e).
Discussion
This study provides important insights into the immune regulation of
the intestinal epithelial barrier with clinically meaningful
implications for precision medicine. By mapping IL-22-responsive
transcriptional networks in colonic epithelial organoids, evaluating
the expression patterns of these genes in colonic tissue, and probing
preclinical models of disease, we identify IL-22-mediated regulation of
CXCR2^+ neutrophils as a functionally and prognostically important
pathogenic pathway in UC.
The distribution pattern and magnitude of enrichment of IL-22
responsive transcriptional footprints, sampled prior to treatment
initiation, could differentiate patients according to their probability
of responding to ustekinumab. Although the purpose of this study was
not to identify new biomarkers, our data pave the way for exploiting
our experimental approach to develop new molecular profiling tools,
which could stratify patients as potential responders or
non-responders. Precision medicine approaches are much needed in UC to
improve patient outcomes, particularly as there are now multiple
therapeutic options available^[191]36,[192]37. Unfortunately, fewer
than 50% of patients achieve long-term durable remission with any of
the treatment options available, including anti-TNF, anti-integrin,
anti-IL12p40 or JAK inhibitors^[193]4,[194]35,[195]38–[196]40. In this
study, UC patients with enrichment scores <0 were more than four times
as likely to achieve remission following treatment with ustekinumab in
comparison with patients with enrichment scores >0, or placebo-treated
patients, and more than twice as likely to achieve remission in
comparison with unstratified patients. The notion of harnessing
transcriptional signatures to guide treatment strategies is highly
attractive^[197]36 and is already exploited in other areas of medicine.
To realize the potential of these exciting observations future work
will focus on developing robust analytical platforms that may be more
appropriate for routine clinical care, including PCR-based
technologies.
Analysis of transcriptional modules linked to low and high enrichment
of IL-22 responsive transcriptional programs, which were
associated with non-response to ustekinumab provided insights into
potential mechanisms of ustekinumab resistance. IL-1β was strongly
predicted as a potential upstream regulator of the transcriptional
changes observed in patients with high IL-22 enrichment scores.
Moreover, despite minor differences in the expression of IL-23
subunits, there was a marked increase in the expression of IL1B, and to
a lesser extent IL6 and TL1A in patients with high IL-22 enrichment
scores. Since IL-1β, IL-6 and TL1A are all capable of inducing IL-22
production, it is possible that pathway redundancy and
IL-23-independent induction of IL-22 might contribute to ustekinumab
resistance in ulcerative colitis.
Causal network analyses of transcripts modulated by IL-22 pointed to a
number of potentially important molecular mechanisms of ustekinumab
resistance. The most strongly implicated biological pathway in patients
with high IL-22 enrichment scores were related to immune cell
trafficking and most notably neutrophil recruitment. Others included
activation of innate-immune pathways, such as TREM1, which has been
linked to resistance to anti-TNF therapy^[198]41. There was also
augmented mucosal expression of metalloproteinases in patients with
high IL-22 enrichment scores and ustekinumab resistance, a process
which has also been linked to resistance to biological therapy,
by direct digestion of the therapeutic monoclonal antibody structure
through protease activity^[199]42.
Our data address outstanding questions about the function of IL-22 in
UC and provide important evidence contrary to current perceptions,
indicating that transcriptional programs regulated by IL-22 in UC are
likely to be pathogenic. The IL-22-regulated transcriptome was highly
conserved between man and mouse and was highly enriched in the colon of
both human and mouse colitis. IL-22 mediated regulation of epithelial
function was closely shared with other pro-inflammatory cytokines, such
as IFNγ and TNF. Immune cell recruitment to sites of inflammation is
regulated by local production of chemokines by the inflamed tissue, and
by selective expression of chemokine receptors by immune cells. Our
data identify the colonic epithelium as a central communication hub in
the regulation of immune cell recruitment to the gut. Different
cytokines instructed induction of different chemokine modules by the
epithelium, reflecting important qualitative differences in the types
of immune responses that are supported by different effector cells.
IL-22 was an especially potent inducer of CXC-family neutrophil-active
chemokines in colonic organoids, and notably, these chemokines were
highly enriched in diseased mucosa of patients with active UC.
Importantly, the transcriptional footprint of IL-22 strongly correlated
with colonic neutrophil recruitment in the epithelial layer. Moreover,
patients failing to respond to ustekinumab therapy had significantly
more neutrophils infiltrating the mucosa in comparison with responders,
consistent with excessive neutrophil recruitment being an important
driver of poor outcomes and treatment resistance. A recent study
looking at overlapping transcriptional features between mice and humans
identified a conserved transcriptomic signature that strongly
associated with poor outcomes and treatment refractoriness in UC also
identified neutrophil-related biological pathways as markers of
resistance^[200]28. There was significant overlap in the
transcriptional signature identified by this group, and transcripts
that we identified as being IL-22-responsive. As well as corroborating
the potential importance of excessive neutrophil
accumulation/activation as markers of poor outcomes and treatment
resistance in UC, our data provide potential mechanistic explanations
for these observations, identifying IL-22 as a potentially important
regulator of the “treatment resistance” transcriptional program
identified by the Czarnewski study.
Our data also support the notion that IL-22-mediated induction of
CXC-family chemokines is functionally important in the recruitment of
CXCR2^+ neutrophils to the colon in chronic colitis. Blockade, genetic
deletion of IL-22, or blockade of CXCR2, the common ligand for the CXC
family of neutrophil-active chemokines, significantly attenuated
disease in the TRUC model of chronic colitis.
We also defined the signaling requirements for this functionally
important colitogenic pathway. Although IL-22 induction of chemokines
was maintained in the absence of Map3k8, induction was abolished in the
absence of Stat3. With the advent of JAK inhibitors, including agents
selectively targeting JAK1, which interacts with STAT3 downstream of
the IL22 receptor, it is tempting to speculate that a key therapeutic
target of this intervention will be alleviation of IL22-induced
CXC-family chemokine induction.
In summary, our study provides further insights into the biology of
IL-22 in human disease and highlights how transcriptional networks
regulated by IL-22 are functionally and clinically important in UC,
impacting patient trajectories and responsiveness to biological
intervention. Refinement of this approach could herald a new paradigm
for tailoring therapies in UC.
Methods
The research work described in this paper complies with all ethical
regulations relevant to human and animal research. Ethical approval for
human samples used for colonoids were provided by King’s College
London, Guy’s and St Thomas’ NHS Foundation Trust and King’s College
Hospital. The national research ethics committee for England reviewed
and approved the study protocol (IRAS id:190309). All patients provided
samples after informed consent. No compensation was provided. Ethical
approval for the immunohistochemistry work on paraffin-embedded tissue
of patients was provided by the Newcastle Academic Health Partners
Bioresource (Newcastle and North Tyneside 1 REC:12/NE/0395 &
10/H0906/41).
All mice were handled according to local (King’s College London) and
national guidelines, and all our experimental protocols were reviewed
and approved by our local ethics review committee. All animal
experiments were conducted in accredited facilities (King’s College
London, Biological services Unit) in accordance with the UK Animals
(Scientific Procedures) Act 1986 (Home Office license number PPL
70/7869).
A list of reagents is provided as supplementary information
(Supplementary Table [201]3).
Colonoids
Human colonic crypts were isolated from serial colonic biopsies (x2
ascending colon, x2 transverse, x2 descending, x2 rectosigmoid) taken
from six adult individuals (median age: 48, range [33,67], female:3),
without past medical history or regular medication who attended for
routine colonoscopy in view of abdominal symptoms without a diagnosis
of IBD and did not have macroscopic or microscopic evidence of
inflammation. All patients provided informed consent (NRES/IRAS id:
15/LO/1998). Subsequent establishment of human colonoids was performed
as described by Sato et al.^[202]43. The crypts were cultured in growth
medium containing advanced Dulbecco’s modified Eagle’s medium/F12,
penicillin/streptomycin (100 U/ml), 10 mM HEPES, 2 mM Glutamax,
supplements N2 (1×) and B27 (1×), 50 ng/ml mouse epidermal growth
factor (all from Life Technologies), 1 mM N-acetylcysteine
(Sigma-Aldrich), 50% v/v Wnt3a conditioned medium, 10% v/v R-spondin-1
conditioned medium, 100 ng/ml mouse recombinant noggin protein
(Peprotech),10 nM gastrin (Sigma-Aldrich), 500 nM A83-01 (Bio-techne),
10 μM SB202190 (Sigma-Aldrich) and 10 mM Nicotinamide (Sigma-Aldrich).
10 μM Y-27632 (Sigma-Aldrich) was added to the culture medium for the
initial 3 days. Medium was changed every 2 days. Differentiation
towards a mature epithelium in human colonoids was achieved with
reduction of Wnt3a to 15% v/v and withdraw of SB202190 and nicotinamide
for 5–7 days. During the last 24 h in differentiation medium human
colonoids were treated with human recombinant IL-22 (10 ng/ml), IL-17A
(50 ng/ml), TNF (10 ng/ml), IFNγ (20 ng/ml), IL-13 (10 ng/ml) and IL-22
(10 ng/ml)/ IL-17A(50 ng/ml) combination. For the experiment presented
in Fig. [203]6 (tofacitinib treated colonoids), tofacitinib was added
with IL-22 at the last 24 h of differentiation at a dose of 0.1 μM.
Mouse colonoids were cultured in the same medium as above but without
gastrin SB202190, Nicotinamide, A83-01 and with the addition of 3 µM
CHIR99021 (Cambridge Biosciences). To differentiate them, Wnt3a was
withdrawn for 3 days. During the last 24 h in differentiation medium
mouse colonoids were treated with mouse recombinant IL-22 (10 ng/ml)
and IL-17A (50 ng/ml).
Next-generation sequencing and analysis
RNA extraction
Colonoids were lysed and RNA was extracted using the RNAeasy kit
(Qiagen). This step was optimized balancing the effectiveness of
elimination of DNA quantified (Qubit dsDNA HS assay kit) versus the
loss of quantity of RNA (Qubit RNA BR assay kit). Optimal DNAse I
concentration was determined to be x5 the standard concentration. Five
hundred nanograms of cDNA was then created using Revertaid cDNA
synthesis kit (ThermoFisher) and diluted to a concentration of
6.25 ng/μl. Harvested colonoids were put in Qiazol and then RNA was
extracted with the RNAeasy kit (Qiagen) as per the manufacturer’s
guidelines. cDNA was created using the Revertaid cDNA synthesis kit
(ThermoFisher). Bioanalyzer analysis revealed excellent quality for RNA
extracted from both colonoids and whole biopsies (RIN score>9).
Library preparation and sequencing
A total amount of 3 μg RNA per sample was used as input material for
the RNA sample preparations. Sequencing libraries were generated using
NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA) following
manufacturer’s recommendations and index codes were added to attribute
sequences to each sample. Briefly, mRNA was purified from total RNA
using poly-T oligo-attached magnetic beads. Fragmentation was carried
out using divalent cations under elevated temperature in NEBNext First
Strand Synthesis Reaction Buffer(5X). First-strand cDNA was synthesized
using random hexamer primer and M-MuLV Reverse Transcriptase (RNase
H-). Second strand cDNA synthesis was subsequently performed using DNA
Polymerase I and RNase H. Remaining overhangs were converted into blunt
ends via exonuclease/polymerase activities. After adenylation of 3′
ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were
ligated to prepare for hybridization. In order to select cDNA fragments
of preferentially 150–200 bp in length, the library fragments were
purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then
3 μl USER Enzyme (NEB, USA) was used with size-selected,
adapter-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C
before PCR. Then PCR was performed with Phusion High-Fidelity DNA
polymerase, Universal PCR primers and Index (X) Primer. At last, PCR
products were purified (AMPure XP system) and library quality was
assessed on the Agilent Bioanalyzer 2100 system. The clustering of the
index-coded samples was performed on a cBot Cluster Generation System
using HiSeq PE Cluster Kit cBot-HS (Illumina) according to the
manufacturer’s instructions. After cluster generation, the paired-end
libraries were sequenced on an Illumina HiSeq platform.
Gene expression quantification and differential expression analysis
Fastq files were firstly processed with in-house Perl scripts to
discard reads with adapter contamination, or at least 10% of uncertain
bases (N), or at least 50% of nucleotides with a Phred quality score
less than 20. Read pairs were aligned to the human genome (GRCh37/hg19)
using TopHat v2.0.12^[204]44. HTSeq v0.6.1 was used to count the read
pairs mapped uniquely and concordantly to each gene^[205]45. The raw
count matrix was screened for genes with low expression levels across
all samples (i.e. average count less than 3), and then with an average
number of read pairs less than 3 were filtered out normalized following
the strategy suggested by Anders et al.^[206]45.
Differentially expressed genes (DEG) were identified through a varying
intercepts hierarchical modeling approach^[207]46–[208]48 implemented
in R^[209]48 and Stan^[210]49. Gene counts were modeled as a negative
binomial variable dependent on cytokine treatment as well as covariates
accounting for repeated measurements from the same donor and additional
sample similarities detected by PCA and hierarchical clustering:
[MATH:
μi=β0+βt[i]+βp[i]+βc[i]<
/msub> :MATH]
1
[MATH:
yi~NegativeBinomial(exp(μi),<
/mo>φ)
:MATH]
2
[MATH:
β[t]~Normal(0,σ1) :MATH]
3
[MATH:
β[p]~Normal(0,σ2) :MATH]
4
[MATH:
β[c]~Normal(0,σ3) :MATH]
5
where:
t = number of groups on treatment (Eq. [211]3), p = number of subjects
(Eq. [212]4), c = number of clusters (Eq. [213]5), i = number of
observations (Eq. [214]1), y = gene expression count (Eq. [215]2),
φ = overdispersion parameter (Eq. [216]2), σ[1] = between treatment
standard deviation (Eq. [217]3), σ[2] = between-subject standard
deviation (Eq. [218]4), σ[3] = between cluster standard deviation
(Eq. [219]5).
The quality of the estimated statistical model was assessed through
posterior predictive simulations that compare replicated datasets to
the actual data. The output p values were corrected for multiple
testing with the Benjamini and Hochberg method^[220]50. Pathway
analysis of DEG lists was performed with Ingenuity Pathway Analysis
(IPA, Qiagen)^[221]16. Protein–protein interaction (PPI) analysis was
undertaken in Cytoscape^[222]51 utilizing the STRING database^[223]52
to generate a PPI network of the IL-22 regulated DEG and assess
complexity by the M-Code algorithm.
Gene set enrichment analysis
To test the activation of each of the cytokine-regulated
transcriptional programs we used gene set variation analysis
(GSVA)^[224]53 and single sample gene set enrichment analysis (ssGSEA)
to probe whole transcriptional profiles of previously reposited
datasets and the dataset generated in the context of the ustekinumab
and golimumab trials programs.
Gene set enrichment analysis (GSEA)^[225]54 was performed using the R
Bioconductor package clusterProfiler^[226]55 to test which known
pathways are significantly impacted following cytokine treatment. To
this end, the genes tested for differential expression between treated
and untreated samples were first ranked by decreasing log fold
expression changes, and then their enrichment was evaluated against the
hallmark gene sets and KEGG and REACTOME pathway annotations from the
MSigDB database^[227]56. Normalized enrichment scores and empirical P
values were estimated using default parameters, and multiple testing
correction was carried out using the Benjamini–Hochberg method.
UNIFI trial program
The UNIFI trial was a randomized placebo-controlled phase 3 clinical
trial evaluating the efficacy and safety of ustekinumab
([228]NCT02407236) and has already been reported^[229]4. In this study,
we report transcriptional data from biopsies, which were correlated to
clinical, endoscopic and biomarker data available from the UNIFI cohort
(Supplementary Table [230]1, Demographics). Colonic biopsies were
sampled at defined time points (15–20 cm from anal verge) in a subset
of patients and were immediately transferred to RNALater (Qiagen) and
stored at −80 °C prior to RNA extraction. Whole genome transcriptomics
were performed on the Affymetrix HG U133 PM array. Clinical data was
recorded prospectively according to the trial protocol. Outcomes
reported include: clinical remission (defined as a total Mayo score of
≤2 and no subscore >1) and deep remission [which required both
histologic improvement (defined as neutrophil infiltration in <5% of
crypts, no crypt destruction, and no erosions, ulcerations, or
granulation tissue) and endoscopic improvement] at week 8. The analysis
presented is based on all patients receiving ustekinumab regardless of
dose (130 mg and 6 mg/kg).
Reposited datasets
The following reposited datasets of transcriptomic profiling of
patients and healthy controls were accessed and analyzed for this
manuscript: [231]GSE59071 (n = 108)^[232]57, [233]GSE23597
(n = 45)^[234]58, [235]GSE16879 (n = 24)^[236]59, [237]GSE92415
(n = 59) and [238]GSE73661 (n = 60)^[239]60.
Human colonic biopsy immunohistochemistry
Immunohistochemistry was performed on formalin-fixed and
paraffin-embedded (FFPE) colonic biopsies obtained by colonoscopy from
adult patients with histologically active UC (n = 5), histologically
inactive UC (n = 5) and control subjects with no history of UC and no
histological inflammation (n = 5). Written informed consent was
obtained in accordance with research and ethics committee (REC)
approval (Newcastle Academic Health Partners Bioresource: Newcastle and
North Tyneside 1 REC:12/NE/0395 & 10/H0906/41). 4μm sections of FFPE
tissue were stained using a Discovery Ultra autostainer (Ventana
Medical Systems, Tucson, AZ) with optimized concentrations of
polyclonal anti-IL22RA (1:750 dilution, Novus Biologicals). Slides were
developed with 3,3′-Diaminobenzidine (DAB) and counterstained with
hematoxylin. Multispectral scanning of stained slides at 10x
magnification was undertaken using a Vectra 3.0 Automated Quantitative
Pathology Imaging System (PerkinElmer, Hopkinton, MA). InForm Cell
Analysis software (v2.0, PerkinElmer) allowed image deconvolution using
a spectral library trained on single-stained colonic biopsies for both
DAB and Hematoxylin counterstain. Intestinal epithelium and lamina
propria were identified by training a tissue segmentation algorithm,
which was then applied to all cases, and the accuracy of segmentation
was optimized by manual correction. Individual cells in both tissue
compartments were identified using a cell segmentation algorithm, based
upon the identification of nuclei (hematoxylin). The relative
expression of IL22RA1 (membranous/cytoplasmic) was scored in each case
and using a binary approach (positive/negative); visual cues were used
to distinguish positive staining compared to background, and thresholds
were assigned. These data were exported and compiled in MATLAB (v2016b
MathWorks, Natick, MA).
Experimental models of IBD
Mice
All mice were housed in specific pathogen-free (SPF) conditions and
handled according to local (KCL) and national guidelines. All
experimental protocols were reviewed and approved by our local ethics
review committee and experiments were conducted in accredited
facilities in accordance with the UK Animals (Scientific Procedures)
Act 1986 (Home Office license number PPL 70/7869). Balb/c Il22^−/− mice
were provided by Pfizer^[240]15. WT C57Bl/6 and Rag1^−/−mice (on
C57Bl/6 background) were purchased from Charles River Laboratories.
Il10^−/− mice were provided by Professor Werner Muller, Faculty of Life
Sciences, University of Manchester^[241]61.
Preclinical models of colitis
TRUC, TRUC Il22^−/−
Balb/c Tbx21^−/−Rag2^−/− double KO (TRUC) mice (n = 30) develop a
communicable, microbiota driven colitis^[242]15,[243]29.
Tbx21^−/−Rag2^−/−Il22^−/− (TRUC Il22^−/−) triple KO mice (n = 30) were
generated by backcrossing Balb/c Tbx21^−/−Rag2^−/− double KO (TRUC)
mice with Balb/c Il22^−/− mice that were provided by Pfizer.
aCD40/DNBS
Two hundred microliters of 3 mg DNBS (Sigma-Aldrich) resolved in 50%
EtOH were administered rectally while mice were under isoflurane
anesthesia. Mice (n = 3) were monitored daily for weight loss, general
signs of distress and adverse disease symptoms. Any mice presented with
these features were humanely culled on welfare grounds; otherwise mice
were culled 3 days post administration for further analysis.
DSS
Three per cent DSS (MW 3600–50,000, MP Biomedicals, LLC) was
administered orally in drinking water for 5 or 6 days to C57Bl6 mice
and animals were culled at day 7 or 8 respectively. Mice (n = 3) were
monitored daily and scored for weight loss, rectal bleeding and feces
consistency. Disease activity index was calculated as the sum of the
above scores divided by 3^[244]62. All animals were daily thoroughly
observed for general signs of distress or adverse symptoms and any mice
presented with these features were humanely culled on welfare grounds.
Il10^−/− mice
Il10^−/− mice (n = 3) were introduced to HT and TRUC microbiota by oral
gavage. Mice were monitored twice per week for weight loss, general
signs of distress and adverse symptoms. Any mice presented with these
features were humanely culled on welfare grounds; otherwise mice were
culled 4 weeks post gavage for further analysis.
TCT
0.5 or 2 × 10^6 naive CD4^+ T cells (defined as live
CD4^+CD25^−CD44^loCD62L^hi cells) were FACS sorted from spleens of
8-week-old C57Bl6 WT female or male donor mice, and injected (in 200 μl
of sterile PBS) intraperitoneally into 8–10 week old C57Bl6 Rag1^−/−
recipients. Purity checks were performed at the end of every sort and
cells were always found more than 97% pure. Recipient mice (n = 3) were
monitored twice per week for weight loss, general signs of distress and
adverse symptoms. Any mice presented with these features were humanely
culled on welfare grounds; otherwise, mice were culled 4–6 weeks post
adoptive transfer for further analysis.
In vivo treatments
Neutralizing anti-IL-22 mAb (clone IL22-01) and recombinant IL-22
(rIL-22) were developed and provided by Pfizer. Two hundred micrograms
of IL22-01 (per mouse) were administered ip. every 3 to 4 days. One
hundred micrograms of rIL-22 (per mouse) were administered ip. at days
0, 4, 8 and 12, while mice were culled at day 14. Anti-CXCR2 (clone
242216, R&D Systems) was administered ip. at a dose of 100 μg per mouse
at days 0, 3, 7, 10 and 14, while mice were culled at day 15.
Isolation of colonic LP leukocytes (cLPMCs)
Mice were euthanized by either cervical dislocation or by a rising
concentration of carbon dioxide gas, and then dissected in a laminar
flow cabinet under aseptic conditions. Colons were opened
longitudinally, cleaned thoroughly with ice-cold PBS and cut into
1–2 mm pieces and washed with 10 ml 5 mM EDTA, 1 mM Hepes in HBSS
(Gibco) in a shaking water bath (300 rpm) at 37 °C for 20 min. Tissue
was then vortexed vigorously for 10 sec and passed through a 100 µM
cell strainer and collected in C-tubes (Miltenyi) in complete RPMI
(Gibco) containing 10% fetal calf serum, 0.25 mg/ml Collagenase D
(Roche), 1.5 mg/ml Dispase II (Roche) and 0.01 μg/ml DNase (Roche) and
put in a shaking water bath (300 rpm) at 37 °C for 40 min. Before and
after the 40 min incubation C-tubes were vigorously shaken for 30 s.
Solutions were then passed through 100 µM cell strainers and washed
with ice-cold PBS. Cells were resuspended in 10 ml of the 40% fraction
of a 40:80 Percoll (GE Healthcare) gradient and carefully placed on top
of 5 ml of the 80% fraction in 15 ml tubes. Percoll gradient separation
was performed by 20 min centrifugation at 800 × g at room temperature
without break. LP cells were collected from the interphase of the
gradient and washed with ice-cold PBS. Cells were resuspended in 1 ml
PBS, counted and immediately used for further experiments.
Flow cytometry
Single cell suspensions were washed with ice-cold PBS and
centrifugation at 400 × g, 4 °C for 5 min prior to all staining. Cells
were then resuspended in 200 μl PBS containing Fc block (aCD32/CD16,
eBioscience) at 1:100 dilution and incubated on ice for 10 min.
Antibodies against all surface markers were added at appropriate
dilutions as well as LIVE/DEAD Fixable Dead Cell Stain (Invitrogen)
used in 1:1000. Samples were mixed by vortex and incubated for another
20 min on ice in the dark. After the incubation, cells were washed with
ice-cold PBS and centrifugation at 400 × g, 4 °C for 5 min and then
fixed with 400 μl of 4% PFA and incubated at RT for 15 min in the dark.
After fixation, cells were washed again with ice-cold PSB, resuspended
in 150–200 μl PBS and stored at 4 °C in the dark awaiting sample
acquisition. A representative gating strategy for LP neutrophils is
provided in Supplementary Fig. [245]12. All samples were acquired on a
BD LSRFortessa (BD Biosciences) at the Biomedical Research Council
(BRC) Flow Core (15th Floor, Tower Wing, Guy’s Hospital). Data were
analyzed using FlowJo software (Treestar).
Cell sorting
To obtain a pure population of naive CD4^+ T cells from the spleen,
splenic single cell suspensions were first treated with ACK buffer for
red blood cell lysis, enriched for CD4^+ cells using
immunomagnetic-based cell separation and then stained with mAbs against
CD4, CD25, CD44 and CD62L and LIVE/DEAD Fixable Dead Cell Stain
(Invitrogen, UK) as described above. Naive CD4^+ T cells were defined
as live CD4^+CD25^−CD44^loCD62L^hi cells. Purity checks were performed
after every sort and purity was always found to be above 97%. To obtain
pure populations of colonic NCR- ILC3s, cLPMCs were stained with mAbs
against CD45, CD90, CD127, KLRG1 and NKp46 and Live/Dead dye as
described above. NCR- ILC3s were defined as live
CD45^+CD90^+CD127^+KLRG1^−NKp46^− cells (Supplementary Fig. [246]13).
All sorts were performed on BD Aria I, BD Aria II or BD Aria Fusion (BD
Biosciences) at the BRC Flow Core (15th Floor, Tower Wing, Guy’s
Hospital).
Cell cultures
Sorted NCR- ILC3s isolated from the colon of TRUC or TRUC Il22^−/− mice
were cultured for 24 h in complete RPMI (Gibco) containing 10% FCS in
the presence or absence of 10 ng/ml IL-23 and 10 ng/ml IL-1β unless
stated otherwise. For the co-culture experiments NCR- ILC3s isolated
from the colon of TRUC or TRUC Il22^−/− mice were activated for 48 h
with 20 ng/ml IL-2, 50 ng/ml IL-7, 10 ng/ml IL-23 and 10 ng/ml IL-1β
prior to being co-cultured with colonoids.
ELISA
Cytokine concentrations in supernatants (S/Ns) of either stimulated
cell cultures or explant cultures were measured by ELISA. At the
endpoint, S/Ns were harvested and stored at −20 °C pending further
analysis. ELISA kits were purchased from eBioscience and ELISAs were
performed according to the manufacturer’s protocols. Cytokine
concentrations were determined within the linear phase of a standard
curve made with known cytokine concentrations provided by the supplier.
Gene expression analysis
Colonoids or sorted cells were lysed in 1 ml TRIsure (Bioline) and
stored at −80 °C pending further processing. Samples were left to thaw
at RT and homogenized by vortex for 10 s. To extract the RNA, 200 μl of
chloroform were added to each sample followed by 10 s vortex and 15 min
incubation at RT. Samples were centrifuged at max speed for 15 min at
4 °C and the clear S/N phase (containing the RNA) was transferred to
new 1.5 ml Eppendorf tubes and then mixed with equal volume of
isopropanol. Samples were vortex and then left at RT for 10 min,
followed by 8 min centrifugation at max speed at 4 °C. RNA pellets were
rinsed with 0.5 ml of 75% EtOH and left to airdry at RT. Depending on
pellet size; RNA was dissolved in 10-100 μl of RNase/DNase free H2O and
stored at −80 °C awaiting further analysis. Concentration of RNA in
each sample was measured using NanoDrop. 11 μl of RNA sample (always
containing the same amount of RNA across all samples of the same
experiment) that was always less that 4 μg RNA, were mixed with 1 μl
oligo dT and incubated at 65 °C for 5 min. At the end of the
incubation, RNA samples were mixed with 8 μl of reverse transcription
mix containing 4 μl Buffer 5×, 1 μl RNase Inhibitor (RI) at 20 U/μl,
2 μl dNTPs and 1 μl Reverse Transcriptase (RT). Reverse transcription
was then accomplished by incubating RNA samples at 42 °C for 1 h
followed by 65 °C for 5 min and then 4oC forever. cDNA samples (20 μl)
were stored at −20 °C until further use. Quantitative PCR was performed
using QuantiTect primers (Qiagen) and Quantitect SybrGreen MasterMix
(Qiagen) on a LightCycler 480 (Roche). Samples were analyzed in
triplicates and relative expression of mRNAs was determined after
normalization against the housekeeping gene Beta-2-Microglobulin (B2M).
Microarray analysis
RNA from colonic tissue fragments (distal region) was extracted using
TRIsure (Bioline) as described above. Contaminating DNA was removed
with the RNase-Free DNase Set (Qiagen) according to the manufacturer’s
protocol. cDNA was synthetized using Ovation PicoSL WTA System V2
according to the manufacture’s protocol (Nugen, USA) and labeled using
Encore BiotinIL module according to the manufacture’s protocol (Nugen,
USA). RNA and cDNA quantity and quality were assessed using the Agilent
RNA 6000 Nano Kit or Agilent RNA 6000 Pico Kit (depending on the amount
of RNA) according to the manufacture’s protocol (Agilent Technologies,
USA). Labeled cDNA were hybridized on a MouseWG-6 v2.0 Expression
BeadChip (Illumina, USA).
Statistical analysis
All graphs were generated and analyzed using GraphPad Prism 8 software.
Data represent median and interquartile range unless stated otherwise.
Statistical analysis was performed using non-parametric Mann–Whitney
test or Kruskal–Wallis test unless stated otherwise. Statistical
significance was indicated using * for p values less than 0.05, ** for
p values less than 0.01 and *** for p values less than 0.001 unless
stated otherwise.
Reporting summary
Further information on research design is available in the [247]Nature
Research Reporting Summary linked to this article.
Supplementary information
[248]Supplementary Information^ (4.3MB, pdf)
[249]Peer Review File^ (3.7MB, pdf)
[250]Reporting Summary^ (463.4KB, pdf)
Acknowledgements