ArticleIL-18-induced HIF-1a in ILC3s ameliorates the
inflammation of C. rodentium-induced colitisGraphical abstractHighlightsd C. rodentium induces production of IL-18 in colon, which
upregulates HIF-1a in ILC3s
d IL-18 production in the colon is boosted by TLR2 signaling
d HIF-1a promotes IL-22 in ILC3s and not in other innate
RORgt+ cells after infection
d IL-18 is responsible for the production of IL-22 by colonic
ILC3s through HIF-1aValle-Noguera et al., 2023, Cell Reports 42, 113508
December 26, 2023 ª 2023 The Authors.
https://doi.org/10.1016/j.celrep.2023.113508Authors
Ana Valle-Noguera,
Lucı´a Sancho-Temin˜o,
Raquel Castillo-Gonza´lez, ...,
Jose´ Marı´a Gonza´lez-Granado,
Julia´n Aragone´s, Ara´nzazu Cruz-Adalia
Correspondence
arancruz@ucm.es
In brief
HIF-1a is a hypoxia-inducible factor
regulating several immune cells in the
response against infections. Valle-
Noguera et al. evaluates the function of
HIF-1a in colonic ILC3s during
C. rodentium infection in mice,
demonstrating that TLR2-induced IL-18
transcriptionally upregulates HIF-1a in
ILC3s, boosting IL-22 secretion to protect
against the infection.ll
OPEN ACCESS
llArticle
IL-18-induced HIF-1a in ILC3s ameliorates
the inflammation of C. rodentium-induced colitis
Ana Valle-Noguera,1,8 Lucı´a Sancho-Temin˜o,1,8 Raquel Castillo-Gonza´lez,1,8 Cristina Villa-Go´mez,1
Marı´a Jose´ Gomez-Sa´nchez,1 Anne Ochoa-Ramos,1 Patricia Yag€ue-Ferna´ndez,2 Blanca Soler Palacios,3 Virginia Zorita,4
Berta Raposo-Ponce,5 Jose´ Marı´a Gonza´lez-Granado,1,7 Julia´n Aragone´s,6,7 and Ara´nzazu Cruz-Adalia1,9,*
1Department of Immunology, Ophthalmology and ENT, School of Medicine, Complutense University of Madrid, Instituto de Investigacio´n
Sanitaria Hospital 12 de Octubre (imas12), Madrid, Spain
2Centro de Investigaciones Biolo´gicas Margarita Salas (CIB-CSIC), Madrid, Spain
3Department of Immunology, Centro Nacional de Biotecnologı´a, Consejo Superior de Investigaciones Cientı´ficas (CNB-CSIC), Madrid, Spain
4Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain
5Centro de Biologı´a Molecular Severo Ochoa (CBM-CSIC), Madrid, Spain
6Hospital Santa Cristina, Fundacio´n de Investigacio´n Hospital de la Princesa, Madrid, Spain
7CIBER de Enfermedades Cardiovasculares, Instituto de Salud Carlos III, Madrid, Spain
8These authors contributed equally
9Lead contact
*Correspondence: arancruz@ucm.es
https://doi.org/10.1016/j.celrep.2023.113508SUMMARYGroup 3 innate lymphoid cells (ILC3s) are vital for defending tissue barriers from invading pathogens. Hypoxia
influences the production of intestinal ILC3-derived cytokines by activating HIF. Yet, the mechanisms gov-
erning HIF-1a in ILC3s and other innate RORgt+ cells during in vivo infections are poorly understood.
In our study, transgenic mice with specific Hif-1a gene inactivation in innate RORgt+ cells (RAG1KO HIF-
1a6Rorc) exhibit more severe colitis following Citrobacter rodentium infection, primarily due to the inability
to upregulate IL-22. We find that HIF-1a6Rorc mice have impaired IL-22 production in ILC3s, while non-
ILC3 innate RORgt+ cells, also capable of producing IL-22, remain unaffected. Furthermore, we show that
IL-18, induced by Toll-like receptor 2, selectively triggers IL-22 in ILC3s by transcriptionally upregulating
HIF-1a, revealing an oxygen-independent regulatory pathway. Our results highlight that, during late-stage
C. rodentium infection, IL-18 induction in the colon promotes IL-22 through HIF-1a in ILC3s, which is crucial
for protection against this pathogen.INTRODUCTION
Intestinal disorders such as bacterial infections or autoinflam-
matory diseases negatively affect human health. In affluent
nations inflammatory bowel diseases (IBDs) affect over 6
million people, causing significant morbidity in patients. How-
ever, in developing countries, enteric bacterial infections,
including enteropathogenic Escherichia coli (EPEC) and enter-
ohemorrhagic E. coli (EHEC), are a common cause of
morbidity and mortality, especially in young children.1,2 The
complexity of the molecular mechanisms involved in these af-
fections can be studied in vivo with Citrobacter rondentium as
a mouse model of gastrointestinal infections, IBD dysbiosis,
and even tumorigenesis.3 C. rodentium is a gram-negative
mucosal pathogen found naturally in mice; it shares some
pathogenic pathways with human EPEC and EHEC infections4
and promotes colonic inflammation and epithelial barrier
impairment making it a great model to investigate pathogen-
host immune interactions and the pathology of IBDs.5 The
complex physiopathology of gastrointestinal disorders in-
volves several cell populations and numerous signaling path-C
This is an open access article under the CC BY-Nways within the immune response such as interleukin-22 (IL-
22)- and IL-17-expressing CD4+ T cells (Th22 and Th17).
Group 3 innate lymphoid cells (ILC3s) constitute one essential
innate immune population involved in the response to entero-
pathogenic infections and IBD development. This subset be-
longs to the innate lymphoid cells (ILCs), recently discovered
cells that lack rearranged antigen-specific receptors.6–8
Although ILC3s are not the largest population of ILCs in the
colon at steady state, they play significant roles in homeosta-
sis and the defense against extracellular pathogens. ILC3s
can be subdivided into two subsets defined according to their
cell surface expression of natural cytotoxicity receptors,
known as NKp46 in mice7–10: NKp46– lymphoid tissue inducer
(LTi) cell-like ILC3s and NKp46+ ILC3s that differ in localiza-
tion and function. NKp46– LTi-like ILC3s secrete IL-22 and
IL-17, whereas NKp46+ ILC3s primarily produce IL-22, which
is involved in the containment of intestinal commensals and
the innate response to extracellular bacteria such as C. ron-
dentium.11–17 ILC3’s main role in the gut includes maintaining
the intestinal barrier integrity and promoting mucosal heal-
ing,18 a major clinical endpoint in gastrointestinal disordersell Reports 42, 113508, December 26, 2023 ª 2023 The Authors. 1
C-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Figure 1. HIF-1a in immune cells is essential for protection against C. rodentium infection
(A) Schematic image of the generation of BM chimeras Ub-Cre ERT2 (WT) or HIF-1afl/fl Ub-Cre (HIF-1a KO) and the experimental procedure of the C. rodentium
model.
(B) Representative dot plot showing the populations of CD45.1+ and CD45.2+ cells in the peripheral blood of the BM chimeras 4 weeks after BM transplantation.
(C) Hif-1a gene expression in the peripheral blood cells of WT and HIF-1a KO BM chimeras.
(D) Relative weight of chimeras WT and HIF-1a KO after C. rodentium infection. Representative data of three independent experiments with n = 8.
(E) Image of representative colons of non-infected (NI) and infected chimeras WT and HIF-1a KO after 12 days.
(F) Colon length of the BM chimeras WT and HIF-1a KO 12 days after C. rodentium infection.
(G) CFUs of C. rodentium in the feces of the chimeras WT and HIF-1a KO 2, 5, and 12 days after infection. Data represent the means ± SD from four (F) or three
experiments n = 4 (C and G). Statistical analysis: (C) unpaired t test; (D and G) two-way ANOVA; (F) one-way ANOVA with Sidak multiple comparisons. *p < 0.05,
**p < 0.01, ***p < 0.001. Graphs shows individual data.
2 Cell Reports 42, 113508, December 26, 2023
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Figure 2. HIF-1a expressed in RORgt+ cells does not alter the colonic ILC3 percentage and distribution
(A) Aggregated UMAP visualization of FlowSOM-generated lymphoid cells (linage–) of multiparametric flow cytometry data from the colon of the HIF-1a6Rorc and
the littermate control (WT). ILC1, ILC2, ILC3, and NK subsets correlate with light green, orange, red, and blue clusters, respectively, according to the expression of
several markers (RORgt, EOMES, CD127, KLRG1, and GATA3).
(legend continued on next page)
Cell Reports 42, 113508, December 26, 2023 3
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OPEN ACCESSproduced by attaching and effacing pathogens and IBDs.5,19
ILC3s and Ltis require RORgt for their generation and func-
tion. Recent research has identified additional innate non-
ILC3 RORgt+ cells in the gut, which are required for the induc-
tion of microbiota-specific T regulatory cell differentiation,20–23
contributing to intestinal homeostasis in steady state.
The molecular mechanisms regulating ILC3s during the devel-
opment of an infection are currently being investigated. ILC3s
can be directly stimulated by cytokines such as IL-23 and IL-
1b or TNF-like ligand 1A (TL1A) during colitis.19,24,25 IL-18 has
recently been shown to also play an important role during colitis,
secreted by epithelial or innate cells, and necessary for the host
defense against C. rodentium.26 In addition, low oxygen levels
have been shown to modulate ILC3 response through HIF-1a
stabilization.27 To date, there has been no research into whether
HIF-1a in colonic ILC3s is involved in the development of colitis
induced by C. rodentium, or in relation to what molecular mech-
anisms, aside from hypoxia, could be modulating this transcrip-
tion factor during an enteric infection. HIF-1a is not exclusively
modulated by oxygen levels; it can also be modulated through
pathogen-associated molecular patterns (PAMPs), cytokines,
or a combination of factors.28
Herein, we study the role of HIF-1a in colonic ILC3s during
the physiological condition and during the C. rodentium infec-
tion model, using specific conditional knockout (KO) mice
HIF-1afl/flRORgtCre (HIF-1a6Rorc) and RAG1KO HIF-1a6Rorc.
The two types of KO mice presented a more severe
C. rodentium-induced colitis than their control. Interestingly,
we found that an innate non-ILC3 RORgt+ population secreted
IL-22 in the early phase of the infection but that this secretion
was independent of HIF-1a expression (day 5). However, the
secretion of IL-22 by colonic ILC3s was regulated via HIF-1a
at a later stage of infection (day 12). Moreover, we discovered
that, while cytokines IL-1b, IL-23, or TL1A were not responsible
for the induction of IL-22 through HIF-1a, IL-18 transcriptionally
upregulated HIF-1a in ILC3s, thus promoting a significant in-
duction of IL-22 inWT ILC3s but not in HIF-1a KO cells. Surpris-
ingly, we did not find any difference in the production in vivo of
IL-17, neither following C. rodentium infection in the colon of
both types of KO mice nor in vitro after the stimulation of HIF-
1a KO and WT ILC3s by IL-18 and IL-23. C. rodentium can be
sensed through the Toll-like receptor 2 (TLR2) by epithelial or
innate immune cells, which stimulate the secretion of IL-18,
upregulating the expression of HIF-1a in ILC3s and finally acti-
vating the production of IL-22. In summary, our findings estab-
lish an additional molecular pathway, independent from hypox-
ia, which can regulate the secretion of IL-22 by colonic ILC3s
through HIF-1a, as a target gene of IL-18 signaling, during
C. rodentium infection.(B) Flow cytometry analysis of ILC subsets in the colon from the WT and the HIF
(C) Representative images of RORgt+ cells (EGFP, green), CD3 (red), and B220
EGFP+/
 and HIF-1a6Rorc EGFP+/
 mice.
(D) Representative images of the lamina propria from the colon of WT EGFP+/
 an
CD3
) and the white arrow indicates T cells (CD3+).
(E) IL-22 and IL-17 protein secretion by colon explants cultured ex vivo for 24 h. I
with n = 5 (B and E). Statistical analysis: (B) two-way ANOVA with Sidak’s mult
animals.
4 Cell Reports 42, 113508, December 26, 2023RESULTS
The expression of HIF-1a in RORgt+ cells is required for
defense against C. rodentium-induced colitis
HIF-1a is well documented to be expressed in inflamed mucosa,
contributing to intestinal homeostasis primarily through its activa-
tion in epithelial cells.29 To investigate the role of HIF-1awithin the
immune compartment during enteric bacterial infections, we
createdbonemarrow (BM) chimeras, depletingHIF-1a in immune
cells using HIF-1afl/fl Ub-Cre ERT2 mice (referred to as HIF-1a
KO), where gene depletion was achieved through tamoxifen
exposure (Figures 1A–1C). Remarkably, BM HIF-1a KO chimera
mice developed worsened C. rodentium colitis, evident in
increased weight loss (Figure 1D), greater colon shortening
(Figures 1E and 1F), and higher colony-forming units (CFUs) in
feces (Figure 1G) compared with control BM Ub-Cre ERT2
(referred to as WT) chimera mice. To explore the specific role of
HIF-1a in the innate compartment during colitis, we established
mixed BM chimeras by injecting cells from RAG1KO and HIF-1a
KOmice (1:1) or RAG1KOandWT control mice (1:1) into recipient
mice, thereby re-establishingHIF-1aexpressionexclusively in the
innate compartment (Figures S1A and S1B). Significantly, there
were no discernible differences in weight loss (Figure S1C), colon
shortening (Figure S1D), or CFUs (Figure S1E) between both sets
of chimeras. These findings underscore the critical role of HIF-1a
expression in the innate compartment for regulating the develop-
ment of exacerbated colitis. Given these observations, we sought
to investigate the hypothesis that HIF-1a in ILC3 cells plays a
pivotal role in the development of C. rodentium-induced colitis.
To investigate the role of HIF-1a in ILC3s during C. rodentium-
induced colitis, we generated mice with HIF-1a gene deletion in
RORgt+ cells, crossing HIF-1afl/fl mice with Rorc promoter-
driven Cre recombinase mice (Rorc-Cre). These conditional KO
mice (HIF-1a6Rorc) lack the Hif-1a gene in RORgt+ cells,
including ILC3s, other innate RORgt+ cells, and T cells express-
ing RORgt during their development in double-positive thymo-
cytes. We also crossed these mice with RORgt EGFP reporter
mice to isolate RORgt-expressing cells for further analysis.
Quantitative real-time PCR showed that WT HIF-1afl/fl RORgt
EGFP+/
 mice (hereinafter WT EGFP+/
) expressed more
Hif-1a mRNA in RORgt+ cells compared with KO HIF-1a6Rorc
EGFP+/
 mice both in the spleen and the lamina propria of the
colon (Figures S2A and S2B). We conducted comprehensive
high-dimensional analysis (Figures 2A and S2C) and gating
strategies (Figure S2D) to confirm that HIF-1a6Rorc mice did
not exhibit differences in the percentage of colonic ILCs under
steady-state conditions (Figure 2B). We also considered the
presence of other non-ILC3 innate RORgt+ cells,20–23 which
share similarities with myeloid cells, expressing RORgt but not-1a6Rorc.
(cyan) immunostaining on colon tissues showing lymphoid follicles from WT
d the HIF-1a6Rorc EGFP+/
. Orange arrows show innate RORgt+ cells (EGFP+
ndividuals/means ± SD for each sample from three independent experiments,
iple comparisons; (E) unpaired t test. *p < 0.05. Symbols represent individual
(legend on next page)
Cell Reports 42, 113508, December 26, 2023 5
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OPEN ACCESSCXCR6, IL-7R, or CD90 (Figure S2E). This analysis revealed no
differences in the cell number of this population between the
two strains (Figure S2F). In addition, the absolute numbers of
various RORgt-expressing populations in the spleenwere similar
in HIF-1a6Rorc and control mice (Figure S2G). Confocal micro-
scopy analysis demonstrated a comparable distribution of
colonic CD3
RORgt+ cells in both transgenic mice within
lymphoid follicles (Figure 2C) and the lamina propria of the colon
(Figure 2D). However, a significant increase in IL-22 protein
induction was observed in the colons of HIF-1a6Rorc mice
compared with control mice, although no difference was de-
tected in IL-17 levels (Figure 2E). Furthermore, histological anal-
ysis of the colon in these conditional mice did not reveal any
signs of pathology in the baseline condition (Figures S3A
and S3B).
To assess colitis development in these conditional mice
following infection, we employed the C. rodentium colitis model,
which relies on ILC3s during the acute phase.30 Surprisingly,
HIF-1a6Rorc mice displayed more severe colitis compared with
their controls. Despite having elevated baseline IL-22 levels in
the colon (Figure 2E), thesemice exhibited increasedCFUs in their
feces (Figure 3A) and more pronounced colon inflammation, as
assessed by histoscore and flow cytometry (Figures 3B–3D).
However, no significant difference was observed in terms of
weight loss (Figure S3C). Of significance, a notable reduction in
IL-22 protein induction was observed in the colons of HIF-
1a6Rorc mice 12 days post-infection in contrast to the control
mice (Figure 3E), potentially explaining the increased disease
severity in HIF-1a-deficient mice. Conversely, other cytokines
such as IL-17 (Figures 3F and 3G) remained unaffected in these
conditional mice. There were no significant alterations in the
mRNA expression of Il-1b, Il-23p19, and Tnf-a (Figure 3G). Con-
cerning the formation and maintenance of lymphoid structures,
the number of colonic patches in both non-infected and
C. rodentium-infected colons was unaltered in HIF-1a6Rorc
compared with control mice (Figure S3D). Furthermore, no differ-
ences were observed in the localization of ILC3s within colon
lymphoid follicles after 12 days of infection (Figure S3E).
Deficiency of HIF-1a in innate RORgt+ cells impairs IL-22
induction during C. rodentium infection, exacerbating
colitis
To further evaluate the in vivo impact of HIF-1a specifically ex-
pressed in innate RORgt+ cells in the C. rodentium-colitis model,
wecrossed theHIF-1a6Rorcmicewith theRAG1KOmice toobtain
RAG1KOHIF-1a6Rorc mice to eliminate any possible contribution
by HIF-1a in T cells. We observed no differences either in the per-
centage of colonic ILCs (Figure S4A) or in histology analysis inFigure 3. HIF-1a, expressed in RORgt+ cells, is important for protectio
(A) CFUs of C. rodentium in feces from the HIF-1a6Rorc and the littermate contro
(B) Histoscore of the colitis in HIF-1a6Rorc and WT mice 12 days after C. rodenti
(C) Representative image of H&E staining on colon tissues from an infected WT
(D) Absolute number of CD45+ cells isolated from the lamina propria of the colon
(E and F) IL-22 and IL-17 protein secretion by colon explants from C. rodentium-
(G) mRNA relative expression of several cytokine genes in the colon from HIF-1
represent the means ± SD from three independent experiments with n = 5/6. Statis
Mann-Whitney U test; (D–F) unpaired t test. *p < 0.05, **p < 0.01, ***p < 0.001. S
6 Cell Reports 42, 113508, December 26, 2023RAG1KO HIF-1a6Rorc mice compared with the littermate control
RAG1KOWTmice in steady state (Figures S4B and S4C). Subse-
quently,weevaluated the impactofdeficientexpressionofHIF-1a
in ILC3s in the C. rodentium-colitis model, observing that the
RAG1KOHIF-1a6Rorc exhibited lowersurvival rate than their litter-
mate controls (Figure 4A) as well as a significantly greater weight
loss (Figure 4B). Interestingly, we observed that the CFUs in the
feces of the RAG1KOHIF-1a6Rorc mice showed a declining trend
in the first few days of infection, but that effect subsequently
became inverted (Figure 4C). Indeed, when we measured IL-22
expression in the colon, a trend of more protein levels was de-
tected in the colon of the RAG1KO HIF-1a6Rorc mice compared
with the control under physiological conditions (Figure 4D), which
explained the decrease in CFUs observed in the RAG1KO HIF-
1a6Rorc mice the first days of infection. However, induction of
IL-22 12 days after C. rodentium infection became impaired in
these deficient mice (Figure 4D), with results similar to those
observed in theHIF-1a6Rorcmice (Figure3E).Moreover, histology
score clearly showed that the RAG1KO HIF-1a6Rorc mice devel-
oped more severe inflammation in the colon than the control
mice (Figures 4E and 4F). In combination, these experiments
demonstrate a key role of cell-intrinsic HIF-1a in regulating the
production of IL-22 by colonic innate RORgt+ cells, thus prevent-
ing a more severe colitis.
HIF-1a regulates the production of IL-22 in ILC3s after
C. rodentium infection
To ascertain whether the impaired induction of IL-22 in this colitis
model was a consequence of the deletion of HIF-1a in the ILC3s
or in other RORgt+ cells, we assessed in which cell types IL-22
was affected in the HIF-1a6Rorc mice after 2, 5, and 12 days of
infection. On day 2, there was a notable increase in IL-22+ cells
in Lin-CD3
RORgt+ NKp46– IL-7R+ CXCR6+ CD90+/
 popula-
tions (which include Lti and ILC3 NKp46
) in the colon of HIF-
1a6Rorc mice. In addition, the CD3
 RORgt+ IL-7R
 CXCR6–
CD90
 subpopulation (referred to as non-ILC3 innate RORgt+
cells) also showed IL-22 production after 2 days of infection
(Figure 5A). No significant differences were observed in the cell
counts for ILC3s or the non-ILC3 innate RORgt+ cells between
WT and HIF-1a6Rorc mice (Figure 5B). On day 5 of infection,
no difference was observed in the percentage of any ILC3 and
Lti IL-22+ subpopulations (Figure 5C). Regarding the non-ILC3
innate RORgt+ cells, these produced high levels of IL-22
5 days after infection, but no difference was detected between
the HIF-1a6Rorc and WT mice (Figure 5C). As we observed in
the IL-17 production by colon explants, the percentage of IL-
17-positive cells in the ILC3s was similar in these transgenic
mice (Figure S4D). Unlike IL-22 (Figure 5C), the non-ILC3 innaten against C. rodentium infection
l mice (WT) after infection.
um infection.
and HIF-1a6Rorc mouse stained with H&E.
from the HIF-1a6Rorc and WT mice 5 days after C. rodentium infection.
infected HIF-1a6Rorc and WT mice on day 12.
a6Rorc and WT mice non-infected (NI) and 12 days after infection. The data
tical analysis: (A and G) two-way ANOVA, Sidak’s multiple comparison test; (B)
ymbols represent individual animals.
Figure 4. HIF-1a expressed in ILC3 RORgt+ cells protects against severe colitis induced by C. rodentium
(A) Survival rate of RAG1KO HIF-1a6Rorc and RAG1KO WT control mice after C. rodentium inoculation, n = 7.
(B) Relative weight of RAG1KO HIF-1a6Rorc and RAG1KO WT mice after C. rodentium infection.
(C) CFUs of C. rodentium in the feces from RAG1KO HIF-1a6Rorc and RAG1KO WT mice after infection.
(D) IL-22 protein quantification secreted by colon explant from non-infected (NI) or infected RAG1KO HIF-1a6Rorc and RAG1KO WT mice cultured for 48 h.
(legend continued on next page)
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OPEN ACCESSRORgt+ exhibited no intracellular staining in IL-17. No difference
was detected in the absolute number of the different cell subsets
on day 5 (Figure 5D). On day 12 of infection, we detected a sig-
nificant decrease in the percentage of IL-22+ in the Lti and ILC3
subsets (including NKp46
 and NKp46+ ILC3 cells) in the colon
of the HIF-1a6Rorc mice compared with the control mice, but
not in the non-ILC3 innate RORgt+ IL-22+ cells (Figure 5E). How-
ever, no difference was observed in the cell number of the
different innate subsets between the WT and HIF-1a6Rorc mice
(Figure 5F), suggesting that deficiency of HIF-1a inhibited pro-
duction of IL-22, as opposed to the expansion or survival of
the ILC3s. Furthermore, a significant decrease in the percentage
of IL-22+ within the Lti/ILC3 NKp46
 and ILC3 NKp46+ in the co-
lon of the RAG1KO HIF-1a6Rorc mice was also observed
compared with the control mice (Figure 5G). No significant vari-
ance was observed in the absolute cell count among the various
innate subsets on day 12 (Figure 5H). Therefore, the lack of
HIF-1a impairs the production of IL-22 by ILC3s following
C. rodentium infection.
For a detailed investigation of specific ILC3 and ILC1 popula-
tions, we performed flow cytometry analysis on RAG1KO HIF-
1a6Rorc and control mice on day 12 post-infection. Interestingly,
we foundahigher percentageof ILC3NKp46
 subset inRAG1KO
HIF-1a6Rorc mice compared with WT mice, despite observing a
lower proportion of IL-22+ ILC3s, suggesting apotential compen-
satory mechanism (Figure S4E). However, there were no
observed differences in absolute cell numbers (Figure S4F).
Furthermore, the decreased IL-22 production by HIF-1a-defi-
cient ILC3s on day 12 post-infection (Figures 5E and 5G) poten-
tially being attributed to the increased bacterial load and height-
ened inflammation observed in these transgenic mice was
addressed. To eliminate the influence of such environmental sec-
ondary factors, IL-22 production was evaluated in colonic ILC3s
in response to C. rodentium infection in a mixed BM chimera,
where both WT and HIF-1a-deficient ILC3s coexist within the
same mouse environment. To achieve this, BM from CD45.2
HIF-1a6Rorc mice and their CD45.1 WT littermate controls were
transferred into B6 recipient mice. After 12 days of infection, IL-
22 was analyzed in the ILC3 CD45.1 WT or ILC3 CD45.2 HIF-
1a6Rorc from the colon by flow cytometry, showing that the
percentage of IL-22+ cells in ILC3 CD45.2 HIF-1a6Rorc was
significantly lower compared with ILC3 CD45.1WT (Figure S4G).
HIF-1a controls the secretion of IL-22 by ILC3s in
response to TLR2 agonist as well as cytokine IL-18
To establish the molecular mechanisms involved in the induc-
tion of IL-22 through HIF-1a, we incubated colon explants
from the RAG1KO HIF-1a6Rorc mice and the littermate controls
with different signals produced by C. rodentium infection. As
these bacteria are Gram-negative, the innate receptor for bac-
terial lipopolysaccharide TLR4 could be contributing to the host
response.31 Moreover, TLR2 has been shown to play a critical(E) Representative images of H&E staining on colon tissues from RAG1KO HIF-1
(F) Histological score of the colitis inflammation from RAG1KO HIF-1a6Rorc and
means ± SD from three independent experiments with n = 7mice (C and D); from tw
(Mantel-Cox) test; (B–D) two-way ANOVA with Sidak multiple comparisons; (F)
animals.
8 Cell Reports 42, 113508, December 26, 2023role in maintaining intestinal mucosal integrity during C. roden-
tium infection.32 In view of this, we incubated colon explants
in vitro with LPS (TLR4 agonist) or Pam3CSK4 (TLR2 agonist)
for 48 h and IL-22 was then quantified by ELISA. Although IL-
22 protein levels did not increase in the supernatant of colon
explants from the RAG1KO WT mice stimulated with LPS (Fig-
ure 6A), we could detect more IL-22 levels when we stimulated
them with Pam3CSK4 and IL-2 (Figure 6B); on the contrary, this
enhancement was not observed in stimulated colon explants
from the RAG1KOHIF-1a6Rorc mice (Figure 6B), demonstrating
that HIF-1a is critical for the induction of IL-22 in ILC3s by TLR2
signaling. Since murine ILC3s do not express TLR2,33 TLR-
induced cytokines were explored as possible activators of
HIF-1a-dependent IL-22 production. IL-1b or TL1A combined
with IL-23 upregulated IL-22 production in colon explants
from both the RAG1KO HIF-1a6Rorc and the WT mice (Figures
6C and 6D), suggesting that HIF-1a was not involved in the in-
duction of IL-22 by ILC3s via these cytokines. However,
although IL-18 alone or with IL-23 promoted the secretion of
IL-22 by ILC3s from colon explants of the RAG1KO WT mice,
this induction was not observed in the RAG1KO HIF-1a6Rorc
mice, indicating that IL-18 promoted the secretion of IL-22
through HIF-1a in ILC3s (Figures 6E and 6F). IL-23, TL1A, and
IL-2 alone were unable to induce a significant increase in IL-
22 when incubated with colon explants from the RAG1KO WT
or the RAG1KO HIF-1a6Rorc mice (Figures S5A‒S5C).
C. rodentium transcriptionally upregulates HIF-1a in
colonic ILC3s to stimulate the production of IL-22
activated by TLR2-induced IL-18
To demonstrate that C. rodentium can transcriptionally upregu-
late HIF-1a and IL-18 in the colon in vivo, WT mice were orally
infected, and colon RNA was isolated at different time points
during the infection forHif-1a and Il-18 expression quantification.
Hif-1amRNA tended to increase over time, with significant upre-
gulation in the colon of infected WT mice at day 12 compared
with uninfected ones (Figure 7A). This increase in Hif-1a was
also observed in sorted colonic ILC3s from WT mice after
12 days of infection, demonstrating transcriptional regulation of
HIF-1a in this colonic cell population (Figure 7B). Furthermore,
IL-18 was upregulated in the colon of WT mice infected with
C. rodentium, showing a significant increase after 7 days and
maintained up to day 12 of infection (Figure 7C). TLR2 signaling’s
requirement for IL-18 induction was demonstrated by incubating
colon explants fromWTmice ex vivowith a TLR2 agonist; this led
to the production of IL-18 (Figure 7D). An antagonist of IL-18 (IL-
18 binding protein; BP IL-18) importantly inhibited IL-22 produc-
tion induced by TLR2 in colon explants from RAG1KO WT mice,
indicating that IL-18, produced upon TLR2 activation, was
responsible for inducing IL-22 in colonic ILC3s (Figure 7E).
Significantly, Hif-1a mRNA was upregulated in isolated ILC3s
from the spleens of WT EGFP+/
 control mice when stimulateda6Rorc and RAG1KO WT mice on day 12 after C. rodentium infection.
RAG1KO WT mice 12 days after C. rodentium infection. The data represent
o independent experiments with n = 8mice (F). Statistical analysis: (A) log rank
Mann-Whitney U test. *p < 0.05, ***p < 0.001. Symbols represent individual
(legend on next page)
Cell Reports 42, 113508, December 26, 2023 9
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OPEN ACCESSin vitro with IL-18 and IL-23 compared with non-stimulated cells
(Figure 7F). In addition, the mRNA of glucose transporter 1
(Slc2a1), one of the key genes regulated by HIF-1a, was signifi-
cantly upregulated in these IL-18- and IL-23-stimulated cells
(Figure 7G). Importantly, IL-18 alone induced the upregulation
of Hif-1a in these sorted cells (Figure 7H). Indeed, ILC3s from
the spleen of WT EGFP+/
 mice significantly upregulated Il-22
mRNA when stimulated with IL-18 and IL-23 compared with
non-stimulated cells. However, this response was not observed
in the ILC3s from KO HIF-1a6Rorc EGFP+/
 mice (Figure 7I).
Notably, there was no difference in Il-17 mRNA expression in
sorted ILC3s from KO HIF-1a6Rorc EGFP+/
 mice compared
with WT mice when stimulated with IL-18 and IL-23 (Figure 7J).
To confirm that IL-22 induction was specific to colonic ILC3s,
lamina propria cells from the colon of WT mice were incubated
with no stimulus, IL-18 alone, or IL-18 plus IL-23, followed by
flow cytometry analysis of intracellular cytokines. ILC3 WT cells
significantly upregulated IL-22 after IL-18 induction and even
more with IL-18 plus IL-23 (Figure 7K). In contrast, the non-
ILC3 innate RORgt+ population did not upregulate IL-22 after
IL-18 or IL-18 plus IL-23 stimulation in vitro (Figure 7K). These
findings demonstrate that HIF-1a is transcriptionally upregulated
in colonic ILC3s following C. rodentium infection, inducing IL-22
production while not affecting IL-17 in ILC3s via TLR2-induced
IL-18.
DISCUSSION
Our research uncovers a pathway for IL-22 production by
colonic ILC3s during acute C. rodentium-induced colitis. This
pathway involves IL-18 upregulation in the colon due to enteric
pathogen infection, promoting IL-22 production by colonic
ILC3s through HIF-1a. Previously, mTOR1 inhibition (Rptor6Rorc
mice, KO of Raptor in the RORgt+ cells) was shown to reduce
ILC3 numbers in the small intestine,34,35 affecting their response
to C. rodentium infection in the colon, although colonic ILC3
numbers remained unaffected, suggesting other mechanisms
at play.34 Hypoxia boosts ILC3 activation and proliferation,
notably observed in the MNK3 cell line.27 In HIF-1a-deficient
RORgt mice, small intestine ILC3 numbers decreased, while
ILC1 numbers increased, preserving the RORgt-ILC3 profile.27
Strikingly, these mice were more susceptible to Clostridium
difficile, although ILC3s were thought to have a minor role,
whereas loss of ILC1s increased susceptibility.36 Indeed, IL-
17-producing gd T cells, also expressing RORgt, contribute to
C. difficile protection37; consequently, the role of HIF-1a in
RORgt+ cells in C. difficile susceptibility may involve gd T cellsFigure 5. Deficiency of HIF-1a impairs the production of IL-22 by ILC3
(A and B) Representative flow cytometry dot plots and the percentage of IL-22+ c
ILC3 Nkp46+, the non-ILC3 innate RORgt+ cells IL-17R
, CXCR6
, CD90
) or abs
the HIF-1a6Rorc and control mice (WT) 2 days after C. rodentium infection.
(C and D) Percentage of IL-22+ cells (C) in the different cell populations or absolu
(E and F) Percentage of IL-22+ cells (E) in the different subsets or absolute numb
(G and H) Percentage of IL-22+ cells (G) in the different populations or absolute nu
control mice RAG1KOWT after 12 daysC. rodentium infection. Data represent me
Statistical analysis: (A, C, E, and G) unpaired t test; (B, D, F, and H) two-way
****p<0,0001. Symbols represent individual animals.
10 Cell Reports 42, 113508, December 26, 2023but requires further investigation. Unlike the HIF-1a-deficient
RORgt mice, the loss of HIF-1a, in Nkp46+ cells, prevents
ILC3-to-ILC1 conversion, protecting against dextran sodium sul-
fate colitis-induced intestinal damage.38
Herein, we generated transgenic HIF-1a6Rorc mice as an
approach for studying the in vivo contribution of HIF-1a in
RORgt+ cells in the colon. The colonic ILC populations remained
stable in steady state, contrasting prior findings in RAG2KO
RptorDRorc mice.34 Intriguingly, the absence of Hif-1a boosted
IL-22 production by ILC3s under steady-state conditions. How-
ever, upon C. rodentium infection, HIF-1a6Rorc mice exhibited
increased susceptibility, likely due to inadequate IL-22 upregula-
tion after 12 days, leading to higher bacterial load and epithelial
damage. Surprisingly, IL-17 levels resembled those in WT mice.
Similarly, in RAG1KO HIF-1a6Rorc mice, IL-22 secretion was
dysregulated 12 days after C. rodentium-induced colitis onset.
This resulted in increased susceptibility and severe disease,
highlighting the impact of HIF-1a deficiency in innate RORgt+
cells, encompassing ILC3s or other non-ILC3 innate RORgt+
populations. Cytometry analysis revealed that HIF-1a deficiency
hindered IL-22 production by ILC3s after 12 days of infection,
while other innate RORgt+ cells surprisingly produced IL-22 early
during C. rodentium infection. Hence, these findings suggest a
dual role for HIF-1a in colonic ILC3s, potentially suppressing
basal IL-22 production during steady-state conditions and
stimulating it during late-stage bacterial infection. Under basal
conditions, microbiota-produced butyrate stimulates IL-22 in
CD4+ T cells and ILC3s by increasing mTOR, STAT3, AhR, and
HIF-1a levels.35 The findings contradict expectations for HIF-
1a6Rorc mice, suggesting that HIF-1a may counteract basal IL-
22 secretion in ILC3s through other pathways, for example,
involving RANK, VIPR2, or RET.15,39–44 Further studies are
needed for confirmation.
In the late infection stage, we found that HIF-1a regulates IL-22
secretion in colonic ILC3s via IL-18, demonstrated through ex vivo
experiments using RAG1KO HIF-1a6Rorc mice and their controls.
Curiously, previous research showed IL-18-induced HIF-1a
in vitro in a mouse melanoma cell line.45 Our study confirms this
connection specifically in ILC3s, whereC. rodentium infection up-
regulates HIF-1a in colonic tissue and ILC3s, especially when
exposed to IL-18 or IL-18 with IL-23. Remarkably, HIF-1a and
IL-22 mRNA upregulation in ILC3s occurs after 6 h, indicating
direct binding to the IL-22 promoter, similar to CD4+ T cells.46
This suggests that HIF-1a in ILC3s is regulated by both post-
translational changes from hypoxia and an O2-independent
mechanism, as seen in monocytes stimulated with LPS.47 More-
over, IL-18 was upregulated in C. rodentium-infected mice, ands
ells (A) in the different innate subsets expressing RORgt (Lti and ILC3 Nkp46
,
olute numbers (B) detected in isolated colonic lamina propria cells (LPCs) from
te numbers (D) after 5 days of C. rodentium infection.
ers (F) 12 days after C. rodentium infection.
mbers (H) in isolated colonic LPC cells from the RAG1KO HIF-1a6Rorc and the
ans ± SD from one representative of three independent experiments with n = 5.
ANOVA with Sidak multiple comparisons. *p < 0.05, **p < 0.01, ***p<0.001,
Figure 6. ILC3s release IL-22 in response to TLR2 agonist or IL-18 through HIF-1a
(A) IL-22 protein secretion by colon explants fromRAG1KOHIF-1a6Rorc and RAG1KOWT control mice incubated ex vivowith TLR4 agonist for 48 h andwith non-
stimulus (NS).
(B) IL-22 protein secretion by colon explants from RAG1KO HIF-1a6Rorc and RAG1KO WT mice stimulated ex vivo with TLR2 agonist and IL-2 for 48 h.
(C) IL-22 protein secretion by colon explants from RAG1KO HIF-1a6Rorc and RAG1KO WT mice incubated ex vivo with IL-1b and IL-23 for 48 h.
(D) IL-22 protein secretion by colon explants from RAG1KO HIF-1a6Rorc and RAG1KO WT mice incubated ex vivo with TL1A and IL-23 for 48 h.
(E) IL-22 protein secretion by colon explants from RAG1KO HIF-1a6Rorc and RAG1KO WT mice ex vivo stimulated with IL-18 for 48 h.
(F) IL-22 protein secretion by colon explants from RAG1KO HIF-1a6Rorc and RAG1KO WT mice incubated ex vivo with IL-18 and IL-23 for 48 h. Data repre-
sentation from three independent experiments with minimum n = 5. Statistical analysis: paired t test. *p < 0.05, **p < 0.01. Symbols represent individual data from
one animal with and without stimulus.
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OPEN ACCESSit increased IL-22 in WT ILC3s but not in HIF-1a KO ILC3s, while
no difference was observed in the upregulation of IL-17, thus
demonstrating an independent regulation of both cytokines in
this infection model. The highest IL-22 peak occurred on day
12, after the IL-18 peak, indicating the role of IL-18 in IL-22 pro-
duction in ILC3s. Similar findings were observed during Toxo-
plasma gondii infection in the ileum.48 Moreover, another study
found that human ILC3 proliferation and IL-22 secretion are IL-
18 dependent via NF-kB in human tonsils.49
Since TLR2 plays an essential role in maintaining mucosal
integrity during C. rodentium-induced colitis32 and C. rodentium
can activate TLR4 in host cells,31,50 we studied whether theactivation of both receptors could induce IL-22 by ILC3s through
HIF-1a in colon explants. Our results showed that a TLR2
agonist with IL-2 significantly boosted IL-22 secretion in colon
explants from RAG1KO WT mice but not in those from RAG1KO
HIF-1a6Rorc mice, suggesting that TLR2 signaling via HIF-1a trig-
gers IL-22 production by ILC3s. Sincemouse ILC3s typically lack
TLR2 and direct regulation by its ligands,33 we ruled out direct
TLR2 modulation of ILC3s. TLR2 can be expressed by innate
and epithelial cells51 and their activation is associated with IL-18
production.26,52,53 A defective IL-18 production in IL-18
/
 mice
causes greater susceptibility to C. rodentium infection.54 During
bacterial infections and PAMPs exposure, intestinal epithelialCell Reports 42, 113508, December 26, 2023 11
(legend on next page)
12 Cell Reports 42, 113508, December 26, 2023
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OPEN ACCESScells activate inflammasomes, releasing IL-18.55 TLR2stimulation
induces proinflammatory cytokines, including IL-18, in human
dendritic cells.53 Our findings indicated that a TLR2 agonist
notably promoted IL-18 secretion in colon explants from WT
mice. Importantly, an IL-18 antagonist inhibited TLR2-induced
IL-22 production in ex vivo colon explants from RAG1KO WT
mice, suggesting that TLR2-induced IL-18 secretion drives IL-
22 production by ILC3s. However, further research is needed to
identify the primary source of IL-18 following C. rodentium infec-
tion in TLR2-dependent signaling.
In summary,wehave elucidated amolecular pathway involving
TLR2-triggered IL-18 release that transcriptionally regulates HIF-
1a expression and IL-22 secretion by ILC3s in the colon during
late-stage C. rodentium infection. In addition, we observed IL-
22 secretion by non-ILC3 innate RORgt+ cells, which, in this
context, is independent of HIF-1a expression. These findings
hold promise for advancing research in colitis, enteropathogen
infections, IBDs, and cancer progression.
Limitations of the study
Regrettably, this study could not ascertain the production of IL-
22 in the different subsets of sorted RORgt+ cells from the colon
of infected mice, primarily due to technical intricacies. Interest-
ingly, further exploration is needed to analyze the role of the
characterized non-ILC3 innate RORgt+ cells in the control of
the C. rodentium infection.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
d KEY RESOURCES TABLE
d RESOURCE AVAILABILITYFig
pro
(A)
(B)
(C)
(D)
(E)
IL-1
(F a
(H)
(I a
and
(K)
wit
rep
and
**pB Lead contact
B Materials availability
B Data and code availability
d EXPERIMENTAL MODEL AND STUDY PARTICIPANT DE-
TAILS
B Mice
B BM chimeras
B Citrobacter rodentium-induced colitisure 7. C. rodentium infection causes TLR2-induced IL-18 producti
moting IL-22 expression
Gene expression of Hif-1a in the colon from uninfected and infected WT mice
Hif-1a relative expression in sorted ILC3s from WT mice non-infected (NI) an
IL-18 protein secretion by colon explants from the uninfected and C. rodentiu
IL-18 protein secretion by colon explants from WT mice incubated ex vivo wi
IL-22 protein secretion by colon explants from RAG1KO WT mice incubated f
8 (S + BP IL-18).
nd G) Gene expression ofHif-1a (F) or Slc2a1 (G) in sorted ILC3s from the splee
Gene expression of Hif-1a in sorted splenic ILC3s of WT EGFP+/
 ex vivo inc
nd J) Gene expression of Il-22 (I) or Il-17 (J) in sorted splenic ILC3s of the WT E
IL-23 for 6 h. The levels of mRNA are normalized to the stimulated WT.
Percentage of IL-22+ cells in ILC3s or in the non-ILC3 innate RORgt+ subsets
h IL-18 or IL-18 plus IL-23 overnight, quantified by flow cytometry. Statistically
resent the means ± SD from three (A, B, and H–J), two (C, K, and L), or four (D
F–H) paired t test; (E, K, and L) RM one-way ANOVA test; (C, I, and J) one-
< 0.01, ***p < 0.001.d METHOD DETAILS
B Counting of colonic patches
B Isolation of colonic LPCs
B Flow cytometry, antibodies, and ELISA
B Flow cytometry gating strategy
B Ex vivo organ culture
B Isolation of splenic ILC3s
B Histology
B Immunofluorescence
B RNA isolation and qPCR
d QUANTIFICATION AND STATISTICAL ANALYSIS
SUPPLEMENTAL INFORMATION
Supplemental information can be found online at https://doi.org/10.1016/j.
celrep.2023.113508.
ACKNOWLEDGMENTS
The authors wish to thank the Flow Cytometry Core Facility of the CBMSO and
the CIB, the Histology and Microscopy Facilities of CNB, and the Genomic Fa-
cility ofCAI atUCMand ‘‘ParqueCientı´fico deMadrid’’ for the technical support
provided. The graphical abstract was created using Biorender software. The
present research was supported by the ‘‘Ramon y Cajal’’ Program (RYC-
2017-21837), the grant nos. RTI2018-093647-B-I00, CNS2022-135365, and
PID2021-122780OB-I00 awarded to A.C.-A. by the ‘‘Ministerio de Ciencia e In-
novacio´n’’, Agencia Estatal de Investigacio´n (AEI). JMGG’s laboratory is sup-
ported by grants from the Instituto de Salud Carlos III (ISCIII) (PI20/00306). All
these grants were co-funded by the European Regional Development Fund
(ERDF) ‘‘A way to build Europe’’. A.V.-N. is a recipient of an FPI fellowship
(PRE2019-090341) from the ‘‘Ministerio de Ciencia e Innovacio´n.’’ R.C.-G. is
a recipient of a Juan de la Cierva grant (FJC2021-047282-I) from the Ministerio
de Ciencia e Innovacio´n. C.V.-G. is supported by ‘‘Programa Investigo Comu-
nidad de Madrid’’ (09-PIN1-00009.8/2022) and L.S.-T. is supported by the
PID2021-122780OB-I00 project by the ‘‘Ministerio de Ciencia e Innovacio´n.’’
Currently, L.S.-T. has been awarded an FPU grant (FPU22/02155) from the
‘‘Ministerio de Educacio´n y Formacio´n Profesional.’’
AUTHOR CONTRIBUTIONS
A.V.-N. performed and analyzed most of the experiments and helped to draft
the manuscript. M.J.G.-S. performed experiments and analyzed some of the
data. L.S.-T., C.V.-G., and R.C.-G. performed experiments, designed the fig-
ures, and revised the manuscript. A.O.-R. performed some of the colon
explant experiments. B.S.P. analyzed the localization of ILCs by means of a
confocal analysis. V.Z. performed the experiments of the BM chimeras.on in the colon, which transcriptionally upregulates Hif-1a in ILC3s,
after 3, 7, and 12 days.
d after 12 days infection.
m-infected WT mice after 3, 5, 7, and 12 days.
th IL-2 and TLR2 agonist (Pam3CSK4) for 24 h and with non-stimulus (NS).
or 48 h ex vivo with Pam3CSK4 and IL-2 (S, stimulus) and blocked with the BP
n ofWT EGFP+/
mice incubated ex vivowith or without IL-18 and IL-23 for 6 h.
ubated with or without IL-18 for 6 h.
GFP+/
 and HIF-1a6Rorc EGFP+/
mice incubated ex vivo with or without IL-18
of LPCs from HIF-1a6Rorc or WT mice, unstimulated (NS) or incubated ex vivo
significant when comparing unstimulated ILC3 WT cells with stimulation. Data
–G) independent experiments. Statistical analysis: (A and B) unpaired t test; (D
way ANOVA with Sidak multiple comparisons. n.s., not significant; *p < 0.05,
Cell Reports 42, 113508, December 26, 2023 13
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OPEN ACCESSB.R.-P. analyzed the ILC populations in the colon using the dimensionality
reduction algorithm and assisted in themulti-parameter setting. P.Y.-F. helped
in the sorting of ILC3s and the multiparametric analysis using the Cytoflex cy-
tometer. J.M.G.-G. provided some protocols, expertise, and reagents. J.A.
provided the HIF-1afl/fl Ub-Cre ERT2 mice for the BM chimera experiments
and reviewed themanuscript. A.C.-A. conceptualized the study, designed, su-
pervised, analyzed the experiments, and wrote the manuscript. All authors
contributed to the article and approved the submitted version.
DECLARATION OF INTERESTS
The authors declare no competing interests.
Received: April 18, 2023
Revised: October 24, 2023
Accepted: November 13, 2023
Published: November 28, 2023
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OPEN ACCESSSTAR+METHODSKEY RESOURCES TABLEREAGENT or RESOURCE SOURCE IDENTIFIER
Antibodies
CD45.2 PerCP-Cy 5.5 Mouse anti-mouse Clon:104 BD Biosciences RRID: AB_394528
CD127 Anti-Mouse PE Cy-5 Clone: A7R34 Thermo Fisher Scientific RRID: AB_468792
CD335 (Nkp46) Anti-mouse PE-Cyanine7 Clone: 2941.4 Thermo Fisher Scientific RRID: AB_2573442
CD4 anti-mouse FITC Clone: GK1.5
CD4 anti-mouse Alexa Fluor 700
TONBO biosciences
Thermo Fisher Scientific
RRID: AB2621665
RRID: AB_493999
CD90.2 anti-mouse BV650 Clone: 53–2.1 BD Biosciences RRID: AB_2740170
KLRG1 Anti - Mouse APC-eFLUOR 780 Clon: 2F1 Thermo Fisher Scientific RRID: AB_2573988
CD3 Anti-mouse APC/Cyanine7 Clone:
17A2 Isotype: Rat IgG2b
CD3 Anti-mouse PE-Cyanine 5 Clone: 145-2C11
Biolegend
TONBO biosciences
RRID: AB_2242784
RRID: AB_2621815
CD19 Anti-mouse FITC Clone: 1D3 TONBO biosciences RRID: AB_2621682
CD5 anti-mouse BV421 Clone: 53–7.3 Biolegend RRID: AB_2562173
CCR6 anti-mouse APC Clone:29-2L17 Biolegend RRID: AB_1877147
CXCR6 Anti-mouse PE/Cyanine7 Clone: SA051D1 Biolegend RRID: AB_2721670
Ly-6G and Ly-6C (GR-1) Rat anti-mouse
BV421 Clone: RB6-8C5
BD Biosciences RRID: AB_2737736
F4/80 Rat anti-mouse BV421 Clone: T45-2342 BD Biosciences RRID: AB_2734779
GATA-3 Alexa Fluor 488 Mouse Clone: L50-823
GATA-3 Mouse BV421 Clone: L50-823
BD Biosciences
BD Biosciences
RRID: AB_1645302
RRID: AB_2738152
RORgt Mouse/Anti-Mouse BV421
RORgt Anti-Hu/Mo PE
BD Biosciences
Invitrogen
RRID: AB_2687545
RRID: AB_1834470
EOMES Anti-Mouse PE-eFluor 610 Clone: Dan11mag Thermo Fisher Scientific RRID: AB_2574613
CD16/CD32 (Fc block) Clone. 2.4G2 BD Biosciences RRID: AB_394656
IL-22 anti-mouse PE Clone: 1H8PWSR Thermo Fisher Scientific RRID: AB_10597428
IL7R anti-mouse/rat PE/Cyanine5 Clone: A7R34 Biolegend RRID: AB_1937261
IL-17A Anti- Mouse/Rat APC Clone: eBio17B7 Thermo Fisher Scientific RRID: AB_763580
TCRb anti-mouse BV421 Clone: H57-597 Biolegend RRID: AB_10933263
TCRgd anti-mouse BV421 Clone: GL3 Biolegend RRID: AB_10896753
Rat IgG2a Isotype control Clone: 2A3 BioXcell RRID: AB_1107769
Mouse anti-GFP Roche Applied Science.
Sigma-Aldrich
RRID:AB_390913
Goat anti-Mouse Alexa 488 Thermo Fisher Scientific RRID: AB_2534088
CD3 Monoclonal Antibody (17A2) Thermo Fisher Scientific RRID: AB_467053
CD3 antibody Abcam RRID: AB_305055
B220 anti-mouse Clone: RA3-6B2 StemCell 100–0422
Goat anti-Rat Alexa 647 Thermo Fisher Scientific RRID: AB_141778
Goat anti-rabbit Cy3 Jackson ImmunoResearch RRID: AB_2338009
Chemicals, peptides, and recombinant proteins
Recombinant mouse IL-23 protein (Sf21-derived)
Lot DFXU0720061 > 95% purity
R&D 1887-ML
Recombinant mouse IL-1b protein 1 Stem Cell 78035.1
Recombinant mouse IL-18 protein 10ug (E-coli derived) R&D 9139–IL/CF
Recombinant mouse TL1A protein Recombinant
Mouse TNFSF15 (carrier-free) 10ug
Biolegend 753002
Pam3CSK4 1mg TOCRIS 4633/1
(Continued on next page)
16 Cell Reports 42, 113508, December 26, 2023
Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
Lipopolysaccharides from Escherichia Coli O111:B4 (LPS) Sigma-Aldrich L2630
Brefeldin A Cell signaling 9972S
Stop Golgi BD Biosciences 554724
BlockAidTM Blocking Solution Life technologies B10710
ProLong mounting medium ThermoFisher Scientific P36984
MacConkey agar Oxoid CM007B
Sodium thiosulfate Sigma Aldrich 1.065.122.500
Ammonium citrate Sigma Aldrich RES20400-A7
TRIzol Sigma Aldrich T9424
PCR Power SYBR Green Applied Biosystems 4367659
DAPI Sigma Aldrich 32670
EvaGreen qPCR Mix Plus ROX Solis Biodyne 08-24-0000S
Critical commercial assays
Foxp3/Transcription Factor Staining Buffer Set Thermo Fisher Scientific 00-5523-00
ELISA IL-18 Mouse Uncoated kit Thermo Fisher Scientific 88-50618-88
ELISA IL-22 10 3 96 Tests Thermo Fisher Scientific 88-7422-88
ELISA IL-17 Mouse Uncoated kit Thermo Fisher Scientific 88-8711-88
RNeasy Micro Kit Qiagen 74004
MojoSortTM Mouse CD4 T cell Isolation Kit Biolegend 480033
Experimental models: Organisms/strains
B6-SJL (Ptprca Pepcb/BoyJ) expressing CD45.1 allele Jackson Laboratory #:002014 RRID:IMSR_JAX:002014
B6.129-Hif1atm3Rsjo/J (HIF1aflox) Aragones J’s laboratory- from
Jackson Laboratory
#:007561
RRID:IMSR_JAX:007561
HIF-1afl/fl Ub-Cre ERT2 Aragones J’s laboratory
B6.Cg-Ndor1Tg(UBC
cre/ERT2)1Ejb/1J (WT Ub-Cre ERT2) Aragones J’s laboratory- from
Jackson Laboratory
#:007561
RRID:IMSR_JAX:007001
B6.FVB-Tg(Rorc-cre)1Litt/J (RORgtCre mice) Jackson Laboratory #:022791
RRID:IMSR_JAX:022791
B6.129P2(Cg)-Rorctm2Litt/J (RORgt EGFP mice) Jackson Laboratory #:007572
RRID:IMSR_JAX:007572
HIF-1afl/fl RORgtCre (HIF-1a6Rorc) This paper N/A
B6.129S7-Rag1tm1Mom/J (RAG1KO) Veiga E’s laboratory from
Jackson Laboratory
#:002216
RRID:IMSR_JAX:002216
RAG1KO HIF-1afl/fl RORgtCre (RAG1KO HIF-1a6Rorc) This paper N/A
HIF-1afl/fl RORgtCre EGFP (HIF-1a6Rorc EGFP+/
) This paper N/A
Software and algorithms
CytExpert software Beckham Coulter RRID:SCR_017217
Flowjo software BD Biosciences RRID:SCR_008520
GraphPad Prism software Dotmatics RRID:SCR_002798
OMIQ software Dotmatics N/A
Biorender software Biorender RRID:SCR_018361
Fiji Open Source RRID: SCR_002285
Article
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OPEN ACCESSRESOURCE AVAILABILITY
Lead contact
Further information and requests for resources and reagents should be directed to, andwill be addressed by, the LeadContact, Cruz-
Adalia A (arancruz@ucm.es).Cell Reports 42, 113508, December 26, 2023 17
Article
ll
OPEN ACCESSMaterials availability
The mouse lines generated in the present study will be made available on request by the Lead Contact without restriction.
Data and code availability
d All original data reported in this paper are available from the lead contact upon request.
d This paper does not report original code.
d Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.
EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS
Mice
HIF-1afl/flRORgtCre (HIF-1a6Rorc) mice were generated by crossing B6.129-Hif1atm3Rsjo/J mice with B6.Cg-Ndor1Tg(UBC-cre/
ERT2)1Ejb/1J mice. The Rorcgfp/+ mice9 were crossed with HIF-1a6Rorc to generate the HIF-1a6Rorc EGFP+/
 mice. RAG1KO
mice were crossed with HIF-1a6Rorc mice to generate the RAG1KO HIF-1a6Rorc mice. The Hif-1afloxed-UBC-Cre-ERT2 mice
were generated by crossing B6.129-Hif1atm3Rsjo/J mice with B6.Cg-Ndor1Tg(UBC-cre/ERT2)1Ejb/1J mice, which harbor two
loxP sites flanking exon 2 of the murine Hif-1a and B6.Tg (UBC-Cre/ERT2) mice, which ubiquitously express a tamoxifen-inducible
CRE recombinase (Cre-ERT2). The Hif-1afloxed-UBC-Cre-ERT2mice were kindly provided by our collaborator Dr. Julia´n Aragone´s
(UAM). For Hif-1a gene inactivation, the mice were given ad libitum access to Teklad CRD TAM400/CreER tamoxifen pellets (The
Harlan Laboratory) for 10–15 days or an intraperitoneal injection of 100 mL (20 mg/mL) tamoxifen for 5 days followed by standard
mouse chow diet. Animals were distributed by age, sex, and weight. Females and males were used interchangeably, but within
the in vivo infection experimental groups, individuals of the same sex were consistently grouped for comparison. All mice are of
the C57BL/6 strain and were bred and housed in a climate-controlled environment at the CNB and CIB in accordance with the
sanitary recommendations of the Federation of European Laboratory Animal Science Associations (FELASA). All experimental
procedures were approved by the Animal Care and Ethics Committee of the CNB, CIB, and the Complutense University of Madrid
(UCM) and by the regional authorities (project nº PROEX 146/18 and PROEX 143.6/20).
BM chimeras
BMWT Cre or HIF-1a KO Cre chimeras were generated by transplanting with 53 106 BM cells from the WT Ub-Cre ERT2 CD45.2 or
the HIF-1afl/fl Ub-Cre ERT2 CD45.2 mice, respectively, into lethally g-irradiated (2 doses of 6 Gy) WT C57/BL6 CD45.1 mice. Mixed
BM chimeras were generated by injecting a mixture of 53106 BM cells from the RAG1KO and WT Ub-Cre ERT2 or the RAG1KO and
HIF-1afl/fl Ub-Cre ERT2 mice in a proportion of 1:1 respectively, into lethally irradiated WT C57/BL6 CD45.1 mice. After 4 weeks, the
reconstituted mice were subsequently treated with tamoxifen. BM WT Cre and HIF-1afl/fl KO mice mRNA was isolated from periph-
eral blood to verify the expression of HIF-1a by qPCR. Furthermore, CD4+ T cells from the spleen of the mixed BM chimeras WT Ub-
Cre:RAG1KO andHIF-1afl/fl KO: RAG1KOmice isolated bymouseCD4+ T cell isolation kit were processed to check the expression of
HIF-1a by qPCR. Mixed BM chimeras were generated by injecting a mixture of 103106 BM cells from the CD45.2 HIF-1a6Rorc and
CD45.1 control mice in a proportion of 1:1, into lethally irradiated WT C57/BL6 mice. After 5–6 weeks, the proportion of CD45.1+ and
CD45.2+ was analyzed in blood to decide whether to proceed to infect with C. rodentium.
Citrobacter rodentium-induced colitis
To induce colitis with C. rodentium, mice aged 8–12 weeks were orally gavaged with 1 3 1010 CFU/mL C. rodentium strain kindly
provided by Dr. Eric Vivier. Survival, body weight, CFUs counts, and tissue histology were assessed as previously described.56,57
Analysis of CFUs was determined via serial dilutions on MacConkey agar plates supplemented with 0.68% sodium thiosulfate
and 0.08% ammonium citrate from mechanically homogenized fecal pellets on days 2, 5 and 12 after C. rodentium infection.58
We performed colon explants and collected a piece of the distal colon to quantify cytokines by ELISA and qRT-PCR respectively
on day 12 of the infection. LPCs were isolated for cell sorting, flow cytometry analysis or for being cultured 4 h with brefeldin A to
analyze intracellular cytokines.
METHOD DETAILS
Counting of colonic patches
Colons from infected or uninfected mice were collected, washed, and opened longitudinally. Afterward, they were incubated by
shaking in phosphate buffered saline solution (PBS) containing 10% Fetal Bovine Serum (FBS), 5 mM EDTA, and 14 mM HEPES
at 37C for 30 min. After washing with PBS, CLPs were counted by backlighting.
Isolation of colonic LPCs
The intestinal lamina propria cells were isolated as described by.59 In short, mouse colons were removed and cut longitudinally and
transversally into 1.5 cm pieces, which were incubated by shaking in PBS containing 10% FBS, 5 mM EDTA, and 14 mM HEPES at
37C for 30min. Then, the tissueswere thendigested inRPMI1640containing10%FBS, 25mMHEPES, and300UI/mLof collagenase18 Cell Reports 42, 113508, December 26, 2023
Article
ll
OPEN ACCESStype VII at 37C for 45 min. The digested tissues were homogenized by passing through 70 mm cell strainers. Mononuclear cells were
then harvested from the interphase of a 70% and 40% Percoll gradient after spinning at 2,100 rpm for 20 min at room temperature.
Flow cytometry, antibodies, and ELISA
ILC staining was performed as previously described by.59 Antibodies against CD45.2, CD3, CD19, Gr-1, F4/80, CD90.2, GATA-3,
CD16/CD32, CD127, NKp46, KLRG1, CD4, RORgt, Eomes, IL-17, IL-22, IL-7R, CXCR6, CD5, TCRb, TCRgd, CCR6, RORgt and
IL-17 were used. For cytokine production, cells were stimulated ex vivo by IL-23 (40 ng/mL), IL-1b (100 ng/mL), and Brefeldin A
(5 mg/mL) or Stop Golgi (1:1500) for 4 h. For intracellular staining, cells were fixed and permeabilized with eBioscience Foxp3/
Factor Staining Buffer Set following the manufacturer’s guidelines. For induction of IL-22 through IL-18 stimulation, LPCs were culti-
vated overnight with or without IL-23 (40 ng/mL) and/or IL-18 (100 ng/mL) followed by 4 h of ex vivo Stop Golgi stimulation (1:1500).
Flow cytometry analysis and cell sorting were performed on FACSCAria (BD Biosciences), Cytoflex (Beckham Coulter) and Cytek
Aurora instruments and analyzed with FlowJo, CytExpert or OMIQ softwares. IL-22, IL-17, and IL-18 in supernatants collected at
0, 3, 5, 7, and/or 12 days after infection were measured by ELISA according to manufacturer’s recommendations.
Flow cytometry gating strategy
Th22/Th17 gating strategy: FSC, SSC; singlets, Live Dead
, CD45+, Linage
 (F4/80, CD19, Gr-1), CD3+, CD4+, RORgt+. ILC1 gating
strategy: FSC, SSC; singlets, LiveDead
, CD45+, Linage
, CD3
, RORgt-, Nkp46+, CD127+, Eomes-.NKcells gating strategy: FSC,
SSC; singlets, Live Dead
, CD45+, Linage
, CD3
, RORgt-, Nkp46+, CD127-, Eomes+. ILC2s gating strategy: FSC, SSC; singlets,
Live Dead
, CD45+, Linage
, CD3
, RORgt-, Nkp46-, KLRG1+, GATA-3+. ILC3s Nkp46+ gating strategy: FSC, SSC; singlets, Live
Dead
, CD45+, Linage
, CD3
, CD4
, RORgt+, CD90+, IL7R+, CXCR6+, Nkp46+. ILC3s Nkp46-/Lti gating strategy: FSC, SSC; sin-
glets, LiveDead
, CD45+, Linage
, CD3
, CD4
, RORgt+, CD90+, IL7R+,CXCR6+Nkp46-. Lti CD4+ cells gating strategy: FSC,SSC;
singlets, Live Dead
, CD45+, Linage
, CD3
, RORgt+, CD90+, IL7R+, CXCR6+, Nkp46-, CD4+. Lti cells gating strategy: FSC,
SSC; singlets, Live Dead
, CD45+, Linage
, CD3
, RORgt+, Nkp46-, CCR6+. Non-ILC3 Innate RORgt cells gating strategy: FSC,
SSC; singlets, Live Dead
, CD45+, Linage
, CD3
, CD4
, RORgt+, CD90
, IL7R-, CXCR6-.
Ex vivo organ culture
Colon tissue was harvested from mice, opened longitudinally, and washed in PBS twice. Two biopsy punches (4 mm, Kai medical)
from the distal colon were cultured in 400 mL of complete medium (RPMI supplemented with 100 U/mL penicillin, 100 mg/mL strep-
tomycin and 10% FBS) for 24 or 48 h at 37C. Colons were stimulated with Pam3CSK4 (0.2 mg/mL) + IL-2 (10 U/mL), LPS (1 mm/ML),
IL-1b (10 ng/mL), IL-23 (40 ng/mL), TL1A (200 ng/mL), IL-18 (100 ng/mL) and the BP IL-18 (1 mg/mL).
Isolation of splenic ILC3s
Single-cell suspensions were prepared with spleens from the KO HIF-1a6Rorc EGFP+/
 and the WT EGFP+/
 control mice. Splenic
cells were then stainedwith CD45.2, CD3, CD19, F4/80, andGr-1. The ILC3s population was also isolated according to its expression
of EGFP. Sorted ILC3s were incubated in vitro with or without 40 ng/mL IL-23 and/or 100 ng/mL IL-18.
Histology
Colons were cleaned and cut in their distal part. Tissues were fixed in 4% paraformaldehyde and processed for wax embedding.
Cross sections of the colon (5 mm) were cut, mounted on slides and routinely stained with hematoxylin and eosin (H&E). Colitis
severity was assessed by researchers blinded to sample identity with criteria adapted from X. Wu et al. 2007.60 This assessment
involved a combined score of immune cell infiltration and epithelial damage (crypt loss, hyperproliferation and ulcers). Each param-
eter graded 0-3 adding on a total score of 12.
Immunofluorescence
Colonic tissues were cut longitudinally, cleaned in PBS and formed into a ‘Swiss roll’ and embedded in OCT. Tissues were frozen and
stored at 
80C, before being sliced into 5-mm-thick sections with a cryostat and fixed in PFA 4% for 20 min. Afterward, tissue
sections were permeabilized with PBS containing 0.05% BSA and 0.2% saponin (PBS-Staining buffer) for 30 min at room temper-
ature. After blocking them with PBS-Staining buffer, mice serum (1/100), and Fc block (1/200) for 30 min at 37C, they were rinsed
in PBS and treated with Blockaid for 1 h at room temperature. Subsequently, slides were incubated with anti-EGFP (1/50), anti-CD3
(1/100), and anti-B220 (1/100) for 1 h at 37C, diluted in the PBS-Staining buffer. After rinsing with PBS, they were stained with goat
anti-mouse 488 (1/500), goat anti-rabbit Cy3, and goat anti-rat Ig Alexa 647 in PBS-Staining buffer for 1h 30min at room temperature.
Finally, tissue sections were stained with DAPI (1/500) in PBS-Staining buffer for 30min at 37C, washed in PBS1X andmounted with
ProLong mounting medium. Images were acquired on a Confocal multispectral Leica TCS SP8 system microscope and Confocal
multispectral system Leica STELLARIS 5.
RNA isolation and qPCR
Colonic RNA was purified with TRIzol and reverse transcribed into cDNA. qPCR was performed with QuantStudio 5 Real-time PCR
(Applied Biosystems) with PCR Power SYBR Green or MasterMix qPCR ROX PyroTaq EvaGreen in triplicates. Samples wereCell Reports 42, 113508, December 26, 2023 19
Article
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OPEN ACCESSnormalized to the expression of gene encoding 18S. Primers for Hif-1a, Il-22, Il-18, Slc2a1, Il-17, Il-1b, Il-23p19, Tnf-a, and 18S
(Table S1) were employed.
RNA from sorted splenic ILC3s, cultured with or without 40 ng/mL IL-23 and/or 100 ng/mL IL-18 for 6 h, or from colonic ILC3s of
infected mice, was extracted with a RNeasy Micro Kit and was reversely transcribed to cDNA. The qPCR of the genes IL-22, Il-17,
Slc2a1 andHif-1awas performed in ‘‘Parque tecnolo´gico deMadrid’’, using SYBRGreen Supermix. Samples were normalized to the
expression of gene encoding 18S.
QUANTIFICATION AND STATISTICAL ANALYSIS
All statistical analyses were performed with GraphPad Prism software. To determine significant differences (*p < 0.05) parametric
data were analyzed by two-way ANOVA and 1-way ANOVA followed by Sidak multiple comparisons test, or paired or unpaired
Student t-test. For histoscore, 2-tailed Mann– Whitney U test was used. For survival curves, statistics were calculated with the
log rank (Mantel-Cox) test.20 Cell Reports 42, 113508, December 26, 2023