Interleukin 11 expression causes murine inflammatory bowel disease

Interleukin 11 (IL11) is a profibrotic cytokine, secreted by myofibroblasts and damaged epithelial cells. Smooth muscle cells (SMCs) also secrete IL11 under pathological conditions and express the IL11 receptor. Here we examined the effects of SMC-specific, conditional expression of murine IL11 in a transgenic mouse (Il11SMC). Within days of transgene activation, Il11SMC mice developed loose stools and progressive bleeding and rectal prolapse, which was associated with a 65% mortality by two weeks. The bowel of Il11SMC mice was inflamed, fibrotic and had a thickened wall, which was accompanied by activation of ERK and STAT3. In other organs, including heart, lung, liver, kidney and skin there was a phenotypic spectrum of fibro-inflammation, together with consistent ERK activation. To investigate further the importance of stromal-derived IL11 in the inflammatory bowel phenotype we used a second model with fibroblast-specific expression of IL11, the Il11Fib mouse. This additional model largely phenocopied the Il11SMC bowel phenotype. These data show that IL11 secretion from the stromal niche is sufficient to drive inflammatory bowel disease in mice. Given that IL11 expression in colonic stromal cells predicts anti-TNF therapy failure in patients with ulcerative colitis or Crohn’s disease, we suggest IL11 as a therapeutic target for inflammatory bowel disease.

characterized key organs that may be affected by SMC pathobiology in Il11 SMC mice to better understand the role of SMC-derived IL11.

Expression of Il11 in smooth muscle cells results in ill health and early mortality
We generated the Il11 SMC mouse model that overexpresses IL11 specifically in Myh11 +ve smooth muscle cells: Conditional transgenic mice with mouse Il11 inserted into the Rosa26 locus (Rosa26-Il11-Tg) [8] were crossed with smooth muscle-specific Myh11-cre/ERT2 mice [15] (Fig. 1a, b). We then injected tamoxifen (tam) three times at day 0, 3 and 5 into 6-week old Il11 SMC mice to induce recombination in Myh11 +ve cells and monitored survival and body weight for 14 days. Following tam-induced Il11 expression in SMCs, mice started dying from day three onwards, with only 37% of Il11 SMC mice surviving to day 14. This was significantly different from the survival of either vehicle-treated Il11 SMC animals or tam-treated Cre SMC control mice, which were unaffected and both had 100% survival (both P < 0.001; Fig 1d and Supplementary Fig 1b). Starting from day four onwards, tam-treated Il11 SMC mice progressively lost weight as compared to vehicle-treatment and tam-treated Cre SMC controls (both P < 0.001 ; Fig 1e and Supplementary Fig 1d). Following two weeks of tam-induced Il11 expression, Il11 SMC mice were significantly smaller in body weight and length as compared to tam-treated Cre SMC controls (both P < 0.001; Supplementary Fig 1f, g) and vehicle-treated Il11 SMC mice (P = 0.002 and P < 0.001 respectively ; Fig 1f, g). In contrast, the indexed weight of the heart, lung and kidney in tam-treated Il11 SMC animals was significantly elevated (P Heart < 0.001; P Lung < 0.001; P Kidney = 0.006) when compared to vehicle treated mice (Fig 1h). We did not observe differences in liver weight or colon length in veh or tam treated Il11 SMC animals (data not shown). indicates the presence of the wild-type transgene (bottom gel). d Survival curve of Il11 SMC mice treated with tam (n = 35) and corn oil vehicle (veh; n = 12) mice following tamoxifen initiation at day 0 and followed until day 14. Survival curves were compared using the logrank Mantel-Cox test. e Body weight changes (expressed as percentage of day 0 body weight) in Il11 SMC mice treated with tam or veh (n = 8 per group). Green arrows denote individual injections. Statistical analyses by two-way ANOVA with Sidak multiple comparisons; data expressed as mean ± standard deviation. f-g Collated body weights (left) and body lengths (right) of Il11 SMC mice treated with tam or veh measured at d14 post initial tamoxifen dose (n = 12-13 per group). h-j Organ weights of the heart, lung and kidney normalized to body weight in Il11 SMC mice treated with tam or veh (n = 12-13 per group). All comparisons were conducted in mice 14 days post-veh and tam treatment. Statistical analyses by two-tailed unpaired t-test; data expressed as median ± IQR, whiskers representing the minimum and maximum values.

Il11 expression causes severe inflammatory bowel disease associated with fibrosis
The most obvious and striking feature of Il11 SMC mice treated with tam was progressive rectal prolapse and pale loose stool formation from as early as day three after gene induction (Fig 2a and Supplementary 1c). Gross anatomical inspection of the gastrointestinal tract revealed injection and swelling of the small and large intestines of tamtreated Il11 SMC mice when compared to veh-treated controls (Fig 2b). Intestinal inflammation was specifically indicated by an increase in fecal calprotectin, a biomarker used to monitor disease activity in human colitis, of tam-treated Il11 SMC mice when compared to veh treatment (P < 0.001; Fig. 2b, c). Masson's trichrome staining of the colon indicated a very large increase in collagen deposition (P < 0.001 ; Fig 2d, e). Histology also showed a significant increase in the thickness of the smooth muscle-dominant muscularis propria (P = 0.040 ; Fig 2f). Quantitative hydroxyproline assessments revealed an increase in colonic collagen content in Il11 SMC mice after tam treatment (P < 0.001 ; Fig 2g), confirming the histological data.

Il11 expression in smooth muscle cells activates non-canonical Il11 signaling pathways
Given that smooth muscle cells are expressed in the walls of most organs, including the vasculature, bronchi, gastrointestinal and abdominal organs, we sought to confirm the expression of Il11 in Il11 SMC mice in specific organs. We performed western blotting across tissues harvested at 14 days after tamoxifen administration. This confirmed that Il11 protein was significantly upregulated at the protein level across all tissues tested (P colon = 0.034; P heart = 0.002; P lung = 0.039; P liver < 0.001; P kidney = 0.004; and P skin = 0.004; Fig. 3 and IL11 is a member of the IL6 family of cytokines, which are considered to signal via the Janus Kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) pathway [16].
However, we recently showed that the IL11 effect, both in vitro in fibroblasts and in vivo at the tissue level, is also dependent on non-canonical signaling via extracellular signalregulated kinase (ERK) [8][9][10]. To investigate both canonical and non-canonical signaling pathways after Il11 expression, we performed western blotting of phosphorylated (p) STAT3 or ERK1/2 and total protein levels and derived indices of kinase activation by normalising phosphorylation amounts to total protein levels (Fig 3 and Supplementary Figure 3). At baseline, ERK was phosphorylated at low levels in most tissues except for the skin. Upon IL11 expression, we detected a strong and significant activation of ERK in all tissues (P colon = 0.002; P heart = 0.004; P lung = 0.049; P liver = 0.056; P kidney = 0.001; and P skin < 0.001; Supplementary Figure 3). STAT3 phosphorylation was unchanged in the heart, lung and liver but was elevated in the colon and skin (P = 0.05 and 0.001 respectively; Supplementary Figure 3). In contrast, total levels of STAT3 appeared to be increased in the liver and kidney of tam-treated Il11 SMC animals (Figure 3d and e). Overall, while both pathways were affected, ERK signaling was consistently activated across tissues tested whereas STAT3 was not.

IL11 destroys tissue integrity and promotes collagen deposition
To investigate the effect of Il11 expression in SMCs on tissue composition beyond the colon, we performed histological analyses of the heart, lung, liver, kidney and skin. Masson's trichrome staining was used to visualize collagen and quantify extracellular matrix deposition. In the heart, we observed collagen deposition in the perivascular region (P = 0.002 ; Fig 4a, b). We also observed vascular hypertrophy (P = 0.019; Fig 4c) and mild ventricular hypertrophy in the absence of dilatation (data not shown). Hydroxyproline assay of the whole heart confirmed cardiac fibrosis (P = 0.026 ; Fig 4d). In the lung, Ashcroft scores of pulmonary histological images showed lung damage after tam-induced Il11 expression (P The effect of Il11 expression on the liver was overall mild and characterized by perisinusoidal fibrosis (Fig 4h-j). Renal tissue structure was also affected only mildly, with limited fibrosis occurring around the blood vessels (Fig 4k-m). The effect of IL11 on the skin of tam-treated Il11 SMC animals was more profound and both the dermal and epidermal thickness was significantly increased (Fig 4n-p; P = 0.041 and P = 0.001 respectively).
Dorsal skin sections showed that epidermal and dermal cell infiltrates were increased and the adipose tissue layer in the hypodermis was largely depleted. Confirming Masson's trichrome staining of skin sections, we observed increased collagen deposition in the skin of tam-treated Il11 SMC using the hydroxyproline assay (P < 0.001 ; Fig 4q).

IL11 secretion from smooth muscle cells drives fibrogenic gene expression
To extend the observation of multi-organ fibrosis from our histology studies, we assessed the RNA expression of fibrogenic genes. Reverse transcription-polymerase chain reaction (RT-PCR) was performed using RNA from colonic, ventricular, pulmonary, hepatic, renal and skin tissue of vehicle-or tam-treated Il11 SMC mice. Collagen, type I, alpha 1 (Col1a1) RNA was significantly upregulated in all tissues (P colon = 0.005; P heart = 0.005; P lung < 0.001; P liver = 0.016; P kidney = 0.022; P skin = 0.016; Fig 5), confirming the effect of Il11 expression on global organ fibrosis that we observed on the protein level (Fig 4). Additional markers for fibrosis such as collagen, type I, alpha 2 (Cola1a2), collagen, type III, alpha 1 (Col3a1), fibronectin 1 (Fn1), tissue inhibitor of metalloproteinase 1 (Timp1) and matrix metallopeptidase 2 (Mmp2) were also assessed via RT-PCR (Fig 5a-f). These genes were elevated in most tissues of tam-treated Il11 SMC mice. Timp1 transcripts were significantly upregulated in the heart (P < 0.001), lung (P = 0.004), liver (P = 0.003), kidney (P = 0.003) and skin (P = 0.017), which is a recognised feature of pathological ECM remodeling [17]. Statistical analyses by two-tailed unpaired t-test; data expressed as median ± IQR, whiskers representing the minimum and maximum values.

IL11 secreted from smooth muscle cells causes widespread inflammation
In addition to fibrosis, SMC-driven diseases are often characterized by tissue inflammation.
To better understand whether IL11 secretion from SMCs can contribute to this pathology, we performed RT-PCR experiments of inflammatory marker genes across multiple tissues.
Interleukin 6 (IL6) also signals via gp130, similar to IL11, but its specific IL6 receptor subunit is expressed on a different subset of cells, most of which belong to the immune system [8].
IL6 is also a well-established therapeutic target for inflammatory diseases such as rheumatoid arthritis [18]. Upon tam-induced Il11 expression in Il11 SMC mice, we found Il6 mRNA to be significantly upregulated across all tissues tested (P colon = 0.001; P heart < 0.001; P lung = 0.015; P liver = 0.007; P kidney < 0.001; and P skin = 0.003; Fig 6).
In the colon, we also detected increased RNA expression of the inflammatory chemokine C-C motif chemokine ligand 2 (Ccl2) (P = 0.017), whereas C-C motif chemokine ligand 5 (Ccl5) was not significantly elevated but trended upwards (P = 0.141). Interestingly, these inflammatory chemokines are upregulated in the colonic mucosa of IBD patients [19,20].
However, CCL2 transcripts, and not CCL5 transcripts, were found to be expressed in vesselassociated cells such as SMCs in IBD [19]. Given that Il11 SMC mice express Il11 in SMCs, it is consistent that the transcript expression of the chemokine expressed in this particular cellular niche in the colon is most affected. In the skin, all three inflammatory markers tested were highly upregulated. This points to an inflammatory gene expression signature in the skin that is reminiscent of that seen in systemic sclerosis, since IL6, CCL2, and CCL5 are elevated in the serum of patients [21,22]. Of note, CCL2 levels were correlated with the extent of skin fibrosis in systemic sclerosis, a pathogenic feature also triggered by IL11 expression in SMCs (Fig. 4, 5) [21].

Fig 6. Relative gene expression of inflammatory genes in organs from tam-treated
Il11 SMC mice. a-f Relative mRNA expression of interleukin 6 (Il6), C-C motif chemokine ligand 2 (Ccl2), C-C motif chemokine ligand 5 (Ccl5) normalized to Glyceraldehyde 3phosphate dehydrogenase (Gapdh) expression in the colon, heart, lung, liver, kidney and skin respectively. All comparisons were conducted in 14 days post-veh (black) and tam (red) initiated mice. Statistical analyses by two-tailed unpaired t-test; data expressed as median ± IQR, whiskers representing the minimum and maximum values.

Fibroblast-selective expression of Il11 recapitulates the colonic inflammatory phenotype seen in Il11 SMC mice
We have previously described a model of Il11 expression in fibroblasts (Il11 Fib ) that drives fibrosis in the heart, kidney, and lung [8,9]. To examine further the effect of Il11 expression in stromal cells on the colon, we studied colonic phenotypes in this second model of Il11 expression from the stromal niche (Fig 7a). Gross examination of the gastrointestinal tract of Il11 Fib mice revealed macroscopic appearances consistent with inflammation of the colon to a similar extent as in Il11 SMC mice (data not shown). The total gastrointestinal gut length of Il11 Fib mice was unchanged overall but the colon length alone was reduced (P = 0.030; Fig   7b-c), which is a feature of experimental colitis in mice [23]. Inflammation of the gut was apparent in the Il11 Fib model as fecal calprotectin was significantly elevated (P = 0.003). In this model, as compared to Il11 SMC mice, we detected Il6 but not Ccl2 or Ccl5, upregulation in the colon (Fig 7d, e). Histological examination revealed marked colonic dilation and increased SMC thickness (Fig 7f, g). In contrast to the Il11 SMC model of Il11 expression, colonic fibrosis as determined by histology, hydroxyproline assay or ECM gene expression, was not significantly different between tam-treated Il11 Fib and controls (data not shown).
Taken together, fibroblast-driven Il11 expression recapitulates primarily the SMC-driven inflammatory, but not the fibrotic, phenotype in the mouse.

Discussion
In humans, IL11 is highly upregulated in the colonic mucosa of patients with either ulcerative colitis or Crohn's disease who do not respond to anti-TNF therapy, with recent single cell RNA-seq studies localizing IL11 to inflammatory mucosal stromal cells [24][25][26]. To better understand the effect of IL11 in the colon, recombinant human IL11 has been used in rodent models of IBD [27][28][29][30] and it was suggested that IL11 may have a protective role in the bowel. However, a caveat with these studies is that human IL11 was administered to rodents despite the fact that human IL11 does not activate some types of mouse cells [8]. Hence, there is a need to assess the effects of species-specific IL11 in the mouse, which we undertook in this study by expressing murine Il11 in SMCs or fibroblasts in adult mice.
To enable our studies, we developed the Il11 SMC mouse as a tool to study the effect of murine IL11 secreted from SMCs, an established source of IL11 in the vasculature, airway, and colon [11,31,32]. Surprisingly, expression of Il11 in SMCs was sufficient to induce severe colonic inflammation and rectal prolapse within 3 days, which was followed by early mortality in Il11 SMC animals. We also documented increased colonic muscle thickness, which is a characteristic of the dextran sulphate sodium-induced colitis model [33].
We explored further the IL11 effect in the bowel using an additional model that expresses mouse Il11 in a second stromal cell type: the fibroblast. This complementary model also develops severe diarrhea and inflammation of the colon, reinforcing the data generated in the Il11 SMC mice. These data show that Il11 expression alone in stromal cells is sufficient to cause an IBD phenotype and challenges the earlier data, based on the use of human IL11 in the mouse. The effect of IL11 on the vasculature will be discussed elsewhere. Considered together with patient studies that show IL11 to be highly upregulated in the colonic mucosa of patients with ulcerative colitis or Crohn's disease [24][25][26], our results highlight IL11 as a promising therapeutic target for IBD, particularly in the context of anti-TNF therapy resistance.

Mouse models
All experimental procedures were approved and conducted in accordance to the SingHealth Institutional Animal Care and Use Committee (IACUC). All mice were from a C57BL/6JN genetic background and they were bred and housed in the same room and provided food and water ad libitum.

Smooth muscle-specific Il11 transgenic model
To direct transgene expression in smooth muscle cells, we crossed the heterozygous Rosa26-Il11 (Gt(ROSA)26Sor tm1(CAG-Il11)Cook ) mouse [8] to the hemizygous SMMHC-CreERT2 (B6.FVB-Tg(Myh11-cre/ERT2)1Soff/J) mouse [15] available from the Jackson Laboratory For genotyping of mice genomic DNA, we performed polymerase chain reaction (PCR) on the tail biopsies which were obtained at the time of weaning. Genotyping was conducted in two sequential PCRs, for Myh11-Cre and Rosa26-Il11 genes separately. Agarose gel electrophoresis was subsequently conducted to confirm the respective product sizes for genotyping. Genotyping primer sequences are listed in Supplementary Table 1.

Fibroblast-specific Il11 transgenic model
To model fibroblasts-selective secretion of IL11 in vivo, we crossed the heterozygous Rosa26-IL11 mice with Col1a2-CreER mice [34] to generate double heterozygous Col1a2-CreER:Rosa26-Il11 mice (referred to as Il11 Fib ) [9]. For Cre-mediated Il11 transgene induction, Il11 Fib mice were intraperitoneal injected with 50 mg kg -1 tamoxifen at 6 weeks of age for 10 consecutive days and the animals were sacrificed on day 21. Wildtype littermates (designated as control) were injected with an equivalent dose of tamoxifen for 10 consecutive days as controls. Both female and male mice were used.
Colon length was measured from the caecum to the anus. The most distal half was taken for histology and the adjacent part was portioned and immediately snap frozen in liquid nitrogen for downstream molecular work (hydroxyproline assay, western blot analysis and quantitative polymerase chain reaction assessment). The excised heart was halved from the base to mid ventricle for histology and the remainder separated into 3 portions for molecular work. The left lung was isolated for histology and the right lung separated into 3 portions for molecular work. The right lobe of the liver was excised for histology and the left lobe separated into 3 portions for molecular work. The left kidney was fixed for histology and the right kidney separated in thirds for molecular work. The dorsal skin was harvested and halved for histology and molecular work.

Hydroxyproline assay
The amount of total tissue collagen was quantified using colorimetric detection of hydroxyproline using the Quickzyme Total Collagen assay kit (Quickzyme Biosciences) performed according to the manufacturer's protocol. All samples were run in duplicate and absorbance at 570 nm was detected on a SpectraMax M3 fluorescence microplate reader using SoftMax Pro version 6.2.1 software (Molecular Devices).

Fecal calprotectin (S100A8/A9) levels
To characterize inflammation in the gut, we investigated levels of fecal calprotectin in the Il11 SMC and Il11 Fib mice using the mouse S100A8/A9 heterodimer duoset ELISA kit (DY8596-05, R&D systems). Calprotectin is a biomarker for inflammatory activity and has been clinically applied as a diagnostic tool for inflammatory bowel diseases [35,36]. Stool samples were collected in a 1.5 ml tube and diluted with 50x (weight per volume) of extraction buffer (0.1 M Tris, 0.15 M NaCl, 1.0 M urea, 10 mM CaCl2, 0.1 M citric acid monohydrate, 5 g/l BSA (pH 8.0)) with the assumption of fecal density to be 1 g/ml. Samples were homogenized until no large particles were present. Homogenate was transferred into a fresh tube and further centrifuged at 10,000 g at 4 °C for 20 minutes. The supernatant was assessed for S100A8/A9 levels by ELISA as per the manufacturer's instructions.

RT-qPCR
Total RNA was extracted from snap-frozen tissues using RNAzol RT (R4533, Sigma-Aldrich) followed by Purelink RNA mini kit (12183025, Invitrogen) purification. The cDNA was prepared using iScript cDNA synthesis kit (1708891, Bio-Rad) as per the manufacturer's instructions. Quantitative RT-PCR gene expression analysis was performed on duplicate samples using fast SYBR green (Qiagen) technology using the ViiA 7 Real-Time PCR System (Applied Biosystem). RT-qPCR primers are listed in Supplementary Table 2.
Expression data were normalized to Gapdh mRNA expression levels and the 2 -ΔΔCT method was used to calculate the fold change.

Immunoblotting
Western blots were carried out on total protein extracts from mouse tissues. Frozen tissues were homogenized and lyzed in radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors (Roche) followed by centrifugation. Equal amounts of

Histology
Tissues from Il11 SMC and Il11 Fib mice were fixed in 10% neutral-buffered formalin for 24-48 hours, tissue processed and paraffin-embedded. Sections were obtained at 5 µm and stained with Masson's trichrome staining for collagen. Brightfield photomicrographs of the sections were randomly captured by a researcher blinded to the treatment groups using the Olympus BX51 microscope and Image-Pro Premier 9.2 (Media Cybernetics).
Photomicrographs of the colon taken at 200X magnification were used to calculate muscle wall thickness. The distance between the inner and outer circumference of the muscularis propria was measured using the incremental distance tool at a calibrated step size of 25 µm on Image-Pro Premier 9.2 (Media Cybernetics). A total of 75 to 250 measurements across three to five photomicrographs per section were taken and averages reported per photomicrograph. Muscle thickness was reported as an average across 3 cross-sections of the colon per animal.
Photomicrographs of the dorsal skin were captured in 3 fields per section at 100X magnification and used to calculate epidermal and dermal thickness. The epidermis was measured from the stratum basale to the stratum granulosum using hand-drawn line segments on Image-Pro Premier 9.2 (Media Cybernetics). The dermis was measured from the dermal-epidermal junction to the hypodermis. Measurements were recorded using the incremental distance tool at a calibrated step size of 50 µm on Image-Pro Premier 9.2 (Media Cybernetics). A total of 75 to 200 measurements across three photomicrographs per section were taken and averages reported per photomicrograph. Overall epidermal and dermal thickness was reported as an average across the 3 fields per animal.
Fibrosis quantification was conducted as referenced [37]. Color deconvolution version 1.5 plugin using the Masson Trichrome vector on ImageJ (version 1.52a, NIH) and thresholding was applied for area quantification. Perivascular fibrosis was measured as a ratio of the fibrosis area to the vessel area. Vascular hypertrophy was quantified as the ratio of media wall area to the lumen area.

Statistical analysis
Data are presented as mean ± standard deviation or median ± range as stated in figure legends. Statistical analyses were performed on GraphPad Prism 8 software (version 8.1.2).
Comparison of survival curves was analyzed with the log-rank Mantel-Cox test. Bodyweight progression was analyzed with two-way ANOVA with Sidak multiple comparisons. A comparison of mice strains for all other parameters was analyzed with a two-tailed unpaired t-test. The criterion for statistical significance was established at P < 0.05.