Mmp17-deficient mice exhibit heightened goblet cell effector expression in the colon and increased resistance to chronic Trichuris muris infection

Intestinal epithelial homeostasis is maintained by intrinsic and extrinsic signals. The extrinsic signals include those provided by mesenchymal cell populations that surround intestinal crypts and is further facilitated by the extracellular matrix (ECM), which is modulated by proteases such as matrix metalloproteinases (MMPs). Extrinsic signals ensure an appropriate balance between intestinal epithelial proliferation and differentiation. This study explores the role of MMP17, which is preferentially expressed by smooth muscle cells in the intestine, in intestinal homeostasis and during immunity to infection. Mice lacking MMP17 expressed high levels of goblet-cell associated genes and proteins, such as CLCA1 and RELM-β, which are normally associated with immune responses to infection. Nevertheless, Mmp17 KO mice did not have altered resistance during a bacterial Citrobacter rodentium infection. However, when challenged with a low dose of the helminth Trichuris muris, Mmp17 KO mice had increased resistance, without a clear role for an altered immune response during infection. Mechanistically, we did not find changes in traditional modulators of goblet cell effectors such as the NOTCH pathway or specific cytokines. We found MMP17 expression in smooth muscle cells as well as lamina propria cells such as macrophages. Together, our data suggest that MMP17 extrinsically alters goblet cell maturation which is sufficient to alter clearance in a helminth infection model.


Introduction
The intestinal tract is lined by a single layer of intestinal epithelial cells.This intestinal epithelium comprises multiple cell types, each with distinct functions, that work together to provide a barrier against harmful microorganisms, while at the same time allowing for nutrient and water absorption (1).To fulfill those functions the intestinal epithelium is constantly replenished by a pool of LGR5+ stem cells located at the base of epithelial invaginations known as intestinal crypts, with a turnover rate of 3-5 days (2,3).This remarkable regenerative capacity enables the intestinal epithelium to rapidly respond to injury or infection.
Goblet cells play a critical role in maintaining the intestinal homeostasis by producing key components of the mucus layer, such as MUC2, CLCA1, and FCGBP (4)(5)(6)(7).The mucus serves as a physical barrier, preventing the attachment and penetration of bacteria (8) and other pathogens, including helminths (9).Additionally, the mucus contains proteins with antimicrobial properties like RELM-b and ANG4, which further bolsters the gut's innate immune defense (10)(11)(12)(13)(14).In response to infections both the amount of mucus and its composition can be altered, with goblet cell specific antimicrobial proteins (AMPs) increasing due to immune-cell derived cytokines that directly induce signaling in intestinal epithelium (15).In fact, an increase in goblet cell numbers is crucial in the defense against pathogenic helminths (9) and during bacterial infections.
To study gut immunity to infection, we use the murine model pathogens Citrobacter rodentium and Trichuris muris.The Gram negative bacterium C. rodentium models human enteropathogenic and enterohemorrhagic Escherichia coli as well as other intestinal diseases (16).C. rodentium colonizes the distal colonic mucosa and is cleared through type 3 immunity (16).T. muris, is a parasitic helminth that colonizes the cecum and proximal colon (17).In C57Bl/6 animals, depending on the infection dose T. muris causes an acute (high dose) or chronic (low dose) infection, which is characterized by a type 2 and type 1 immune response, respectively.
Epithelial homeostasis depends on a niche created by gradients of factors to preserve stemness at the base of crypts while enabling the differentiation of mature and functionally diverse cell types towards the lumen.Critical niche factors include WNTs, R-spondins, Bone morphogenic proteins (BMPs), and prostaglandins, which are expressed by mesenchymal cell subtypes and immune cells such as macrophages that are present in the lamina propria between the epithelium and the smooth muscle layers (18)(19)(20)(21)(22)(23)(24).
In addition to secreted factors, the structure and composition of the extracellular matrix (ECM) within the lamina propria also regulates the intestinal niche (25).Matrix metalloproteinases (MMPs) are a key group of ECM regulators, modifying the structure and function of the ECM both through direct action on ECM components and by cleaving ECM associated-molecules, such as growth factors (26).MMPs have been implicated in wound healing, (chronic) inflammation, and cancer (27)(28)(29).We recently demonstrated that the membrane-bound MMP, MMP17 (also called MT4-MMP) is important in maintaining epithelial homeostasis and its ability to respond to injury.Furthermore, we identified the intestinal smooth muscle as a source of niche factors (30).
In the present work, we show that the loss of Mmp17 induces goblet cell effector genes in the colon and cecum epithelium.We propose that these changes to the epithelium result in improved clearance of low-dose T. muris infection.

Mmp17 KO mice show increased expression of goblet cell-effectors in proximal colon goblet cells
We recently demonstrated that mice lacking the membranebound matrix metalloproteinase MMP17 display impaired intestinal regenerative capacities in both a chemically-induced colitis and whole-body irradiation injury models.In our previous study, we conducted transcriptomic analysis of colon crypts isolated from naïve Mmp17+/+ (WT) and Mmp17-/-(KO) mice using bulk RNA sequencing (RNA-seq) (30).We re-examined this dataset and identified several goblet cell-associated genes among the most upregulated genes in crypts derived from KO animals, particularly Ang4, Clca1, Retnlb, and Muc2 (Figure 1A).Retnlb, Clca1, and Ang4 can be classified as AMPs, which are typically upregulated in the intestine during infections.Immunofluorescence (IF) staining of RELM-b, the protein encoded by the Retnlb gene, confirmed increased protein levels and revealed a distinct pattern of RELM-b positive cells in the proximal region of the colon (Figure 1B).Similarly, we observed a higher fluorescent intensity in KO tissue compared to WT tissue in the proximal colon when stained for CLCA1 (Figure 1C).Examination of other AMP genes, including Sprr2a1, Sprr2a2, and Sprr2a3, showed slightly increased levels, albeit not significant (Supplementary Figure 1A) (31).RNA levels of the core mucus protein Fcgbp were also significantly elevated in KO epithelium (Supplementary Figure 1B).The upregulation in goblet cell-associated genes in KO animals did not correlate with an increased number of this cell type, as demonstrated by the quantification of total goblet cell numbers per crypt in WT and KO mouse colon using alcian blue staining (Figure 1D).Accordingly, we detected no changes in the expression of the transcription factors Atoh1 and Spdef (Figure 1E), which are involved in NOTCH signaling to regulate goblet cell differentiation.Since our previous data indicated that MMP17 is not expressed in the intestinal epithelium, we sought to determine whether the increase in epithelial goblet cell genes was intrinsic to the epithelium or due to changes in the surrounding niche in vivo.We isolated colonic crypts from WT and KO mice to cultivate colon organoids (colonoids), measured Retnlb expression via qPCR (Figure 1F) and assessed RELM−b protein expression using IF staining (Figure 1G).We observed no differences in RELM-b protein and Retnlb mRNA expression, suggesting that the increased expression of goblet cellassociated genes in KO mouse crypts is not intrinsic to the intestinal epithelium but rather a consequence of the environment in the surrounding niche.In summary, we find that animals lacking MMP17 exhibit elevated levels of goblet cell effector proteins without an increase in goblet cell numbers, that are presumably induced by extrinsic factors in the proximal colon.

Goblet cell effector upregulation is not caused by a change in cytokine levels
Ang4, Clca1, and Retnlb are goblet-cell associated genes that are induced during immune responses, such as bacterial and helminth infections, and are typically stimulated by type 2 and 3 immune cytokines such as IL-4/IL-13 (type 2) and IL-22 (type 3) (10, 11, 14, 15, 32).To investigate whether naïve KO animals inherently exhibit elevated cytokine levels, we measured levels of IL-13, IL-22, and IFN-g in re-stimulated mesenteric lymph node lymphocytes (mLNL) in vitro and homogenized colon tissue using ELISA (Figure 2A).We also isolated mRNA from whole proximal colon tissue and measured gene expression of the same cytokines (Figure 2B).Our analysis revealed no significant differences in the levels of IL-13 and IL-22 protein (mLNL, colon tissue) or gene expression (proximal colon tissue) between WT and KO mice.Of note, in all cases, we did observe a subtle increase in the levels of IFN-g/Ifng, but only with a significant elevation in colon tissue (Figures 2A, B).While IFN-g is not known to induce goblet cell effectors or their differentiation in intestinal epithelium (15), it has been reported that RELM-b can stimulate IFN-g production in Tcells (33).Therefore, we propose that the modest increase in IFN-g levels may be a result of RELM-b upregulation rather than the inverse.To further rule out the possibility that the observed increase in goblet cell effector proteins was due to IL-4/IL-13 or IL-22 signaling, which are known to induce such effectors, we also analyzed the levels of phosphorylated (p)STAT6 and pSTAT3, which are downstream signaling molecules of IL-4/IL-13 signaling and IL-22 signaling respectively.We observed no significant difference in protein levels of pSTAT6 (Figures 2C, E) or pSTAT3 (Figures 2D, E) between WT and KO colon crypts, thus excluding IL-4/IL-13 and IL-22 signaling as the cause for the altered epithelial expression.In conclusion, the heighted levels of goblet cell effectors in Mmp17 KO mice are not the result of a heightened level of cytokines during homeostasis.

Increased expression of goblet cell effector proteins does not influence the course of Citrobacter rodentium infection
Goblet cell effector proteins, which are secreted into the intestinal lumen as part of the mucus layer, form a crucial line of defense against pathogens in the intestine.We hypothesized that elevated levels of the effectors we observe in mice lacking MMP17, might offer an advantage in clearing intestinal infections, as both ANG4 and RELM-b have been described to have anti-bacterial properties (11,14).Consequently, we infected WT and KO mice with C. rodentium, a gram-negative bacterial pathogen that primarily infects the colon, causing mild colitis and diarrhea.Generally, mice with a C57BL/6 background exhibit resistance to C. rodentium and can clear the pathogen in 2 to 4 weeks, depending on the infectious dose (34,35).Both WT and KO-infected mice cleared the infection between 11-20 days.Although the average duration for clearance was shorter in KO mice (14.7 dpi) compared to WT mice (16.7 dpi), these differences were not significant throughout infection (Figure 3A, Supplementary Figure 2A).Furthermore, we observed equal peak bacterial burdens at day 12 post-infection, averaging at 10 8 colony-forming units (CFU) per gram (g) of fecal matter for all mice (Figure 3B).We also did not observe differences in bacterial burden in liver or spleen (Supplementary Figure 2B).The immune response to C. rodentium is characterized by the induction of IL-22 and IL-17A which contribute to the clearance (36).We noted similar levels of IL-22 and IL-17A in both WT and KO animals at day 12 indicating there was no difference in mounting a type 3 response (Figure 3C).Crypt hyperplasia, resulting from increased epithelial proliferation is a hallmark of C. rodentium infection.As we previously discovered that epithelial proliferation was nearly absent in KO animals after chemical-induced colitis-type injury in the colon (30), we measured the crypt length and Ki67 staining to assess proliferation.We observed no changes in either crypt length or Ki67+ staining (Figures 3D, E).RELM-b levels, measured by fluorescence intensity at day 12 post infection were not significantly different between WT and KO mice (Supplementary Figures 2C, D

Increased expression of goblet cell effector proteins does not influence the course of a high dose Trichuris muris infection
Clearance of parasitic worms (helminths) in the gut relies on a "weep and sweep" response induced by type 2 cytokines (IL-4, IL-5, IL-13), which includes increased epithelial turnover, upregulated mucus production by goblet cells, and enhanced muscle contraction, collectively working to expel the parasites (37).Goblet cell effectors CLCA1, ANG4, and RELM-b are induced during infection with the helminth T. muris (9,32,38,39).CLCA1 plays a role in the formation of the two distinct mucus layers in the colon and is upregulated upon T. muris infection (40), while RELM-b can reduce the viability of T. muris (10) and ANG4 expression has been correlated with the expulsion of T. muris (14,41).Given the heightened levels of these effector proteins during homeostasis, we hypothesized that KO animals would be better equipped to combat T. muris infection compared to WT animals.
Male mice on a C57BL/6 background exhibit a dose-dependent immune response to T. muris.Depending on the infective dose of T. muris, the immune system is polarized towards a susceptibilityassociated type 1 immune response (low dose) or a resistanceassociated type 2 immune response (high dose) (37).Initially, we infected both WT and KO male mice with a high dose of T. muris eggs.After 14 days all mice developed an infection, which was mostly cleared after 21 days (Figure 4A).During T. muris infection, increased epithelial proliferation leads to crypt hyperplasia.Histological analysis showed comparable crypt length at both day 14 and day 21 (Figure 4B), and Ki67 staining indicated equivalent increased proliferation at day 21 (Figure 4C) in both WT and KO animals.As observed during C. rodentium infection, the previously described lack of regenerative ability of the KO epithelium does not impair increased epithelial proliferation during infection.RELM-b is elevated in both WT and KO and counts of RELM-b+ cells reveal a reduced difference between WT and KO mice on day 21 post infection (Figure 4D).To assess the immune response, we stimulated mLN with E/S antigen and measured the cytokine levels for IFNg, IL-6, IL-10, IL-4, IL-5, and IL-13 (Figure 4E).Increased levels of IL-13 and IL-5 in re-stimulated mLN cells as well as serum IgE indicated a typical and equal type 2 immune response in both WT and KO animals ( Figures 4E, F).Mice with a type 2 cytokine profile produce high levels of IgG1, while mice with increased levels of type 1 cytokines exhibit elevated levels of IgG2.Although there was no difference in IgG1 levels, Mmp17 KO mice showed significantly higher, through variable, levels of IgG2 despite having comparable type 1 cytokine levels to WT animals (Figure 4G).In conclusion, while the expression of AMPs and mucus proteins during T. muris infection has been shown to be essential (31), the higher baseline levels in Mmp17 KO mice do not influence the course of high dose T. muris infection.Numerical data are means ± SEM.Data was analyzed using Student's unpaired t-test (two-tailed).ns, not significant.
Mmp17 KO mice are resistant to low-dose T. muris infection without inducing a type 2 immune response Low-dose T. muris infection in C57BL/6 mice leads to a chronic infection due to the induction of a non-protective type 1 immune response (42).We found that T. muris-infected Mmp17 KO mice are resistant to chronic infection.Compared to WT mice, KO mice exhibited significantly reduced worm burdens (Figure 5A) with 46% (6 out of 13 mice) completely clearing the worms after 35 days, while all WT mice remained infected.At the endpoint (day 35), we observed no morphological differences.Measurement of crypt length and Ki67 positive percentage of the crypt revealed no changes in epithelial proliferation (Figures 5B, C).Although we observed increased levels of RELM-b in the proximal colon in the naïve state (Figures 1A, B), we do not see any differences in RELM-b+ cells in the proximal colon of infected mice when comparing WT and KO (Figure 5D).Interestingly, this appears to be the case for all infection models we tested.While we observed significant differences in goblet cell effector expression in healthy KO animals, these differences are reduced during infections as they are strongly induced by the immune response (Figures 4D, 5D).
Infected WT mice exhibited a type 1 cytokine profile consistent with a chronic infection, showing significantly increased levels of IFN-g, in addition to IL-6 and IL-10, while cytokine levels in KO animals remained low (Figure 5E).It is generally assumed that a type 2 immune response is essential for clearing T. muris; however, we did not observe increased levels of type 2 cytokines like IL-4, IL-5 or IL-13 in the KO (Figure 5E) at the time of sampling.To confirm the lack of a local type 2 response in the KO colon, infected colon tissue samples were analyzed for Ifng, Il10, Il13, and Il4 mRNA levels (Figure 5F).Furthermore, tissue mRNA levels of Tgfb1 and Foxp3 suggested no involvement of regulatory T-cells in the downregulation of type 1 cytokines in the KO (Figure 5G).Whilst the lack of a detectable Th2 immune response in the KO may reflect the sampling point, and thus a consequence of lower worm burdens at the end point, the significantly lower total serum IgE compared to WT (Figure 5H) suggests that the enhanced resistance to infection seen in the KO mouse is not accompanied by an elevated Th2 immunity.Alcian blue/PAS staining of paraffin sections from infected colon tissue revealed no difference in mucus pH, hinting towards similar mucus quality between WT and KO mice (Supplemental Figures 2A, B).This data collectively demonstrates that Mmp17 KO mice can clear low-dose T. muris infection without mounting a type 2 immune response which is typically seen as requisite for parasite clearance.Thus, we propose that the changes in the naïve state contribute to the enhanced ability of the KO to clear the infection.

Mmp17 KO mice show increased goblet cell maturation in cecum crypts
T. muris primarily colonizes the cecum and proximal colon (37,43).As we observed heighted levels of goblet cell effectors in the proximal colon at steady state, we were curious if the cecum of naïve Mmp17 KO mice exhibited similar changes.Consequently, we examined the cecum of KO mice, assessing RELM-b and CLCA1 levels and analyzing goblet cell numbers and morphology.Consistent with our findings in the proximal colon (Figure 1D), we detected no differences in goblet cell numbers when comparing WT and KO cecum (Figures 6A, B).However, we observed an increase in goblet cell size in the upper part of the crypt (Figure 6C), suggesting enhanced goblet cell maturation.Upon evaluating goblet cell effectors, both RELM-b and CLCA1 levels were markedly increased in the KO cecum, as demonstrated by IF staining (Figures 6D, E).Moreover, while RELM-b+ cells were predominantly located towards the base of the cecum crypts, CLCA1 signal was mainly present in the upper crypt in WT animals, suggesting that CLCA1 is produced by mature goblet cells (7).In contrast, in KO mouse cecum, we observed significantly more CLCA1 positive cells toward the lower half of the crypts (Figure 6E).
In accordance with Figure 1, our results suggest an increased goblet cell maturation state in KO mice, which did not correlate with elevated cytokines levels (Figure 1).The change in the zonation of goblet cell effector expression could imply that an altered gradient of niche factors controlling goblet cell maturation might be the cause for our observations.

Mmp17 is expressed in upper-crypt lamina propria cells
We have previously reported that the primary cell type expressing Mmp17 in the colon at steady state are smooth muscle cells, particularly those in the muscularis mucosa, a thin layer of muscle located right beneath the epithelium, which exhibit high levels of Mmp17 expression (30).Using the reporter gene bgalactosidase in Mmp17 +/-mice, we observed lower, yet distinct expression in other non-epithelial cells located near the upper part of the crypt, that do not express the smooth muscle marker alphasmooth muscle actin (Figures 7A, B).It has been previously shown that Mmp17 can be expressed by monocytes and macrophages in the vasculature (44,45).To investigate whether gut macrophages express Mmp17, we co-stained with the macrophage marker F4/80 and indeed found cells that appear to be positive for both bgalactosidase and F4/80 (Figure 7A).In addition, we found a population of low b-galactosidase expressing cells in the vicinity of PDGFRa+ mesenchymal cells (Figure 7B).These mesenchymal cells in the intestine play a crucial role in forming the intestinal niche, for instance, by maintaining the BMP gradient through the expression of BMP agonists and antagonists (18).Thus, in addition to the reported expression of Mmp17 in smooth muscle cells, we find a population of upper-crypt colon lamina propria cells expressing Mmp17.We propose that the lack of Mmp17 expression in those cells is altering the intestinal niche resulting in enhanced goblet cell maturation in Mmp17 KO mouse colon.

Discussion
In the Mmp17 KO mouse model, we observed an elevated expression of goblet cell effector genes, such as Ang4, Clca1, and Retnlb, in cecum and proximal colon (Figures 1, 5).
While previous studies have shown that mice lacking RELM-b are highly susceptible to C. rodentium infection, to our knowledge there have been no investigations into the effect of increased RELMb levels.Our study did not find any evidence that suggest that the elevated homeostatic levels of ANG4, CLCA1, and RELM-b are beneficial during C. rodentium infection, as WT and KO mice cleared the infection at the same time, and peak infection numbers were comparable.In a previous study, we reported that Mmp17 KO mice were highly susceptible to DSS-induced colitis (30).However, in the case of C. rodentium infection, which induces only mild colitis in C57BL/6 mice, we observed no elevated tissue damage in KO mice.This may be due to less overall damage caused by C. rodentium-induced colitis, and thus the deficiencies in repair might not be significant in this model.Moreover, both WT and KO mice showed crypt hyperplasia with a comparable average crypt length, indicating a similar proliferation rate.Resistance to intestinal helminths has been mainly attributed to the type of immune response elicited, with a type 2 response typically leading to clearance, while a type 1 response results in chronic infection (37).However, our findings suggest that the ability to clear the infection may also be influenced by other factors.Mmp17 KO mice, infected with a low dose of T. muris eggs, were able to clear the infection, while WT mice developed a chronic infection (Figure 5).The KO mice did not show the typical increase in cytokines of a chronic (type 1) infection (IFNg, IL-6, IL-10), nor did they have increased levels of IL-4, IL-13, or IL-5, which would be indicative of a type 2 immune response.Further, no differences in epithelial or immune responses were observed in the infected mice.Thus, enhanced resistance is not accompanied by an elevated Th2 immune response.Interestingly a similar disconnect between resistance and Th2 immunity has been reported before in the context of goblet cell derived mucins, with MUC5ac deficient mice failing to expel T. muris despite mounting a strong Th2 response (46).This could indicate that the changes in the epithelium of KO mice at steady-state may be responsible for the improved clearance of T. muris infection in KO mice.
Notably, altered expression of genes involved in mucus production and processing, as well as AMPs were observed in the cecum and proximal colon of KO mice, which are the areas of the intestine that are colonized by T. muris.Specifically, there was an increase in goblet cell effectors such as RELM-b and ANG4, both upregulated during helminth infection and RELM-b being able to directly affect the viability of T. muris and other parasitic helminths (14,47).Additionally, CLCA1, which plays an important role in mucus processing and helps form the two distinct inner and outer mucus layers of the colon by processing mucin 2 (MUC2), is also described to be upregulated during T. muris infection (40).While CLCA1 is not essential in the steady state to form a functional mucus barrier, it is crucial during nematode infections (48).Thus, one possible mechanism for the improved clearance of T. muris infection in KO mice is changes in the mucus composition, which could prevent some larvae invading the epithelium.However, worm burdens at day 14 post infection were equivalent in WT and KO evidencing equivalent establishment.Thus the significantly lower worms burdens seen at day 35 implicate mucus as an aid in flushing worms out in concert with other clearance mechanisms like increased epithelial proliferation and muscle contractions (49).The increase in goblet cell effectors could also contribute to the enhanced clearance of T. muris in KO mice by exerting direct effects on the worms themselves, lowering their viability and hindering their movement.Finally, a change in mucus composition may also affect the commensal microbiota, which in turn interacts with helminths and thus may contribute to the persistence of infections (50).
Mucus and goblet cell effector genes, such as Ang4, Clca1, and Retnlb are typically induced during infections by cytokines, especially IL-4, IL-13 and IL-22.However, we see no indication of increased cytokine levels in the naïve colon or re-stimulated mesenteric lymph node cells that could cause this change (Figure 1G).Furthermore, we did not observe any signs of goblet cell hyperplasia, which is often associated with cytokine-mediated activation (Figures 1C, 5).As we could not find detectable differences in cytokine levels in the Mmp17 KO colon to explain this changed goblet cell expression pattern there must be another mode of induction.The colon epithelium is regulated by growth factor gradients that help preserve the stem cells at the bottom of intestinal crypts while allowing for mature cells with different functions in the upper part of the crypt.One of those gradients is BMP signaling, kept low by BMP-antagonists at the bottom of the crypt and increasing towards the top.In the Mmp17 KO mouse colon epithelium we previously reported an increase in SMAD4, suggesting increased BMP-signaling (30).Although initially thought off as primarily affecting enterocyte maturation, Beumer et al. show that BMP signaling can also induce changes in goblet cell expression (51).Another indication of an altered gradient is the fact we find more CLCA1+ cells in the lower cecum crypts.CLCA1 is a marker for upper crypt goblet cells that are among the main mucus producers in the colon.The presence of CLCA1 towards the bottom of the crypt may indicate a more mature goblet cell state.
While the primary cells expressing Mmp17 in the intestine are smooth muscle cells, we also observed other non-epithelial cells that were b-galactosidase positive.Although the membranous staining of macrophages (F4/80) and mesenchymal cells (PDGFRa) made it difficult to be distinctive, these cells likely include macrophages and might include other cells as mesenchymal cells (Figure 7).Further investigations are necessary to describe the full extent of Mmp17expressing cell types in the colon and its mechanism to regulate the intestinal niche.Nevertheless, this study contributes to highlighting the importance of MMPs and different lamina propria cells as important regulators of the intestinal niche.

Mmp17 KO mice
Mmp17 KO mice in the C57BL/6 background have been described previously (45).Mice were handled under pathogenfree conditions in accordance with CoMed NTNU institutional guidelines.Experiments were performed following Norwegian legislation on animal protection and were approved by the local governmental animal care committee.
Citrobacter rodentium infection studies were approved by Norwegian Food Safety Authority (FOTS ID: 11842) and were performed in accordance with the guidelines and recommendations for the care and use of animals in research.
Trichuris muris infections were performed at the University of Manchester and were approved by the University of Manchester Animal Welfare and Ethical Review Board and performed within the guidelines of the Animals (Scientific Procedures) Act, 1986.Mice were maintained at a temperature of 20-22°C in a 12h light, 12h dark lighting schedule, in sterile, individually ventilated cages with food and water ad lib.Mice were culled via exposure to increasing carbon dioxide levels, an approved schedule 1 method as specified within the guidelines of the Animals (Scientific Procedures) Act.

Citrobacter rodentium infection
C. rodentium infection was performed following established protocols (52).In brief, a GFP-expressing C. rodentium strain was cultured in Luria-Bertani (LB) medium supplemented with 30 µg/ ml chloramphenicol at 37°C.Male and female mice, aged 6-10 weeks, were infected with a dose of 10 8 -10 9 CFU via oral gavage in a total volume of 0.1ml sterile PBS.The mice were monitored daily after the oral administration of C. rodentium for weight loss and pain associated behaviors such as hunchbacked posture, piloerection, and rectal prolapse.Fresh fecal pellets were collected, weighed, and homogenized in sterile PBS using a FastPrep homogenizer from MP Biomedicals.Dilutions of the homogenate were plated on agar plates supplemented with 30 µg/ml chloramphenicol.The plates were incubated at 37°C overnight and the number of colonies was assessed.At the end of each experiment (12 days post infection or until clearance), mice were euthanized, and organ samples were collected for histology, RNA isolation and CFU analysis.

T. muris infection
The parasite maintenance, E/S products generation, and eggs preparation was performed following previously established protocols (53).Mice were orally administered with an infection dose of 150 viable T. muris eggs in a volume of 200µl.
Upon, both the caecum and the proximal colon were harvested, blinded, and subsequently stored at -20°C.For quantitative assessment of worm burdens, the caecum and colon were thawed, cut open longitudinally and rinsed to remove gut contents.Curved forceps were used to scrape off the gut mucosa, thereby removing both epithelia and worms.The gut contents as well as the excised mucosa were then examined for T. muris numbers using a dissecting microscope (Leica S8 APO).

Mesenteric lymph node cells re-stimulation and cytokine bead array
mLNL were processed into a cell suspension, then cultured at a density of 5x10 6 cells/ml were cultured in RPMI 1640 supplemented with 4 h E/S antigen (50µg/ml) for 48h at 37°C and 5% CO 2 .Following incubation, supernatants were collected and frozen at −20°C.
For cytokine quantification, the Cytometric Bead Array (CBA) Mouse/Rat soluble protein flex set system from BD Bioscience was used following the manufacturer's protocol.A FACS Verse (BD Biosciences) was used for cell acquisition and the data was analyzed utilizing FCAP Array v3.0.1 software (BD Cytometric Bead Array).

Mesenteric lymph node lymphocyte restimulation and cytokine ELISA
The mesenteric lymph nodes were dissected from naïve or C. rodentium infected mice and kept in sterile PBS.The lymph nodes were passed through 70 mm cell strainer to form a single cell suspension.The cell viability was assessed using Trypan blue and a Countess automated cell counter (Invitrogen).A 96 well plate was pre-incubated with a 5 µg/ml solution of anti-mouse CD3e (100314, Biolegend) in sterile PBS at 37°C for 2 h.The antibody solution was then removed, and the isolated lymph node cells were added at 2×10 5 cells per well together with soluble anti-mouse CD28 (102112, Biolegend) at a final concentration of 2 µg/ml.After incubating the cells for 4 days at 37°C, 5% CO 2 , the cellsupernatant was collected for cytokine analysis by ELISA.

Total IgE ELISA
Serum samples from T. muris infected mice were analyzed for total IgE antibody levels.96 well plates were pre-coated with purified anti-mouse IgE (2ug/ml, Biolegend, Clone: RME-1) in 0.05M carbonate/bicarbonate buffer and incubated overnight at 4°C.Post-incubation, the plates were washed using PBS-T.Nonspecific binding was blocked with 3% BSA (Sigma-Aldrich) in PBS for 1 hour at room temperature.After the blocking buffer was removed, 1:10 diluted serum was added to the wells and incubated for 2 h at 37°.Following a washing step, HPR-conjugated goat antimouse IgE (1µg/ml; Bio-rad) was added to the wells and incubated for 1 h.After an additional washing step, the plates were developed using the TMB substrate kit (BD Biosciences, Oxford, UK), with the reaction being terminated with 0.18M H2SO4, upon sufficient coloration.Absorbance was measured at 450 nm, with a reference of 570 nm subtracted, with a Versa max microplate reader (Molecular Devices) with SoftMax Pro 6.4.2.

IgG ELISA
Serum of T. muris infected mice was analyzed for parasite specific IgG1 and IgG2c antibody levels.First, 96 well plates were pre-coated with 5µg/ml T. muris overnight E/S antigen overnight.After washing, non-specific binding was blocked using 3% BSA (Sigma-Aldrich) in PBS.Post blocking and subsequent washing, the plates were incubated with the serum samples at 2 fold dilutions ranging from 1:20 to 1:2560.The parasite specific antibody was detected using biotinylated IgG1 (BD Biosciences) or IgG2c (BD Biosciences) antibodies together with Streptavidin-POD (Roche).After washing, the plates were developed with the TMB substrate kit (BD Biosciences, Oxford, UK) following the manufacturer's instructions.Upon sufficient coloration, the reaction was stopped using 0.18 M H 2 SO 4 .Absorbance was measured at 450 nm, with reference of 570 nm subtracted, with a Versa max microplate reader (Molecular Devices) with SoftMax Pro 6.4.2.

Sample preparation for histology
After excising the proximal colon tissue of T. muris infected mice, the tissue was fixed for 24 hours in 10% neutral buffered formalin (Fisher) containing 0.9% sodium chloride (Sigma-Aldrich), 2% glacial acetic acid (Sigma-Aldrich) and 0.05% alkyltrimethyl-ammonium bromide (Sigma-Aldrich) before storing it in 70% Ethanol (Fisher) until further processing.For naïve and C. rodentium infected samples, colon or cecum samples were dissected, and washed with ice-cold PBS.The colon was rolled into "swiss rolls" as previously described or a 5 mm piece of the proximal colon was taken (54).The tissues were fixed in 4% formaldehyde for 24h at Room Temperature.For paraffin embedding, the fixed tissues were dehydrated through a graded series of ethanol, then cleared in xylol and infiltrated with paraffin in a dehydration automat (Leica ASP300 S) using a standard protocol.Specimens were embedded in paraffin.Alternatively, proximal colon tissue was embedded in OCT and snap-frozen on dry ice and stored at -80C until processing.

Histological stainings
Paraffin embedded tissues were cut into 4µm sections and placed on glass slides.The slides were heated at 60°C for 20 min prior to deparaffinization in Neo-Clear Xylene substitute (Sigma-Aldrich).Rehydration was performed through a decreasing ethanol gradient.
For Hematoxylin and eosin stain, the samples were incubated for 1 min in hematoxylin solution.The tissues were then washed with water until the water ran clear and incubated in Eosin Y solution for 3 min.
For Alcian blue stain, the slides were incubated in Alcian blue solution for 30 min.After thoroughly washing the slides with water, the tissues were counter stained with nuclear fast red solution for 5 minutes.
For Alcian blue/PAS stain, the tissues were stained in 1% Alcian blue in 3% acetic acid (pH2.5)for 5 min, 1% periodic acid for 5 min, Schiff's reagent for 15 min and finally in Mayer's hematoxylin for 1 minute.After each staining step the slides were washed in distilled water for 5 minutes.
After the staining, the tissues were washed, then dehydrated using an alcohol gradient, cleared in Xylene/Neo-Clear and mounted.

Tissue immunostainings
The paraffin-embedded or frozen OCT-embedded tissues were cut into 4 mm or 12 µm sections and placed on glass slides.Paraffinembedded tissue sections underwent deparaffinization, rehydration, and antigen retrieval using citrate buffer at pH-6.0.Frozen sections were permeabilized in −20°C cold methanol for 10 min before continuing with blocking.Tissues were then blocked and permeabilized with PBS containing 0.2% Triton X-100, 1% BSA, and 2% normal goat serum (NGS) or normal donkey serum (NDS) for 1 hr at RT in a humidified chamber.

Colon crypt isolation
Colon crypts for RNA sequencing and organoid culture were isolated using a previously published method (30).Briefly, the entire colon was flushed with cold PBS and then cut into 2-4 mm pieces.These pieces were washed with cold PBS until the supernatant appeared clear and subsequently transferred into cold chelation buffer (PBS containing 2 mM EDTA) for incubation at 4°C under gentle agitation for 1 h.The chelation buffer was then removed, and the tissue pieces were resuspended in PBS.Crypts were separated from the tissue by vigorously pipetting with a BSA-coated 10 ml pipette or by shaking the tube.The tissue pieces were allowed to settle at the bottom of the tube for approximately 1 min, after which the supernatant was collected into a BSA coated 50 ml tube.The pipetting/shaking process was repeated using fresh PBS until most crypts had detached.The collected supernatant was centrifuged at 200 x g for 3 min, and the resulting crypt pellet was resuspended in basal crypt medium (BCM, see below) and counted.For subsequent experiments, the crypts were either used for RNA isolation, protein extraction or colonoid culture, seeded into 40 µl drops of Matrigel in a P24 well plate (300 crypts/well) and cultured in WNR medium (medium composition is described below) with Rock inhibitor (10 µM) for 24 h at 37°C and 5% CO 2 .

Organoid immunostaining
Organoid immunostaining was performed using a previously described protocol (15).In brief, organoids were fixed with 4% paraformaldehyde with 2% sucrose for 30 min at room temperature.After washing with sterile PBS, the organoids were permeabilized with 0.2% Triton X 100 in PBS for 30 min at room temperature.Blocking was performed for 1 hour at room temperature in PBS containing 1% BSA, 2% NGS, and 0.2% Triton X 100.Primary antibodies (RELMb (1:200, Peprotech 500-p215)) were diluted in the same blocking buffer and incubated with the organoids over night at 4°C.After washing, the organoids were incubated with the appropriate Alexa Fluor secondary antibodies, UEA-1 (1:500, Vector Laboratories, RL-1062), and counterstained with Hoechst 33342 (1:1000, MERCK, H6024) over night at 4°C in the dark.After incubation, the organoids were washed and 250 µl Fluoromount G was added to each well.Imaging was conducted with a Zeiss Airyscan confocal microscope, using a 10× and 20× objective lens.Images were analyzed using ImageJ software.

RNA seq of colon crypts
RNAseq data from colon crypts and muscle from Mmp17 KO mice was obtained and analyzed as described in our previous publication (30).In brief, colon crypts were isolated using the colon crypt isolation protocol above.The crypts were placed in lysis buffer (RLT, provided in RNeasy® Mini Quiagen Kit, 74104) and RNA was isolated following manufacturer instructions (RNeasy® Mini Kit, Qiagen, 74104).RNA quantification was done by spectrophotometry (ND1000 Spectrophotometer, NanoDrop, Thermo Scientific) and 25 µl with an RNA concentration of 50 ng/µl were used for RNA seq.The library preparation and sequencing was performed by the NTNU Genomic Core facility using a Lexogen SENSE mRNA library preparation kit to generate the library and Illumina NS500 flow cells to sequence the samples at 2 x 75 bp paired end.The STAR aligner was used to align reads to the Mus musculus genome build mm10.Feature Counts was used to count the number of reads that uniquely aligned to the exon region of each gene in GENCODE annotation M18 of the mouse genome.Genes with less than 10 total counts were filtered out and DESeq2 with default settings was used for differential expression analysis.