ABSTRACT Gasdermin C is one of the least studied members of the gasdermin family of proteins, known for their critical involvement in pyroptosis and host defense. Furthermore, evidence for

the role of Gasdermin C in the intestine is scarce and partly controversial. Here, we tested the functional role of Gasdermin C in intestinal homeostasis, inflammation and tumorigenesis. :

We studied Gasdermin C in response to cytokines in intestinal organoids. We evaluated epithelial differentiation, cell death and immune infiltration under steady state conditions in a new

mouse line deficient in Gasdermin C. The role of Gasdermin C was analyzed in acute colitis, infection and colitis-associated cancer. Gasdemin C is highly expressed in the intestinal

intestinal infection and colitis-associated cancer. Gasdermin C genes are upregulated by type 2 immunity, yet appear dispensable for the development of intestinal inflammation, infection and

colitis-associated cancer. SIMILAR CONTENT BEING VIEWED BY OTHERS TRANSCRIPTION FACTOR ELF-1 PROTECTS AGAINST COLITIS BY MAINTAINING INTESTINAL EPITHELIUM HOMEOSTASIS Article Open access 08

March 2025 CD47 IS A NEGATIVE REGULATOR OF INTESTINAL EPITHELIAL CELL SELF-RENEWAL FOLLOWING DSS-INDUCED EXPERIMENTAL COLITIS Article Open access 23 June 2020 CDX2 REGULATES IMMUNE CELL

INFILTRATION IN THE INTESTINE Article Open access 04 August 2021 INTRODUCTION The gasdermins (GSDM) are a superfamily of proteins that currently includes GSDMA, GSDMB, GSDMC, GSDMD, GSDME

(also known as DFNA5) and PJVK (also known as DFNB59). Out of these, GSDMD is the best characterized member of the group. Several members of the GSDM family mediate a form of inflammatory

cell death known as pyroptosis1. In its canonical form, pyroptotic death is mediated by inflammasome assembly, followed by the cleavage of GSDMD by activated Caspase-1. The N-terminal domain

of cleaved GSDMD perforates the cell membrane, forming oligomeric pores. In addition to the secretion of IL-1β and IL-18, the formation of pores allows the entry of water, which causes cell

swelling and osmotic lysis. With the exception of DFNB59, all the proteins in the GSDMs superfamily are characterized by an N-terminal pore-forming domain. However, pyroptosis is not the

only cell death mechanism in which GSDMs are involved. In fact, during the last years, a number of reports have shown that GSDMs can execute other modes of programmed cell death, including

apoptosis and NETosis2. Recently, a role of selected GSDMs in gut homeostasis and intestinal inflammation has also been reported. GSDMD is upregulated in IBD patients, however there are

discrepancies regarding its protective or deleterious effect on intestinal inflammation. In one of these reports, it was shown that GSDMD-derived pores promotes colitis via IL-18 secretion

from IECs3. In striking contrast, a different publication showed that in experimental colitis, GSDMD plays a protective role in macrophages but not in IECs4. Moreover, GSDME deficiency in

nonhematopoietic cells alleviated experimental colitis in mice5. Despite belonging to the GSDM family, GSDMB seemingly plays a rather pyroptosis-independent role. Interestingly, GSDMB acts

by promoting epithelial restitution and repair via FAK phosphorylation6. In the gut, among the Gasdermin family, Gasdermin C (GSDMC) has only recently begun to attract attention. The role of

this protein in health and disease is far from understood. It is noteworthy that, in contrast to the human GSDMC, the mouse genome contains four copies of the _Gsdmc_ gene, arranged in

tandem (_Gsdmc1-4_). Overall, the evidence for the role of GSDMC in the gut is scarce and, in some cases, controversial. Du et al., reported that N6-adenomethylation of _Gsdmc_ is essential

for the survival of mouse colonic Lgr5+IECs via a mechanism involving disruption of mitochondrial membrane potential and cytochrome c release. In support for this role, they also showed that

GSDMC knockdown induces massive apoptosis in human colonic organoids. Hence, the authors propose that GSDMC functions as a critical mitochondrial stabilizer7. In contrast, no change in the

stem cell compartment was reported by Zhao et al. Using mice with a specific deletion of _Gsdmc1-4_ in IECs, the authors detected no differences in the transcriptome of these mice under

steady state conditions, although they presented Gasdermin C as a driver of type 2 inflammation and a critical effector in the response to _Heligmosomoides polygyrus_helminth infection8. In

conclusion, further work is needed to elucidate the role of GSDMs family in the gut. In this study, we comprehensively analyze the role of GSDMC in the gut using newly generated GSDMC

knockout mice and mouse models of intestinal inflammation, infection and tumorigenesis. We observed that Gasdermin C expression is highly induced by the type 2 cytokines IL-4 and IL-13 in a

STAT6 dependent manner. Surprisingly, we observed that in our model, knockout of the four _Gsdmc1-4_ genes did not result in any detectable phenotypes in homeostatic or pathological

conditions that we tested. RESULTS GASDERMIN C ISOFORMS ARE PREDOMINANTLY LOCATED IN THE GUT To characterize the role of the GSDMC family of proteins, we first examined the expression

patterns of the different _Gsdmc_ genes, including _Gsdmc1_, _Gsdmc2_,_ Gsdmc3_ and _Gsdmc4_, in different tissues (Fig. 1A). Surprisingly, we found that of all tested organs, the intestine

exhibited the highest expression of all GSMDC isoforms. Of note, _Gsdmc1_, _Gsdmc2_, and _Gsdmc3_ transcripts, were barely detected in tissues other than the intestine. All _Gsdmc_ genes

exhibit a similar expression along the gastrointestinal tract, with the exception of _Gsdmc4_, which shows higher levels of mRNA in proximal small intestinal segments (Fig. 1B). Concerning

the precise cellular source, the analysis of a single-cell transcriptome of intestinal epithelial cells (IECs) revealed an uneven expression of the _Gsdmc1-4 _genes across the different IECs

subsets (Supplementary Fig. 1A)9. In particular, _Gsdmc1_, _Gsdmc2_, _Gsdmc3_, and _Gsdmc4_ exhibited the highest expression in the enterocyte subset, followed by the enterocyte

progenitors. The presence of GSDMC in IECs was also supported by IHC performed on ileum and colon tissues, using a newly designed antibody raised against GSDMC4 (Fig. 1C-D). The procedure

for the generation of this new antibody is depicted in Supplementary Fig. 1B. We next sought to assess GSDMC levels along the intestinal tract using this newly developed tool. We detected

GSDMC4 in IECs across all sections of the intestine, showing consistent levels throughout (Fig. 1E-F). Having identified IECs as a niche for the expression of the _Gsdmc_ family genes, we

subsequently explored how their expression changes in pathological conditions. For this, we analyzed the expression pattern of the different _Gsdmc_ genes (_Gsdmc1_ was excluded due to low

expression) in response to intestinal inflammation induced by chemical insults (TNBS, Oxazolone) or bacterial/apicomplexean infection (_Helicobacter hepaticus_, _Eimeria vermiformis_,

_Citrobacter rodentium_) (Fig. 1G). A similar trend (downregulation) was observed for all _Gsdmc_ genes in intestinal inflammation triggered by _C. rodentium_ and _H. hepaticus_ infection.

In contrast, intestinal inflammation induced by TNBS instillation was not associated with significant changes in _Gsdmc1-4_ expression. Notably, a distinct pattern emerged in

oxazolone-mediated colitis and _E. vermiformis_ infection. While _Gsdmc3_ and _Gsdmc4_ levels remained unchanged, _Gsdmc2_ showed a marked upregulation. Collectively, these findings suggest

both model-specific modulation of the _Gsdmc_ genes and differences in the regulation of individual _Gsdmc_ genes. Finally, given the preferential expression of the _Gsdmc1-4_ in mature

enterocytes and their progenitors, we hypothesized that strategies that alter the process of IEC differentiation may lead to changes in GSDMC expression. Treatment with DBZ, a known

inhibitor of the γ-secretase complex, results in the inhibition of Notch signaling and, as a consequence, a marked imbalance in IEC differentiation toward the secretory lineage. As expected,

in this context, a reduction in the absorptive lineage was associated with a dramatic decrease in all the GSDMC isoforms, observed at both the mRNA and the protein levels (Supplementary

Fig. 2A-B). THE BASAL EXPRESSION OF _GSDMC1-4 _IS DRIVEN BY TYPE 2 CYTOKINE-INDUCED- STAT6 SIGNALING Given the complexity of the changes we observed in _Gsdmc_ gene expression in the

different scenarios evaluated thus far, our subsequent efforts aimed at uncovering the factors responsible for mediating GSDMC modulation in IECs. For this, we cultured intestinal organoids

and quantified the expression levels of _Gsdmc1-4_ genes. Interestingly, while _Gsdmc1-4_ were detected in freshly isolated IECs from the small intestine, the expression was barely

detectable in small intestinal organoids cultured _in vitro_ for 10 days (Fig. 2A). These data hinted at the potential requirement of soluble factors present in the intestinal milieu for the

basal expression of _Gsdmc1-4_. In a previous report, _Gsdmc1-4_ were identified as target genes of IL-410. We extended this to test the ability of other cytokines to upregulate _Gsdmc1-4_

expression. As depicted in Fig. 2B, of all cytokines tested, IL-4 and IL-13 markedly stimulated the expression of all _Gsdmc_ isoforms. Western blotting confirmed the absence of GSDMC2 and

GSDMC3 under steady state conditions and the remarkable induction of both proteins upon IL-4 and/or IL-13 stimulation in small intestinal organoids (Fig. 2C-D and Supplementary Fig. 4A-B).

In line, this effect was also observed _in vivo_, after increasing the systemic abundance of IL-13 (Fig. 2E-F). Overexpression of IL-13 resulted in increased transcription of intestinal

_Gsdmc1-4_, as shown by qPCR analysis for the different isoforms and immunostaining of GSDMC4 in the colon. Signal transducer and activator of transcription factor-6 (STAT6) is essential for

mediating some of the effects induced by IL-4 and IL-1311. To prove the involvement of STAT6 in the effect induced by type 2 cytokines, we took advantage of small intestinal organoids

generated from STAT6−/− mice. In contrast to the expected upregulation of the _Gsdmc1-4_ observed in STAT6 proficient organoids, the expression remained unaltered in STAT6 deficient

organoids stimulated with IL-4 and IL-13 (Fig. 2G). In addition, basal expression of _Gsdmc1-4_ was already reduced under unchallenged conditions. Altogether, our experiments identify IL-4

and IL-13 as the only cytokines tested, capable of modulating _Gsdmc1-4_ expression via a STAT6-mediated mechanism (Fig. 2H). MICE LACKING THE EXPRESSION OF _GSDMC _GENES ARE VIABLE AND SHOW

NO OVERT PHENOTYPE In order to determine the functional significance of th_e Gsdmc1-4_ genes in intestinal homeostasis and disease, we generated a novel mouse line. Using CRISPR-Cas9

technology, a genomic region containing all 4 Gasdermin C genes were deleted to generate the _Gsdmc1-4__−/−_ mice (Supplementary Fig. 2C). Deletion of the _Gsdmc1-4_ was confirmed by western

blotting, qPCR (Fig. 3A and Supplementary Fig. 4C) and immunostaining (Fig. 3B) in both small intestine and colon tissues. After confirming the successful deletion of all _Gsdmc_ genes, we

next aimed to determine the functional role of the _Gsdmc_ genes in IECs, using small intestinal organoids derived from _Gsdmc1-4__+/+_ and _Gsdmc1-4__−/−_ mice. As mentioned before (Fig. 

2A), the expression of _Gsdmc1-4_ is profoundly diminished in cultured IECs, likely due to the absence of factors produced by non-epithelial cells and present in the intestinal

microenvironment. To achieve a high expression of _Gsdmc1-4 in vitro_, we stimulated the organoids with IL-13 and analyzed the effects of the deletion of _Gsdmc1-4_. Neither _Gsdmc1-4__+/+_

nor _Gsdmc1-4__−/−_ organoids showed significant cell death in response to IL-13 stimulation, as measured by propidium iodide binding to DNA (Fig. 3C). Next, the effect of _Gsmdc1-4_

deletion on organoid development was assessed. The organoids developed a similar size and comparable number of buds were counted in organoids derived from _Gsdmc1-4__+/+_ and _Gsdmc1-4__−/−_

mice (Fig. 3D). Moreover, and in agreement with a normal development, in the presence of IL-13, no differences were detected in EdU incorporation, a surrogate marker for cell proliferation;

as well as in Olfactomedin 4 (OLFM4) and ULEX immunostaining, reflecting normal stem and goblet cell proportions, respectively (Supplementary Fig. 2D-E). To explore if _Gsdmc1-4_ deletion

is associated with more subtle changes, we performed bulk RNA sequencing of intestinal organoids derived from _Gsdmc1-4__+/+_ and _Gsdmc1-4__−/−_ mice. However, ablation of _Gsdmc1-4_ did

not result in significant changes in the transcriptome of IECs (Supplementary Fig. 2F). Collectively, our data reveal that Gasdermin C is dispensable for intestinal organoid growth and

differentiation. We then investigated if GSDMC is involved in the interplay between the epithelial and non-epithelial compartments in intestinal homeostasis _in vivo_. Histological analysis

of the small intestine and colon tissues revealed no visible alterations in tissue morphology (Fig. 3E). In agreement with the normal differentiation and development of organoids, stem cell

population and proliferation as defined by _Lgr5_ expression, OLFM4 and KI67 stainings showed comparable patterns between _Gsdmc1-4__+/+_ and _Gsdmc1-4__−/−_ mice (Fig. 3F-H). To further

evaluate cell differentiation, we immunostained for Mucin2 (MUC2) and Doublecortin Like Kinase 1 (DCLK1), markers of goblet and tuft cells, that is, secretory cell subsets in ileum and colon

tissues. The deletion of the _Gsdmc_ genes did not affect the differentiation of the IECs toward the secretory lineage _in vivo_ (Supplementary Fig. 2G-H). In addition to the epithelium, we

studied the impact of GSDMC on the gut immune compartment. CD45+ cell content in the ileum and colon was indistinguishable from control mice (Supplementary Fig. 2I-J). Due to the known

involvement of the Gasdermin family in cell death and immune homeostasis, we determined the presence of cell death in small intestine and colon tissues from _Gsdmc1-4__+/+_ and

_Gsdmc1-4__−/−_ mice. However, in unchallenged mice, apoptotic cells (positive for cleaved Caspase-3 and TUNEL) and presumed non-apoptotic cell death (only TUNEL positive) were scarcely

detected, independently of the genotype (Fig. 3I). THE GSDMC PROTEINS PLAY NO MAJOR ROLE IN THE CONTEXT OF INTESTINAL INFLAMMATION AND HEALING To uncover the role of the _Gsdmc_ family genes

under pathological conditions, we firstly assessed the effect of _Gsdmc1-4_ gene deletion on intestinal inflammation induced by dextran sulfate sodium (DSS). Interestingly, the levels of

intestinal G_sdmc1-4_ transcripts in wildtype mice are altered during the course of DSS-induced intestinal inflammation (Fig. 4A-C). In particular, the levels of all the _Gsdmc_ genes were

drastically reduced in highly inflamed tissue. In addition, _Gsdmc1-4_ reached levels comparable to healthy tissue only after the full recovery following the cessation of DSS administration

in drinking water (Fig. 4B-C and Supplementary Fig. 4D). Given the dynamic expression of _Gsdmc1-4_ during intestinal inflammation and healing, we challenged _Gsdmc1-4__+/+_ and

_Gsdmc1-4__−/−_ mice with DSS for 5 days followed by a recovery phase of 10 days. Both body weight measurements and endoscopic evaluation revealed comparable levels of inflammation and

healing progression (Fig. 4D-E). In support of the macroscopic observation, the comparison of both groups by histology and KI67 staining, a surrogate marker of proliferation, yielded no

differences (Fig. 4F) and neither did the analysis of inflammatory and IECs markers in colon tissue (Fig. 4G). These results show that intestinal inflammation induced by DSS and the

subsequent healing process are not affected by the expression of _Gsdmc1-4_. GSDMC IS NONESSENTIAL FOR _N. BRASILIENSIS _CLEARANCE Gasdermin C has been implicated in type 2 immune responses

against helminthic parasites8. Infection with the nematode _N.brasiliensis _induces a pronounced type 2 response, characterized by massive expansion of Th2 cells in the lung and the small

intestine. IL-4 and IL-13, produced by ILC2s, play a critical role for timely worm expulsion and tissue repair12. Our own experiments suggest that _Gsdmc1-4_ are target genes of Th2

cytokines and, in addition, a recent publication describes the _Gsdmc_ family of genes as an important effector of anti-helminth immunity against another parasite, the helminth

_Heligmosomoides polygyrus_8 _._ Similar to _H. polygyrus_, _N. brasiliensis_ infection induced a marked upregulation of the _Gsdmc1-4_ transcripts (Fig. 4H-I). Furthermore, similar to _H.

polygyrus_ infection, the increased expression occurred in parallel with the detection of the cleaved form of GSDMC (Fig. 4J and Supplementary Fig. 4E). These data prompted us to investigate

whether _Gsdmc_ family genes might be important mediators in the response against the nematode _N. brasiliensis_ (Fig. 4K). However, 8 days post _N. brasiliensis_-infection, microscopic

damage and worm expulsion, determined by counting adult worms in the small intestine, were comparable in _Gsdmc1-4__+/+_ and _Gsdmc1-4__−/−_ mice (Fig. 4L-M). In addition, both mouse strains

had similar numbers of _N. brasiliensis_ eggs in their feces (Fig. 4N). Nematode infection induces goblet cell hyperplasia and augmented mucus secretion13. To assess both outcomes provoked

by the nematode infection, we analyzed goblet cells and mucus production in infected _Gsdmc1-4__+/+_ and _Gsdmc1-4__−/−_ mice. However, MUC2 production and goblet cell markers were

comparable between _Gsdmc1-4__+/+_ and _Gsdmc1-4__−/−_ mice (Fig. 4O-P). Similarly, the mRNA levels of the type 2 cytokines (_Il4_,_ Il5_ and _Il13_), and the alarmins _Il25_ and _Il33_ were

similar (Fig. 4P). Overall, our experiment revealed that GSDMC is dispensable for the inflammatory response and worm expulsion during _N. brasiliensis_ infection. THE _GSDMC _FAMILY OF

GENES ARE DISPENSABLE IN AN EXPERIMENTAL MODEL OF COLITIS-ASSOCIATED COLORECTAL CANCER Finally, we explored the impact of modulating the _Gsdmc_ genes in the AOM-DSS model, the standard

animal model for colitis-associated colorectal cancer. We immunostained for GSDMC4 in AOM-DSS-induced colon tumors. Interestingly, while GSDMC4 was present in IECs in normal mucosa, its

expression was notably diminished in tumor tissue (Supplementary Fig. 3A). Accordingly, western blotting confirmed the downregulation of GSDMC2 and GSDMC3 in tumors compared to normal mucosa

(Supplementary Fig. 3B and Supplementary Fig. 4F). To investigate the functional involvement of GSDMC in intestinal tumorigenesis, we utilized the AOM-DSS model of colitis-associated

colorectal cancer in _Gsdmc1-4__+/+_ and _Gsdmc1-4__−/−_ mice. As observed in acute DSS colitis, body weight was comparable in response to DSS administration (Supplementary Fig. 3C). At the

end of the experiment, tumor number and size (Supplementary Fig. 3D-G) were measured at the macroscopic and microscopic level. The analysis of these parameters showed no differences between

_Gsdmc1-4__+/+_ and _Gsdmc1-4__−/−_ mice. Altogether, our experiment reveals that GSDMC does not play a predominant role in inflammation-dependent intestinal tumorigenesis. DISCUSSION In

recent years, a growing interest in the GSDMs family of proteins has led to a remarkable progress in the mechanistical understanding of pyroptotic cell death and the discovery of novel

physiological functions of their cleaved forms. In our study, we show that the _Gsdmc_ family genes are predominantly expressed along the intestine. In order to study the role of GSDMCs in

intestinal homeostasis and disease, we have developed a new mouse line that is deficient in all _Gsdmc_ family genes. Although there has been increasing interest in GSDMC in recent years,

the current scientific literature on the role of GSDMC in the gut is scarce, inconclusive and even partly contradictory. Notably, there are striking discrepancies regarding the role of GSDMC

in cell death and survival, both in steady state and during intestinal tumorigenesis. A recent publication showed that GSDMC knockdown in human colonic organoids led to impaired organoid

growth and massive apoptosis. Detailed analysis of the organoids revealed an abolished LGR5 expression and the induction of pro-apoptotic pathways. Underscoring the importance of GSDMC in

the gut epithelium, the authors described how GSDMC is required for Lgr5+stem cell survival and colon homeostasis7. In striking contrast, Zhao et al., reported no alterations in the

intestinal epithelium of GSDMC-deficient mice8. The results of our own mouse model are consistent with the latter report, as no evidence of increased cell death was observed under steady

state conditions. Furthermore, our own data revealed no effect of GSDMC for organoid development or epithelial cell death _in vitro_. This discrepancy may highlight differences between human

and mouse GSDMC. In fact, while there is only one _GSDMC_ gene in humans, the mouse genome contains four different _Gsdmc_ genes14. However, the differences in regulation and function

between human and mouse GSDMC require further investigation. In close association with its role during cell death, the available evidence points to a controversial effect of GSDMC in

intestinal tumorigenesis. Interestingly, GSDMC is upregulated in mouse and human colorectal cancer15. Using cancer cell lines, including colon cancer, Zhang et al., reported that

GSDMC-induced pyroptosis mediates the antitumor effect of α-Ketoglutarate, an essential metabolite in the tricarboxylic acid cycle16. Along the same lines, Xi et al., described that the

overexpression of _Gsdmc2_ in HEK293 cells induced robust pyroptosis. Interestingly, the enhanced lytic cell death was exclusive to _Gsdmc2_, as the other GSDMC tested, _Gsdmc4_, was

ineffective10. Surprisingly however, not only did Miguchi et al., report the opposite effect, that is, no GSDMC-mediated cell death, they also observed that silencing _GSDMC _in human

colorectal cancer cell lines significantly reduced cell proliferation _in vitro_ and in tumor growth in a xenograft model _in vivo_15. Accordingly, the authors suggested that GSDMC functions

as an oncogene, rather than a cell death inducer, suggesting it as a promising therapeutic target15. Using an established model of colorectal cancer in mice, namely colitis-associated

colorectal cancer (AOM-DSS), we observed further discrepancies with the aforementioned reports. First, we detected a marked downregulation of all _Gsdmc_ genes in intestinal tumorigenesis.

In addition, and in contrast to the reported effect in human cell lines, the deletion of the _Gsdmc1-4_ did not alter tumor initiation or development in our experiments. Thus, our studies in

this model do not support an important role for GSDMC in proliferation or cell death _in vivo_ or in other mechanisms that alter tumor growth. The AOM-DSS model of colorectal cancer offers

the advantage of recapitulating the tumor microenvironment and immune interactions more accurately than xenograft models or _in vitro_ models. Collectively, no clear picture has emerged so

far of whether GSDMC might promote or suppress cancer development. In view of the conflicting results, it is tempting to speculate that several factors, such as the tissue and cell type

investigated, the species (human vs. mouse) or tumor stage make it difficult to draw conclusions about the role of GSDMC in cancer. In the specific case of colorectal cancer, we cannot

exclude the possibility that GSDMC only exerts a tumor-promoting effect in advanced stages. GSDMC has been reported to be involved in type 2 immune responses and intestinal infection and

inflammation. As shown by others8,10 and our own research, _Gsdmc1-4_ are target genes of the type 2 cytokines IL-4 and IL-13 via STAT6 dependent activation. Type 2 cytokines play a key role

in regulating immunity against helminth parasites. During parasitic infection, IL-4 and IL-13 induce the _Gsdmc1-4 _expression in the intestine. It has been hypothesized that cleaved GSDMC

forms pores in IECs through which cytokines and antiparasitic factors are released8,10. Consistent with the role of GSDMC in anti-helminth response, one study reported that the specific

ablation of _Gsdmc1-4_ genes in intestinal epithelial cells resulted in increased worm burden after infection with _Heligmosomoides polygyrus_. The mechanism proposed in this publication

involved the GSDMC pores in the unconventional secretion of IL-33. This effect appeared to be specific to parasitic infection, as the course of acute intestinal inflammation induced by DSS

was not affected8,17. Consistent with this, no differences in DSS-induced colitis were observed in our newly developed mouse line deficient in GSDMC. However, in sharp contrast to the

aforementioned study, our own studies using the type 2 helminth infection model _Nippostrongylus brasiliensis_, found no changes in the levels of inflammation, IEC markers, mucus production

and worm burden in GSDMC-deficient mice. Thus, GSDMC does not seem to be essential for type 2 driven anti helminth immunity. Both parasites differ in their life cycle and, more importantly,

in the duration of infection and induction of the granuloma response. While _H. polygyrus_ establishes a stable, long-lasting infection with a strong intestinal granuloma response, _N.

brasiliensis _infection involves extraintestinal stages and resembles an acute infection that develops without intestinal granuloma formation18. Whether GSDMC is only relevant in chronic and

more dramatic phenotypes remains to be investigated. Collectively, our data confirm that GSDMC in mice is strongly induced by the type 2 cytokines IL-4 and IL-13 via STAT6 signaling.

However, our data show that GSDMC is dispensable for immune homeostasis of the gut in steady state and also in models of intestinal infection, inflammation and cancer development, despite

being strongly expressed in the intestinal epithelium _in vivo_. In agreement with the data from Zhao et al., our data do not support an overt effect of GSDMC on epithelial homeostasis,

contradicting previous reports by Du et al., In conclusion, the field of GSDMC research is still in its infancy and the differences in the results obtained suggest a complex regulation of

GSDMC, with a number of factors influencing their functions. Further investigations are warranted to help clarify the nature of GSDMC regulation and function in the intestine and related

diseases. METHODS MICE _Gsdmc1-4__−/−_ mice (B6-_Gsdmc1-4_-tm1Agb/J) were generated using CRISPR/Cas9 technology to introduce Cas9/guide RNA (gRNA) ribonucleoproteins into mouse embryos

(Applied Stem Cell). Only heterozygous mice were used for breeding. Males and females littermates were used in all the experiments. Mice were maintained in individually ventilated cages

under conditions of consistent humidity (40–60%) and temperature (18–23 °C), with an equal light: dark cycle (12 h), and had _ad libitum_ access to rodent chow diet and drinking water. In

the dextran sulfate sodium (DSS) experiment, mice were given 2.5% DSS in drinking water for 5 days, followed by a 10 day recovery period. Mini-endoscopy was performed to assess the recovery

after DSS challenge. The γ-secretase-specific inhibitor DBZ was administered intraperitoneally (i.p.) at a concentration of 30 µmol/kg/day, diluted in 0.5% hydroxyethylcellulose, for 7

consecutive days, as described before19. Wildtype animals used in this study were procured from Janvier Labs. Mice were sacrificed on day 8 after the first dose. _Nippostrongylus

brasiliensis_ L3 were recovered from the faeces of infected rats, mixed with activated charcoal, and cultured in humidified chambers at room temperature. After extensive washing in sterile

0.9% saline (37 °C), 500 larvae were injected subcutaneously into mice in 200 µl of saline. Mice were provided with water containing antibiotics (Borgal 24%, Virbac) for the first 7 days. In

the Azoxymethane (AOM)/DSS model, _Gsdmc1-4_+/+ and _Gsdmc1-4_-/-mice were injected i.p. once with AOM (10 mg/kg, Sigma-Aldrich) followed by three cycles of DSS (1.5% (w/v)) for 5 days,

with an interval of two weeks in between. Mini-colonoscopy was performed and tumor number and size (mm) were analyzed in the colon and categorized as previously described20. _In vivo_

expression of IL-13 was performed as described previously21. Wildtype animals used in this study were procured from Janvier Labs. Mice were sacrificed twelve days after vector

administration. The institutional review board and the ethics committee of the University of Erlangen-Nürnberg and the ethics commission of Lower Franconia approved animal experiments. All

experiments were performed in accordance with relevant guidelines and regulations and ARRIVE guidelines. Mice were euthanised by cervical dislocation under isoflurane anaesthesia. IECS

ISOLATION Small intestines were dissected, cut longitudinally and divided into small fragments. The fragments were incubated at 37 °C with 2mM EDTA. After 15 min, the fragments were passed

through a 70 μm mesh filter and the supernatant obtained was centrifuged. The recovered cells were used for RNA preparation. SMALL INTESTINAL ORGANOIDS Small intestinal organoids were

generated as previously described22. In brief, intestinal crypts were isolated from mouse small intestine and resuspended in Matrigel (Corning). After solidification of the 3D-dome, cells

were cultured in Advanced DMEM/F12 (Thermo Fisher Scientific) supplemented with glutamine (2 mM, Invitrogen), HEPES (10mM, Sigma-Aldrich), Penicillin-Streptomycin (100 U/mL and 100 µg/mL),

R-spondin and Noggin conditioned medium, B27 (5x, Gibco), N-acetylcystein ( 1mM, Sigma-Aldrich), Primocin (100 µg/mL, Invivogen) and EGF (50 ng/mL, Immunotools). Organoids from wildtype (WT)

and STAT6−/− mice were stimulated with IL-13 or IL-4 (50 ng/ml, Peprotech) for 24 h. WT organoids were stimulated with IL-1β (50 ng/ml, Immunotools), IL-4 (50 ng/ml, Peprotech), IL-6 (50

ng/ml, Immunotools), IL-10 (50 ng/ml, Biolegend), IL-13 (50 ng/ml, Peprotech), IL-17A (50 ng/ml, Immunotools), IL-22 (100 ng/ml, Invitrogen), IL-33 (50 ng/ml, Biolegend), TGF-β (50 ng/ml,

Immunotools), TNF (50 ng/mL, Immunotools) and IFN-γ (50 ng/ml, eBioscence) for 24 h. IMMUNOHISTOCHEMISTRY (IHC) Formalin-fixed paraffin-embedded tissue sections were deparaffinized.

Heat-induced antigen retrieval was performed in Tris-EDTA buffer, tissue sections were incubated with the following primary antibodies: GSDMC4 (1:200, GeneScript), KI67 (1:200, ab16667,

Abcam), MUC2 (1:200, NBP1-31231, Novus), DCLK1 (1:200, ab31704, Abcam), OLFM4 (1:200, 39141, CST), F4/80 (1:200, 70076, CST), Cleaved Caspase-3 (1:200, 9661, CST). CD4 staining was performed

on cryosections (1:200, 553043, BD Bioscience). TUNEL staining was performed using the In Situ Cell Death Detection Kit, TMR red (Roche). Nuclei were counterstained with Hoechst 33342

(1:1000, Invitrogen). Small intestinal organoids were fixed in 4% PFA, permeabilized with 0.1% Triton X-100 and then incubated with _Ulex Europaeus_ Agglutinin I (UEA-1; 1:750, FL-1061,

Vector) or the primary antibody OLFM4. Nuclei were counterstained with Hoechst 33342. Incorporation of EdU into DNA in tumor organoids was measured using EdU Proliferation Kit (iFluor 488,

Abcam). Immunofluorescence images were acquired using the Leica DMI6000 B inverted fluorescence microscope (Leica Microsystems) or the Leica laser-scanning confocal microscope. GENE

EXPRESSION ANALYSIS Total RNA was extracted from small intestinal organoids, tissue and IECs using a RNA isolation kit (NucleoSpin kit, Macherey Nagel), following the manufacter’s protocol.

cDNA was obtained by reverse transcription using SCRIPT cDNA Synthesis Kit (Jena Bioscience). Real-time PCR was performed using specific QIAGEN QuantiTect Primer Assays. For normalisation,

_Hprt_ was used as a housekeeping gene. TRANSCRIPTOME META-ANALYSIS For analyzing the expression of _Gsdmc1-4_ and _S100a8_ in the different stages of colitis we used the European

Bioinformatics Insitute ArrayExpress, through which the publicly available dataset E-MTAB-9850 was obtained. MRNA SEQUENCING Following RNA extraction and quality assessment, the samples

underwent sequencing on an Illumina Novaseq 6000 platform, producing paired-end reads. The sequences were then aligned to the reference genome using STAR (version 2.7.0d) and quantified with

featureCounts (version 1.6.4). Differential expression analysis between sample groups was conducted with DESeq2 (version 1.24.0). Further analyses, including enrichment and clustering, were

carried out using bioinformatics tools. WESTERN BLOT Tissue and small intestinal organoids were homogenized in Lysis buffer: M-PER for organoids; and T-PER for tissue (Thermo Scientific),

with Pierce protease and phosphatase inhibitor Mini Tablets (Thermo Scientific) and Phenylmethylsulfonyl fluoride (PMSF, Roche) in both cases. Homogenates were centrifuged at 14000_g_ for 20

 min at 4 ⁰C. Protein concentration was determined by Bradford assay. Samples were boiled for 5 min in LDS sample buffer 4x (Invitrogen), separated by SDS–PAGE using a MiniProtean-TGX gel

(4–15% polyacrylamide; Bio-Rad), blotted onto nitrocellulose membranes (Bio-Rad), and probed with the following antibodies: GSDMC2 + 3 (1:1000, ab229896, Abcam), β-actin-HRP (1:10000,

ab49900, Abcam), and GAPDH (1:5000, 2118, CST). Blots were incubated with the HRP-conjugated anti-rabbit secondary antibody. STATISTICAL ANALYSIS Data were analyzed by Student’s t test,

One-Way ANOVA and Two-Way ANOVA using GraphPad Prism. Significance levels are indicated as *_p_ < 0.05, **_p_ < 0.01 and ***_p_ < 0.001. All data are presented as mean values ± SD.

DATA AVAILABILITY Data are available in a public, open access repository. Data are available on reasonable request. The publicly available datasets used in this study are published in Array

Express service of the Molecular Biology Laboratory–European Bioinformatics Institute under accession number: E-MTAB-9850

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https://doi.org/10.1038/s41556-021-00708-8 (2021). Article  PubMed  CAS  Google Scholar  Download references FUNDING This work was Funded by the Deutsche Forschungsgemeinschaft (DFG, German

Research Foundation) - TRR 241–375876048 (A03, B05, C04 and Z03), CRC1181 (C05), CRU5024 (A03) and individual grants under project numbers 418055832 and 510624836. The Interdisciplinary

Center for Clinical Research (IZKF: J68, A76, J96, A93) also supported this project. A.N.H is supported by a Lichtenberg fellowship and “Corona Crisis and Beyond” grant by the Volkswagen

Foundation, a BIH Clinician Scientist grant and German Research Foundation 375876048-DFG-TRR241-A05 and INST 335/597-1, as well as with the ERC-StG “iMOTIONS” grant (101078069). Open Access

funding enabled and organized by Projekt DEAL. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Medicine 1, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU) and

Universitätsklinikum Erlangen, Erlangen, Germany Reyes Gámez-Belmonte, Yara Wagner, Mousumi Mahapatro, Ru Wang, Lena Erkert, Miguel González-Acera, Jay V. Patankar, Markus F. Neurath, Stefan

Wirtz & Christoph Becker * Department of Gastroenterology, Infectiology and Rheumatology, Charité Universitätsmedizin, Berlin, Germany Roodline Cineus & Ahmed N. Hegazy * Deutsches

Rheumaforschungszentrum Berlin (DRFZ), An Institute of the Leibniz Association, Berlin, Germany Roodline Cineus, Saskia Hainbuch & Ahmed N. Hegazy * Institute of Immunology,

Ludwig-Maximilians-Universität München, 80336, München, Germany David Voehringer * Department of Infection Biology, University of Erlangen, 91054, Erlangen, Germany David Voehringer * The

Transregio 241 IBDome Consortium, Berlin, Germany Ahmed N. Hegazy, Markus F. Neurath, Stefan Wirtz & Christoph Becker * Deutsches Zentrum Immuntherapie (DZI), Erlangen, Germany Markus F.

Neurath, Stefan Wirtz & Christoph Becker * Department of Medicine 1, University Medical Centre Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany Christoph Becker

Authors * Reyes Gámez-Belmonte View author publications You can also search for this author inPubMed Google Scholar * Yara Wagner View author publications You can also search for this author

inPubMed Google Scholar * Mousumi Mahapatro View author publications You can also search for this author inPubMed Google Scholar * Ru Wang View author publications You can also search for

this author inPubMed Google Scholar * Lena Erkert View author publications You can also search for this author inPubMed Google Scholar * Miguel González-Acera View author publications You

can also search for this author inPubMed Google Scholar * Roodline Cineus View author publications You can also search for this author inPubMed Google Scholar * Saskia Hainbuch View author

publications You can also search for this author inPubMed Google Scholar * Jay V. Patankar View author publications You can also search for this author inPubMed Google Scholar * David

Voehringer View author publications You can also search for this author inPubMed Google Scholar * Ahmed N. Hegazy View author publications You can also search for this author inPubMed Google

Scholar * Markus F. Neurath View author publications You can also search for this author inPubMed Google Scholar * Stefan Wirtz View author publications You can also search for this author

inPubMed Google Scholar * Christoph Becker View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS Study concept, design, literature search,

experiments, analysis, interpretation of data, critical revision of the manuscript, manuscript drafting: RG-B and CB. Experimentation and analysis: RG-B, YW, MM, RW, LE, MG-A, RC, SH, JVP,

ANH. Material support: DV. Intellectual contributions during manuscript editing, acquisition of funds and supervision: MFN, SW and CB. CB acts as guarantor. All authors reviewed the

manuscript. CORRESPONDING AUTHOR Correspondence to Christoph Becker. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER’S

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Mahapatro, M. _et al._ Intestinal epithelial Gasdermin C is induced by IL-4R/STAT6 signaling but is dispensable for gut immune homeostasis. _Sci Rep_ 14, 26522 (2024).

https://doi.org/10.1038/s41598-024-78336-z Download citation * Received: 19 June 2024 * Accepted: 30 October 2024 * Published: 03 November 2024 * DOI:

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currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative KEYWORDS * Intestinal homeostasis * Gut pathology * Gasdermin