Fibrogenesis in chronic murine colitis is independent of innate lymphoid cells

Abstract Introduction Insight in the pathogenesis of intestinal fibrosis is an unmet medical need in inflammatory bowel diseases. Studies in murine models and human organ fibrosis point to a potential role of innate lymphoid cells (ILC) in chronic intestinal inflammation and fibrosis. Materials and Methods Dextran sodium sulfate (DSS) in drinking water was used to induce chronic colitis and remodeling in C57Bl/6 wild type (WT), RAG‐deficient, RAG−/− common γ chain deficient and anti‐CD90.2 monoclonal antibody treated RAG−/− mice. Inflammation was scored by macroscopic and histological examination and fibrosis was evaluated by hydroxyproline quantification and histology. Results In RAG−/− mice (which have a normal ILC population but no adaptive immunity), chronic intestinal inflammation and fibrosis developed similarly as in WT mice, with a relative increase in ILC2 during repeated DSS exposure. Chronic colitis could also be induced in the absence of ILC (RAG−/−γc−/− or anti‐CD90.2 treated RAG−/− mice) with no attenuation of fibrosis. Importantly, clinical recovery based on weight gain after stopping DSS exposure was impaired in ILC‐deficient or ILC‐depleted mice. Conclusion These data argue against a profibrotic effect of ILC in chronic colitis, but rather suggest that ILC have a protective and recovery‐enhancing effect after repeated intestinal injury.

obstructions) due to excessive extracellular matrix deposition and muscularis propria hyperplasia in the bowel wall. 1,2 Up to 80% of CD and 20% of UC patients undergo surgery during the disease course, with stricture formation being the most common indication for major intestinal surgery in CD. 3,4 Notably, recurrence of inflammation and fibrosis after surgical resection is common. Although fibrosis is most prevalent in CD, a minority of patients with extensive UC of long duration have shortening of the colon with formation of a lead-pipe colon as a result of excessive remodeling. 5 The mechanisms by which fibrosis in the intestine develops are incompletely understood. Therefore, insight in the pathogenesis of intestinal fibrosis is an unmet medical need in IBD care.
Over the last few decades, most studies on IBD have focused on the identification of abnormal adaptive immune responses. More recently, the focus has shifted toward mucosal innate immune responses. Recent data suggested a causal link between defects in the resolution of intestinal inflammation and impaired bacterial clearance, excessive cytokine secretion, altered monocyte-macrophage transition, and activation of tissue innate immune cells. 6,7 Intestinal fibrosis is believed to be a chronic and progressive process triggered by inflammation through complex matrix/ cell/protein interactions, but may be reversible. 8 Innate lymphoid cells (ILC) were discovered as an additional source of mucosal cytokines. 9,10 Three groups of ILC have been defined based on shared expression of surface markers, transcription factors, and effector cytokines with T helper (Th) cell subtypes. Group 1 ILC (ILC1) are implicated in immunity to intracellular pathogens, influenced by interleukin 12 (IL-12), they express T-bet, and produce interferon γ (IFN-γ) and tumor necrosis factor (TNF). Group 2 ILC (ILC2) are involved in type 2 inflammation required for antihelminth defenses and allergic inflammation, influenced by IL-25, IL-33, and TSLP, they express GATA-3, and produce type 2 effector cytokines IL-4, IL-5, IL-9, IL-13, and amphiregulin. Group 3 ILC (ILC3) are involved in antibacterial immunity, chronic inflammation, and tissue repair, influenced by IL-1β, IL-6, and IL-23, they express RORγt, and produce IL-17 and/or IL-22. 11 ILC residing in the gut mucosa are important regulators of homeostasis and have the potential to control tissue remodeling. [12][13][14] The role of ILC in mucosal immune pathology is not clear. In murine models of asthma, ILC2 are a source of IL-4, IL-5, and IL-13 after stimulation with epithelial cell derived IL-25 and IL-33. 15 In murine models of IBD, ILC contribute to the development and persistence of intestinal inflammation by production of IL-17A and IFN-γ in response to IL-23. 16 ILC3 are important players in protecting the epithelial barrier by production of IL-22 in response to epithelial stress, however IL-17 production by these cells can contribute to development of colitis. 17,18 Bernink et al showed that IFN-γ producing ILC1 are increased in resected tissues of patients with CD, suggesting their involvement in human disease. 19,20 ILC might also play an important role in organ fibrosis. In murine models of lung and liver fibrosis, type 2 ILC have been shown to be essential for the development of fibrosis, via an IL-25 and IL-33 dependent pathway, respectively. 12,14 Previously, we and others have shown that chronic dextran sodium sulfate (DSS) colitis induced by repeated cycles of DSS exposure is characterized by transmural inflammation and granuloma formation and followed by remodeling and fibrosis. 21,22 To dissect the involvement of ILC in this process, chronic DSS colitis was induced in wild type (WT), in recombination activating gene (RAG −/− ) deficient mice (which lack adaptive immunity but have a normal ILC population), in anti-CD90.2 injected RAG −/− mice and in RAG −/− common γ chain −/− (RAG −/− γc −/− ) mice which both also lack the ILC population. This enabled us to separately analyse whether adaptive immunity and/or ILC are involved in colitis and fibrosis induction.

| Mice and induction of colitis
To induce DSS colitis, female 6-week-old C57BL/6OlaH WT mice were obtained from Envigo (Horst, The Netherlands). RAG1 −/− tm/tm mice, lacking T and B cells, were obtained from Charles River (MA) (mice in figure 1 and 2) or were bred in our own animal facility (figures 3-8). 21 Female 6-week-old C57BL/6NTac;B10(Cg)-Rag2 tm1Fwa Il2rg tm1Wjl (RAGγc −/− ) mice were obtained from Taconic (New York). All animals were maintained in the Animal Care Facility of the Faculty of Medicine, University of Leuven (Belgium) and according to ARRIVE guideline. Colitis was induced with DSS as previously described. 20 Briefly, 1.5% to 2.25% DSS (35-50 kDa; MP Biomedicals, Illkirch, France) was added to the drinking water to induce colitis. Acute colitis mice received DSS for 7 days. For the study of chronic colitis, mice were exposed to repeated "cycles" of DSS exposure. One cycle was defined as exposure to DSS for 7 days followed by a recovery period of 2 weeks with normal drinking water ( Figure 1A). Mice were exposed to one, two, or three cycles in total. All mice were age-matched at the time of sacrifice. and Disease Activity Index (DAI) was determined based on body weight loss, stool consistency, and presence of gross blood in the stools. Next, inflammation in distal colon was evaluated as previously described. 18 A macroscopic damage score was calculated based on the presence of adhesions, hyperemia, and extent of colonic inflammation. Paraffin embedded, 5 µm-thick longitudinal and transverse sections were stained with hematoxylin and eosin. The microscopic score of inflammation was calculated as previously described. 18 Briefly, this score comprised the sum of changes in mucosal architecture, neutrophil infiltration, mononuclear cell infiltration, goblet cell loss, and epithelial defects. The histological active disease score comprised the sum of neutrophil infiltration and epithelial defects, reflecting acute tissue damage during the last 24 hours before tissue sampling. Tissue sections were scored by an experienced pathologist (GdH) blinded to the experimental conditions. Fibrosis was also evaluated by hydroxyproline quantification and Martius Scarlet Blue (MSB) stainings as previously described. 20,23 In short, areas stained for collagen in the mucosa and submucosa were measured in two cross-section using ImageJ. 20 The thickness of the mucosa and muscularis propria was calculated as mean value of two different measurements per mouse on uniform horizontal cross sections of colon crypts using ImageJ. 22,24 The hydroxyproline assay was performed as previously described to quantify the amount of collagen. 25 2.3 | Antibody-mediated depletion of ILC YTS154 (rat anti-mouse CD90.2, cell clone provided by H. Waldmann, University of Cambridge, Cambridge, England, UK), to deplete CD90.2 expressing ILC, was produced at Bioceros and was used as previously described. 26 Rat IgG1 isotype (clone MCP-11) was obtained from BioXCell (West Lebanon, NH). Antibodies or corresponding isotype controls, diluted in sterile phosphate-buffered saline (PBS), were administered intraperitoneally every 3 days (250 µg/mouse) starting from 3 days before DSS exposure and during the whole experiment (from day 3 until day 63). In control mice, not exposed to DSS, the corresponding volume of sterile PBS was administered.
Flow cytometry was conducted on a BD LSR Fortessa instrument in accordance with standard methods. Calibration was performed before each acquisition by CS&T beads (BD). For fluorescence compensation settings, single color UltraComp eBeads compensation beads were used (Thermo Fisher Scientific). Fluorescence Minus One controls were included.

| Protein quantification
A 0.5 cm piece of affected distal colon was opened longitudinal and incubated for 24 hours at 37°C in complete medium before supernatant collection. Protein concentrations in the supernatants for interleukin 4 (IL-4), IL-5, IL-6, IL-12p70, IL-22, IL-23p19, TNF, and IFN-γ were determined using multiplex meso scale discovery technologies. In short, samples were incubated overnight at 4°C on a U-plex plate coated with biotinylated capture antibodies. After three wash steps in PBS 0.05% tween (Sigma-Aldrich), detection antibodies were added for 1 hour at room temperature before activation of the plate electrodes resulting in a quantitative measurable emission of light.

| Statistical analysis
Statistical analysis were performed using GraphPad Prism 8 (GraphPad, La Jolla, CA). Data are represented as medians (interquartile range) and the individual P values for two group comparison were obtained using Mann-Whitney U testing. The following methods were used: multiple comparison with Dunn's correction between multiple groups, Spearman for correlation testing, and Kaplan-Meier curve for survival analysis. Differences were considered statistically significant at *P < .05, **P ≤ .01, and ***P ≤ .001.

| Ethical considerations
All studies were approved by and performed according to the local ethics committee for animal experimentation of the University of Leuven (P230-2015).

| RESULTS
3.1 | Inflammation in acute and chronic colitis is similar in WT and RAG-1 −/− mice To study whether adaptive immunity is required for development of colitis in the DSS model, we compared WT mice with RAG-1 −/− mice lacking B and T lymphocytes. 27 Acute DSS colitis was induced by DSS ingestion through drinking water ( Figure 1A). Weight loss after 1 week was more pronounced in the RAG-1 −/− mice than in WT mice (P = .006; Figure 1B). Spleen weight did not increase after 1 week of DSS exposure (P > .99 for both backgrounds; Figure 1C). DAI scores were significantly higher in RAG-1 −/− mice exposed to DSS compared with DSS exposed WT mice (P = .026; data not shown). Colon length decreased in both strains after 1 week of DSS exposure (WT: P = .006 and RAG-1 −/− : P = .001), but significantly more in RAG-1 −/− mice compared with WT mice (P = .032; Figure 1D,E), resulting in a significantly higher colon weight/length (W/L) ratio in RAG-1 −/− mice compared with WT mice (P < .001; Figure 1F). No significant differences could be observed in macroscopic damage score, microscopic score of inflammation, and histological active disease score between RAG-1 −/− mice and WT mice ( Figure 1G-I).
For chronic colitis, we used three cycles of DSS exposure in RAG-1 −/− and WT mice. One cycle of DSS exposure consisted of 1 week of DSS exposure followed by a recovery period of 2 weeks. RAG-1 −/− mice lost more weight during three cycles of DSS administration than WT mice ( Figure 1B). Spleen weight, colon length, colon weight, colon W/L ration, macroscopic damage score, microscopic score of inflammation, and histological active disease score were similar in RAG-1 −/− mice compared with WT mice in this chronic model ( Figure 1D-I).
We also analysed the composition of inflammatory cells in the colonic mucosa. Gating of the different myeloid cell populations is illustrated in Figure S1. When compared with controls without DSS exposure, neutrophils (14.40% vs 0.63% of CD11b + cells, P = .004) and monocytes (28.40% vs 8.16% of CD11b + cells P = .004) were increased in WT mice after three cycles of DSS ( Figure S2). In contrast, decreased proportions of eosinophils (18.30% vs 7.94% CD11b + cells, P = .016) and macrophages (57.80% vs 33.51% CD11b + cells %, P = .004) F I G U R E 1 Induction of acute and chronic colitis with DSS in RAG −/− as compared with WT mice. A, Study set-up. Colitis was induced with DSS in drinking water. Acute colitis was induced in WT and RAG −/− mice (n = 7 and 8) by 7 days of DSS administration without recovery. For induction of chronic colitis, WT and RAG −/− mice (n = 20 and 19) were exposed to repeated cycles of DSS exposure. One cycle of DSS comprises 7 days of DSS in drinking water, followed by a recovery period of 14 days with normal drinking water. Inflammatory and fibrotic parameters were compared between both groups of DSS exposed mice, and also to control WT and RAG −/− mice without DSS administration (n = 10 and 10). were observed after three cycles of DSS in WT mice ( Figures S2B and S2D). Similarly, in RAG-1 −/− mice with chronic DSS colitis, neutrophils (9.50% vs 0.61% of CD11b + cells, P = .004) and monocytes (5.78% vs 1.78% of CD11b + cells, P = .028) were increased as compared with RAG-1 −/− mice without DSS exposure, while no significant effect was seen on eosinophils and macrophages ( Figure S2E-H).
In summary, after 1 week of DSS exposure (acute model) or after three cycles of DSS administration (chronic model), systemic and colonic inflammation were similar or slightly more severe in RAG-1 −/− mice lacking T and B lymphocytes vs WT mice.

| Intestinal fibrosis is induced despite the absence of adaptive immunity
Intestinal fibrosis was then evaluated in the acute and chronic colitis models. Collagen deposition was clearly more pronounced in chronic three-cycles DSS colitis compared with acute colitis, in both WT and RAG-1 −/− mice as shown by MSB collagen quantification (P = .003 and P < .001, respectively) and hydroxyproline measurements (P = .003 and P < .001, respectively; Figure 1J-R). However, both fibrosis quantification methods revealed similar collagen deposition in RAG-1 −/− mice versus WT mice in the chronic model (P > .999 for both; Figure 1J,K). Notably, no differences were observed with regard to the thickness of the muscularis propria ( Figure 1L). Altogether, these results suggest that the adaptive immunity is not required for the intestinal fibrogenesis process in chronic DSS colitis. We, therefore, turned our attention to a potential involvement of ILC.
3.3 | Increased IL-13 producing ILC2 after two and three cycles of DSS in WT and RAG-1 −/− mice To study whether ILC are involved in intestinal fibrosis in this model we first studied the distribution of mucosal ILC subsets during acute or repetitive DSS administration in WT mice. The gating strategy for ILC and the identification of subsets based on membrane markers and intracellular RORγt expression are shown in Figure S3. No changes were observed in the total ILC proportion in acute colitis or after two and three cycles of DSS exposure (Figure 2A). The percentage of ILC1 was not different after 1 week of DSS exposure as compared with control mice without DSS exposure (5.42% vs 4.21% of ILC, P > .999; Figure 2B). However, after two or three cycles of DSS in WT mice, we observed a decrease of ILC3 and a relative increase in KLRG-1 + ILC2 in the colon mucosa as compared with control mice without DSS exposure (ILC2: 68.90% and 57.70% vs 48.00% of ILC, P = .012 and P = .023; Figure 2C,D). Among these ILC2, an increased proportion expressed IL-13 in the chronic model as compared with control mice (30.60% vs 6.60% of ILC2, P = .004; Figure 2E). However, the mRNA levels of IL-13 in total colon tissue at sacrifice were not increased (data not shown).

| ILC are not crucial for induction of fibrosis in chronic DSS
To further investigate the contribution of ILC to colitis and fibrosis, ILC were depleted in the chronic DSS model in RAG-1 −/− mice using YTS, an anti-Thy1.2 (CD90.2) depleting monoclonal antibody (mAB). To exclude effects of antibody treatment on CD90.2 expressing epithelial cells and fibroblasts, control RAG-1 −/− mice (not exposed to DSS) were injected at a similar dosing schedule. 25 We did not see any macroscopic or microscopic effect on the colon architecture as a result of treatment ( Figure S4C,D).
After elimination of ILC and chronic DSS exposure, weight loss was similar as compared with isotype and saline treated RAG-1 −/− mice with chronic DSS colitis ( Figure 3A,B). At sacrifice, colon length, colon weight, colon W/L ratio, macroscopic damage score, microscopic score of inflammation, and histological active disease score were not different after ILC depletion as compared with chronic DSS controls ( Figure 3C-H and S4 C-SG). Hydroxyproline levels and thickness of muscularis propria and mucosa of the distal colon (which in this model is the most affected part) were not altered after ILC depletion as compared with saline or isotype injected chronic DSS colitis controls ( Figure 3I-O). We also studied the inflammatory cells in the LP cell suspension isolated from the colonic mucosa. ILC depletion resulted in higher proportion of CD11b + myeloid cells as compared with saline treated chronic DSS colitis controls (76.90% vs 42.40% of CD45 + , P = .041; Figure 4C). Within the myeloid population, we found an increase of neutrophils (YTS: 10.40, saline: 20.50 vs control: 1.25% of CD11b + cells, P = .041 and P = .004) and eosinophils (YTS: 16.25%, saline: 15.30%, isotype: 14.00% vs control: 4.14% of CD11b + cells, P = .001, P = .008, and P = .042) after chronic DSS as compared with mice without DSS exposure ( Figure 4D,E), but no differences were observed related to treatment. Also, no differences in levels of monocytes and macrophages were observed in the different groups ( Figure 4F,G).
Antibody-mediated ILC depletion resulted in a lower production of ILC derived cytokines IL-5 (39.74 vs 418.60 pg/mL, P = .002) and IL-22 (218.30 vs 1619.00 pg/mL, P = .046) and a tendency toward lower production of IFN-γ (3.81 vs 12.92 pg/mL, P = .15), as compared with DSS exposed control groups ( Figure 4I-K). In contrast, levels of IL-33, an epithelial cell derived cytokine, were elevated during chronic DSS exposure and were elevated in absence of ILC as compared with DSS unexposed control mice (10.47 and 11.31 vs 0.37 pg/mL, P = .013; Figure 4H).

| DISCUSSION
This study intended to analyse the involvement of the adaptive immune system and of ILC in a model of chronic colitis, with special focus on fibrosis induction. As inflammation and fibrosis were unaltered in RAG-1 −/ − mice, adaptive immunity is clearly not required for the induction of fibrosis in this model. The redundancy of the adaptive immune system has previously been shown in acute DSS colitis. [26][27][28][29][30] However, here we also show that in absence of the adaptive immune system there is not only unaltered induction of chronic inflammation, but also fibrosis can still be induced to the same extent as in the presence of T and B cells.
We then focused our attention on ILC as potential pathogenic cells. First, we could show that in chronic DSS colitis there is an increased proportion of ILC2 among ILC, both in absence and presence of the adaptive immune system. This was accompanied by a decrease in ILC3. Second, we obtained evidence for increased ILC activity upon DSS exposure, as ILC2 in the distal colon produced IL-13 (a cytokine thought to be important for lung fibrogenesis), and as there was decreased production of IL-5 and IL-22 in the colonic mucosa of anti-CD90 injected mice. As a relative shift from ILC3 predominance toward ILC2 predominance in the colon was observed both in WT and RAG −/− mice, and as the manifestations of ILC2 expansion persist in RAG −/− mice, this indicates that these changes in ILC activity and expansion occur independently of T cell activation. The most likely possibility is that they are the result of epithelial cell triggering and damage by DSS. We could indeed demonstrate that DSS exposure induces IL-33 production, probably by epithelial cells, which is a potent inducer of ILC2 activity and can lead to the activation of the amphiregulin/EGFR pathway. This can potentially explain the expansion of ILC2 during intestinal remodeling in our model. 31,32 Importantly, the high IL-33 production persisted in the absence of ILC after YTS treatment.
To know whether ILC contribute to tissue damage, fibrosis, and/or recovery in colitis, we then used a mAb to deplete ILC in RAG-1 −/− mice. RAG-1 −/− mice have a relative high proportion of ILC among CD45+ cells. To confirm the findings we also used a mouse strain lacking both ILC and adaptive immunity. Importantly, in both models no attenuation of fibrosis was observed neither after depletion of ILC nor in the absence of ILC, thus indicating that their role in fibrogenesis is negligible. However, in the absence of ILC a slower resolution of inflammation highlighted by a slower recovery of weight and higher mortality was observed rather pointing to a protective role of ILC. In contrast to murine models of lung and liver fibrosis, in our model depletion of ILC thus did not result in an attenuation of fibrosis. 12,14 Although the ILC2 in the DSS model produced IL-13 which is a profibrogenic cytokine, IL-13 is not required for fibrosis. We indeed have previously shown that fibrosis in this model can be induced in IL-13 deficient mice. 21,33 Our results are in contrast to the abrogation of acute and chronic innate colitis upon ILC depletion in an anti-CD40 model. 16 The anti-CD40 model is characterized by strong activation of macrophages and dendritic cells which supposedly then interact directly with ILC. 34 In DSS colitis, chemical disruption of the epithelial barrier might bypass the need of ILC by direct activation of fibroblasts. Further studies are required to identify ) and compared with control mice without DSS administration (n = 8 for both). Mice were killed on day 9 (acute) or day 63 (chronic). Colonic lamina propria cells were isolated, and the different types of myeloid cells were identified by staining and flow cytometry. Relative contribution of (A) total CD11b+ cells, (B) neutrophils, (C) eosinophils, (D) monocytes, and (E) macrophages as % of the total CD45+ population. Results of Mann-Whitney U testing to compare control and DSS groups within each background is shown. *P < .05, **P ≤ .01, ***P ≤ .001. Data are shown as individual values with median. Data are pooled from two independent experiments. DSS, dextran sulfate sodium; RAG, recombination activating gene differences in the pathways leading to fibroblast activation in both models. Furthermore, Salmonella-induced infection models of lung and colon fibrosis are prone to develop a type 2 immune response that can contribute to or be responsible for fibrosis, while DSS colitis is marked by a type I response. 13,35,36 Moreover, as ILC2 are scarce is the human intestine, both in normal and in IBD, contribution of these cells to fibrosis is made even more questionable. 35 Interestingly, slower recovery of weight after repeated inflammatory insults by DSS exposure was observed in RAG −/− γc −/− mice, suggesting a protective role for ILC. Protective effects of ILC via IL-22 production have been shown in the recovery phase of acute intestinal injury. 37 As almost no IL-22 was produced upon ILC depletion in our experiments, this might contribute to the delayed recovery in ILC deficient mice, and further points to a protective effect of ILC probably via IL-22 production and eventually other cytokines. However, as recovery was not impaired in ILC-depleted mice, this effect is arguably due to impaired cytokine signaling in RAG −/− γc −/− mice.
We also evaluated expansion of neutrophils, eosinophils, and monocytes in chronic DSS colitis in the different strains. All three cell types were indeed expanded in each of the strains and also after YTS treatment. These inflammatory cells might contribute and/or be essential for the induction of chronic intestinal remodeling in an ILC-independent innate immune process. Upon intestinal injury, neutrophils are the first cells recruited into the intestinal lining as a first line of response. 25 Neutrophils are short-living cells but are still present up to 2 weeks after last DSS exposure in the chronic DSS model, suggesting their contribution not only to active inflammation but also to recovery and remodeling. In IBD patients, neutrophils accumulate around abscesses and contribute to cryptitis, but their presence does not correlate with strictures. 38,39 Importantly, studies in experimental pulmonary fibrosis have shown a neutrophil dependent IL-17A pathway in the induction of lung fibrosis. 40 However, IL-17A neutralization in our model did not result in attenuation of intestinal fibrosis. 21 The observed increase of eosinophils in RAG-1 −/− but not in RAG −/− γc −/− mice after three cycles of DSS can be explained by the absence of the common γc in the latter strain. Carlens et al 41 could show a reduction of eosinophils specifically in the intestine in RAG −/− γc −/− mice, together with a reduction in CCL11 (eotaxin-1). Eosinophils can also be drivers of inflammatory damage and potentially contribute to tissue repair and remodeling in IBD. [42][43][44][45] Eosinophils, sources of IL-4 and IL-13, have been implicated in tissue remodeling in other diseases including eosinophilic oesophagitis, asthma, and hypereosinophilic syndrome. 44,45 More recently, eosinophils were identified as a crucial pathogenic source of intestinal fibrosis in radiation-induced intestinal fibrosis. 46 Furthermore, the role of an IL-33eosinophil pathway was recently shown in pediatric CD strictures and these findings were validated in SAMP1/Yit mice. 47 As administration of DSS leads to a disruption of the epithelial barrier and release of mature IL-33, elevated levels of IL-33 can be a potential driver of inflammation and remodeling in our model. However, IL-33 plays a dual role as IL-33 −/− mice showed an exacerbation of acute DSS colitis due to decreased levels of KC and MIP-2 and an impaired neutrophil resolution of damage, while exogenous IL-33 administration attenuated chronic DSS inflammation 48,49 In conclusion, our results show that DSS-induced chronic murine colitis and fibrosis can develop independently of T cells, B cells, and ILC, pointing to an innate immune response mediated by neutrophils, macrophages, and/or eosinophils, as the drivers of inflammation and fibrosis. Our results do not exclude a contributing/modulatory role of ILC, especially of ILC2, but ILC2 are clearly not essential to the process of experimental intestinal fibrogenesis. On the other hand, ILC3 and their IL-22 production might be protective against epithelial damage. We emphasize that these results were obtained in a particular murine model, which does not necessarily reflect the human situation. However, also in human IBD more data now support a primary role of myeloid cells. 6,7