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• W2808558566 abstract "Research Article15 June 2018Open Access Transparent process Myeloid p38α signaling promotes intestinal IGF-1 production and inflammation-associated tumorigenesis Catrin Youssif Catrin Youssif Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology, Barcelona, Spain Search for more papers by this author Monica Cubillos-Rojas Monica Cubillos-Rojas Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology, Barcelona, Spain Search for more papers by this author Mònica Comalada Mònica Comalada Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology, Barcelona, Spain Search for more papers by this author Elisabeth Llonch Elisabeth Llonch Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology, Barcelona, Spain Search for more papers by this author Cristian Perna Cristian Perna Hospital Universitario Ramón y Cajal, IRYCIS, Madrid, Spain Search for more papers by this author Nabil Djouder Nabil Djouder Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain Search for more papers by this author Angel R Nebreda Corresponding Author Angel R Nebreda [email protected] orcid.org/0000-0002-7631-4060 Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology, Barcelona, Spain ICREA, Barcelona, Spain Search for more papers by this author Catrin Youssif Catrin Youssif Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology, Barcelona, Spain Search for more papers by this author Monica Cubillos-Rojas Monica Cubillos-Rojas Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology, Barcelona, Spain Search for more papers by this author Mònica Comalada Mònica Comalada Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology, Barcelona, Spain Search for more papers by this author Elisabeth Llonch Elisabeth Llonch Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology, Barcelona, Spain Search for more papers by this author Cristian Perna Cristian Perna Hospital Universitario Ramón y Cajal, IRYCIS, Madrid, Spain Search for more papers by this author Nabil Djouder Nabil Djouder Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain Search for more papers by this author Angel R Nebreda Corresponding Author Angel R Nebreda [email protected] orcid.org/0000-0002-7631-4060 Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology, Barcelona, Spain ICREA, Barcelona, Spain Search for more papers by this author Author Information Catrin Youssif1, Monica Cubillos-Rojas1, Mònica Comalada1, Elisabeth Llonch1, Cristian Perna2, Nabil Djouder3 and Angel R Nebreda *,1,4 1Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology, Barcelona, Spain 2Hospital Universitario Ramón y Cajal, IRYCIS, Madrid, Spain 3Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain 4ICREA, Barcelona, Spain *Corresponding author. Tel: +34 934031379; E-mail: [email protected] EMBO Mol Med (2018)10:e8403https://doi.org/10.15252/emmm.201708403 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The protein kinase p38α plays a key role in cell homeostasis, and p38α signaling in intestinal epithelial cells protects against colitis-induced tumorigenesis. However, little is known on the contribution of p38α signaling in intestinal stromal cells. Here, we show that myeloid cell-specific downregulation of p38α protects mice against inflammation-associated colon tumorigenesis. The reduced tumorigenesis correlates with impaired detection in the colon of crucial chemokines for immune cell recruitment. We identify insulin-like growth factor-1 (IGF-1) as a novel mediator of the p38α pathway in macrophages. Moreover, using genetic and pharmacological approaches, we confirm the implication of IGF-1 produced by myeloid cells in colon inflammation and tumorigenesis. We also show a correlation between IGF-1 pathway activation and the infiltration of myeloid cells with active p38α in colon samples from patients with ulcerative colitis or colon cancer. Altogether, our results uncover an important role for myeloid IGF-1 downstream of p38α in colitis-associated tumorigenesis and suggest the interest in evaluating IGF-1 therapies for inflammation-associated intestinal diseases, taking into consideration IGF-1 signaling and immune cell infiltration in patient biopsies. Synopsis The molecular & cellular events involved in the pathogenesis of colitis-associated tumorigenesis are not fully understood. Targeted inhibition of the p38α pathway in myeloid cells could be therapeutically useful in colon diseases associated with inflammatory cell infiltration. Myeloid cells rely on p38α to promote intestinal inflammation and inflammation- associated colon tumorigenesis. IGF-1 functions as an effector of p38α in myeloid cells, which are an important source of IGF-1 in the colon. Genetic or pharmacological inhibition of IGF-1 suppresses inflammatory cell recruitment and reduces colitis-associated colon tumor burden. Samples from ulcerative colitis and colon cancer patients showed a positive correlation between recruitment of myeloid cells with active p38α and activation of the IGF-1 pathway. Introduction Colorectal cancer (CRC) is the third most common cancer and the fourth leading cause of cancer-related mortality. The risk of developing CRC is significantly increased in individuals diagnosed with inflammatory bowel disease (IBD), a chronically relapsing inflammatory disorder (Coussens & Werb, 2002). The link between inflammation and carcinogenesis was postulated more than 150 years ago (Balkwill & Mantovani, 2001). However, in spite of the progress in IBD research, the mechanisms and the pathogenesis underlying colitis-associated cancer (CAC) are only partially understood (Kaplan & Ng, 2017). The co-evolution of mammals with their intestinal flora has led to a situation of tolerance in the gut. The equilibrium between immune activation and suppression (intestinal tolerance) is marked by a situation of controlled “physiological inflammation”, where distinct populations of resident and inflammatory macrophages in the gut maintain a balance and ensure protective immunity when required (Fiocchi, 2008; Mowat & Bain, 2011). Deregulation of this equilibrium, as it happens in IBD, implicates an imbalance between pro- and anti-inflammatory cytokines, impeding the resolution of inflammation and leading to disease perpetuation and tissue destruction (Neurath, 2014). Cytokines play an important role in the repair of the intestinal epithelia, but have been also implicated in tumor promotion (Schneider et al, 2017). Several signaling pathways are involved in macrophage activation, phenotype plasticity, and the regulation of proliferation and survival. One of these pathways relies on the protein kinase p38α (Cuadrado & Nebreda, 2010), which regulates cytokine production and inflammatory responses (Kim et al, 2008; Wagner & Nebreda, 2009). Genetic inactivation of p38α in intestinal epithelial cells has provided evidence for the ability of this signaling pathway to suppress inflammation-associated colon tumorigenesis (Gupta et al, 2014). On the other hand, mice with myeloid cell-specific deletion of p38α show less inflammation in response to dextran sodium sulfate (DSS) when compared to wild-type (WT) mice (Otsuka et al, 2010). However, whether and how myeloid p38α signaling can affect intestinal repair mechanisms and tumorigenesis remains unclear. During colorectal carcinogenesis, colonic epithelial cells accumulate genetic mutations, which are mostly induced by environmental factors and confer a selective growth advantage. Nevertheless, it is clear that an inflammatory microenvironment involving growth factors and cytokines secreted by activated monocytes and macrophages plays a pivotal role in the formation of tumors (Baylin & Ohm, 2006; Chanmee et al, 2014). Therefore, targeting the recruitment of immune cells to the site of inflammation has emerged as a potential strategy to impede tumorigenesis (Grivennikov et al, 2010). Myeloid cells are the predominant leukocytes that infiltrate tumors and are known to support tumor initiation and progression. Since p38α controls the production of leukocyte chemo-attractants and other pro-inflammatory mediators (Kim et al, 2008; Cuadrado & Nebreda, 2010), we investigated the contribution of myeloid p38α signaling to CAC. We found that mice with p38α-deficient myeloid cells show reduced inflammation-associated colon tumorigenesis. In a recent screening for bone marrow-derived macrophage (BMDM)-produced cytokines that are regulated by p38α signaling, we identified IGF-1, a peptidic hormone that is involved in inflammatory cell recruitment and has repair functions with high tumorigenic potential (Mourkioti & Rosenthal, 2005; Roussos et al, 2011; Tonkin et al, 2015). We show that p38α signaling regulates IGF-1 expression in intestinal macrophages and that IGF-1 signaling promotes colitis and inflammation-associated colon tumorigenesis. Our results indicate that myeloid p38α through the production of IGF-1 controls colon inflammation and tumorigenesis. Results Mice with p38α-deficient myeloid cells show decreased susceptibility to colon tumorigenesis To evaluate the role of myeloid p38α in inflammation-associated colon tumorigenesis, we generated mice expressing LysM-Cre and p38α-lox alleles (p38α-ΔMC). Intestinal macrophages were isolated (Fig EV1A), and the efficiency of p38α downregulation was confirmed by qRT–PCR quantification of the floxed exon 2 in p38α mRNA (Fig 1A) and by immunoblotting of p38α in peritoneal macrophages (Fig EV1B). We found that WT mice treated with azoxymethane (AOM) and DSS to induce CAC (Appendix Fig S1A) developed rectal prolapse, which was observed less often in p38α-ΔMC mice (Appendix Fig S1B). Macroscopic tumors were mainly located in the distal to middle colon of both WT and p38α-ΔMC mice (Fig 1B). The overall tumor load was significantly reduced in p38α-ΔMC mice compared to WT mice, mainly due to a decrease in the number of tumors (Fig 1C). Although the average tumor size was similar (Fig EV1C), p38α-ΔMC mice had less tumors larger than 4 mm and no tumors larger than 6 mm (Fig EV1D). In agreement with the reduced tumorigenesis and the lower number of big tumors observed in p38α-ΔMC mice, we also found lower cell proliferation rates evaluated by Ki67 staining in tumors from these mice (Fig EV1E). However, apoptosis determined by TUNEL or cleaved caspase-3 staining did not show significant differences between tumors from WT and p38α-ΔMC mice (Appendix Fig S1C and D). Click here to expand this figure. Figure EV1. Downregulation of p38α in myeloid cells reduces inflammation in the tumor microenvironment A. Representative images of intestinal macrophages isolated from the colonic lamina propria and stained for F4/80 and CD115. Scale bars, 10 μm (F4/80) and 100 μm (CD115). B. Representative immunoblots of p38α in lysates from peritoneal macrophages. C. Average tumor size (n ≥ 11). D. Number of tumors > 4 and >6 mm in AOM/DSS-treated mice (n ≥ 11). E, F. Representative sections from normal colon epithelia and colon tumors stained for Ki67 (n ≥ 6) (E) and phospho-STAT3 (n ≥ 3) (F). Quantifications are shown in the histogram. Scale bars, 100 μm. Data information: Statistical analysis was performed by using Mann–Whitney test for the comparison of two groups or ANOVA using Bonferroni post hoc correction for multiple groups. Data are expressed as the average ± SD. Download figure Download PowerPoint Figure 1. Downregulation of p38α in myeloid cells reduces colitis-associated tumorigenesis Analysis by qRT–PCR of the levels of floxed exon 2 versus exon 12 (as a control) of the mRNA encoding p38α in intestinal macrophages (n ≥ 5). Representative images of colon tumors in AOM/DSS-treated mice. Red arrows indicate macroscopically visible tumors that were measured. Average tumor number and load in AOM/DSS-treated mice (n ≥ 11). The experiment was performed three times. Percentage of Ly6ChiCCR2+ cells in the bone marrow cells that were alive and CD45+CD11b+ from AOM/DSS-treated mice (n ≥ 3). Representative sections from normal colon epithelia and colon tumors stained for F4/80. Quantifications are shown in the histogram (n = 4). The area indicated was used for quantification and was selected for all the immunohistochemistry quantifications of epithelium. Scale bars, 100 μm. Quantifications of a mouse cytokine antibody array that was interrogated using pools of 3-mm tumors derived from AOM/DSS-treated mice either WT or p38α-ΔMC (n = 8/genotype). Arbitrary units (a.u.) are referred to the expression level of each cytokine in tumors from WT mice, which was given the value of 1. Data information: Statistical analysis was performed by using Mann–Whitney test for the comparison of two groups or ANOVA using Bonferroni post hoc correction for multiple groups. Data are expressed as the average ± SD. Download figure Download PowerPoint Myeloid p38α controls the tumor-promoting inflammatory microenvironment Given the important contribution of immune cells to the inflammatory microenvironment, we evaluated the number of inflammatory monocytes in the bone marrow. The C-C chemokine receptor type (CCR) 2 is very important for Ly6Chi monocyte trafficking, and it is well accepted that Ly6Chi monocytes rely on CCR2 to egress from the bone marrow to the inflamed and healthy intestine, where they can give rise to different types of macrophages (Bain & Mowat, 2014). We found significantly less Ly6ChiCCR2+ inflammatory monocytes in the bone marrow of p38α-ΔMC mice compared to WT mice, indicating a weaker inflammatory response in tumor-bearing p38α-ΔMC mice (Fig 1D and Appendix Fig S1E). Therefore, we evaluated the immune cell infiltrate in the tumors. In agreement with the reduced levels of inflammatory monocytes detected in the bone marrow of p38α-ΔMC mice, tumors in these mice showed less macrophage (F4/80+) infiltration compared to the those in WT mice (Fig 1E and Appendix Fig S1F). We further evaluated the phosphorylation status of signal transducer and activator of transcription 3 (STAT3), a potent activator of inflammatory pathways that contributes to oncogenic signaling leading to enhanced cell proliferation and tumor growth (Yu et al, 2009; Sanchez-Lopez et al, 2016). As expected, colon tumors showed enhanced STAT3 phosphorylation compared to the normal epithelium, and interestingly, STAT3 phosphorylation was reduced in tumors from p38α-ΔMC mice compared to WT mice (Fig EV1F). Next, we evaluated the expression of cytokines and chemokines in tumors (Fig 1F and Appendix Fig S1G). In line with the decreased macrophage infiltration and STAT3 phosphorylation levels observed in tumors from p38α-ΔMC mice, these tumors also showed reduced levels of chemokines important for monocyte/macrophage recruitment such as CXCL10, CCL3, and CCL2 (Balkwill & Mantovani, 2001; Zimmerman et al, 2008; Mowat & Bain, 2011), as well as STAT3-activating cytokines, such as G-CSF and IL-27 (Rebe et al, 2013). Since inflammation is an essential component of the AOM/DSS tumorigenesis protocol, we investigated the contribution of myeloid p38α to the DSS response (Appendix Fig S2A). In agreement with a previous report (Otsuka et al, 2010), we found that p38α-ΔMC mice were less susceptible to DSS-induced colitis, as indicated by reduced body weight loss (Appendix Fig S2B) and a lower disease activity index (DAI; Fig 2A). In line with the decreased severity of colitis indicators, the large areas of complete crypt loss and erosions observed in colons from DSS-treated WT mice were strongly reduced in p38α-ΔMC mice. However, WT and p38α-ΔMC mice showed no differences in colon histology prior to DSS administration (Fig 2B and EV2A). We also observed that the numbers of macrophages (F4/80+), leukocytes (CD45+), activated neutrophils (MPO+), and T cells (CD3+) were all reduced in the immune cell infiltrates of colons from DSS-treated p38α-ΔMC mice compared to WT mice (Fig 2C and EV2B–D, and Appendix Fig S2C–E). Figure 2. Myeloid deletion of p38α decreases colitis susceptibility and leukocyte recruitment to the inflamed intestine Disease activity index was recorded daily in DSS-treated mice (n ≥ 9). The experiment was performed three times. Epithelial damage was quantified in untreated mice or mice treated with DSS for 6 days and analyzed at the indicated times (days) using H&E-stained colon sections (n ≥ 5). Representative colon sections from untreated mice or mice treated with DSS for 6 days were analyzed at day 7 for F4/80 staining. Quantifications are shown in the histogram (n ≥ 4). Scale bars, 100 μm. Representative colon sections from untreated mice or mice treated with DSS for 6 days and analyzed at day 7 were stained for phospho-STAT3. Quantifications are shown in the histogram (n ≥ 4). Scale bars, 100 μm. Quantifications of a mouse cytokine antibody array that was interrogated using pools of whole colon extracts derived from DSS-treated mice either WT or p38α-ΔMC at day 7 (n = 8/genotype). Arbitrary units (a.u.) are referred to the expression level of each cytokine in WT mice, which was given the value of 1. Data information: Statistical analysis was performed by ANOVA using Bonferroni post hoc correction. Data are expressed as the average ± SD. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Mice with p38α-deficient myeloid cells show reduced DSS-induced colitis and decreased leukocyte recruitment during intestinal inflammation A. Representative images of H&E-stained colon sections from animals either untreated or treated with DSS for 6 days and analyzed at the indicated days. Scale bars, 100 μm. B–D. Representative colon sections stained for CD45 (B), MPO (C), and CD3 (D) from untreated mice or mice treated with DSS for 6 days and analyzed at day 7 (n ≥ 5). Quantifications are shown in the histogram. Scale bars, 100 μm. Data information: Statistical analysis was performed by ANOVA using Bonferroni post hoc correction. Data are expressed as the average ± SD. Download figure Download PowerPoint Infiltrating immune cells produce cytokines that activate STAT3 and its target genes contributing to tumor-promoting inflammation (Yu et al, 2009). Accordingly, STAT3 phosphorylation was reduced in the colon epithelial cells of DSS-treated p38α-ΔMC mice compared to WT mice (Fig 2D). Moreover, ELISA analysis showed that the inflammatory mediator IL-1β was downregulated in colons from DSS-treated p38α-ΔMC mice compared to WT mice, and TNF-α was also induced at lower levels upon DSS treatment in p38α-ΔMC mice (Appendix Fig S2F). Further analysis of colons at day 7 after DSS treatment showed the downregulation of several other pro-inflammatory cytokines and chemokines in p38α-ΔMC mice, which likely contribute to inflammatory cell recruitment and disease promotion (Fig 2E and Appendix Fig S2G). Downregulation of p38α in myeloid cells reduces IGF-1 signaling during intestinal inflammation and tumorigenesis We have identified IGF-1 as one extracellular factor potentially regulated by p38α signaling in myeloid cells. Both intracellular and extracellular IGF-1 protein levels were reduced in p38α-deficient BMDMs compared with WT BMDMs (Fig 3A). IGF-1 mRNA expression was more potently induced by IL-4 than by LPS (Fig 3B), in agreement with its proposed role as marker for wound-healing macrophages (Tonkin et al, 2015; Spadaro et al, 2017), which contribute to tumor progression (Murray & Wynn, 2011). The implication of p38α signaling in IGF-1 expression by macrophages was confirmed by using chemical inhibitors (Fig 3B and C). Figure 3. p38α regulates IGF-1 production by macrophages Whole protein lysates (left panel) or supernatants (right panel) from bone marrow-derived macrophages (BMDMs) were used to analyze IGF-1 protein levels by ELISA (n = 5). BMDMs were starved for 18 h prior to stimulation with either LPS (10 ng/ml) or IL-4 (10 ng/ml) for the indicated times in the presence or absence of the indicated p38α inhibitors or vehicle (DMSO). The inhibitors were added to the medium 1 h prior to stimulation with LPS or IL-4, and IGF-1 mRNA expression levels were measured by qRT–PCR (n = 3). Arbitrary units (a.u.) are referred to the expression level in DMSO-treated control at 3 h, which was given the value of 1. BMDMs were starved overnight, and the indicated p38α inhibitors or DMSO were added to the medium 1 h prior to stimulation with IL-4 for 6 h. Supernatants were collected and used to measure IGF-1 protein levels by ELISA (n = 3). The expression level in the DMSO-treated control (about 700 pg/ml) was given the value of 1. Data information: Statistical analysis was performed by using Mann–Whitney test for the comparison of two groups or ANOVA using Bonferroni post hoc correction for multiple groups. Data are expressed as the average ± SD. Download figure Download PowerPoint To confirm that p38α downregulation in myeloid cells affects IGF-1 signaling during inflammation and tumorigenesis, we analyzed IGF-1 levels in mice treated with DSS or AOM/DSS. In response to DSS, intestinal macrophages switch from the initial classical activation phenotype to a wound-healing phenotype in the repair phase. Accordingly, we detected a clear reduction in IGF-1 levels in colons from p38α-ΔMC mice compared to WT mice during the repair phase at day 13, whereas no significant differences were observed in untreated colons or during the acute inflammatory phase at day 7 (Fig 4A). Analysis by qRT–PCR also showed lower levels of IGF-1 mRNA at day 13 in colon extracts from p38α-ΔMC mice compared to WT mice (Appendix Fig S3A). Consistently, IGF-1 mRNA levels were also reduced in p38α-deficient intestinal macrophages compared to WT macrophages at day 13 (Fig 4B), and the differences were even clearer than in whole colon extracts. Taken together, our results support a key role for p38α signaling in IGF-1 production by myeloid cells during the repair phase in the inflamed colon. However, we observed no differences in serum IGF-1 levels between WT and p38α-ΔMC mice (Appendix Fig S3B), suggesting that changes in IGF-1 signaling in the intestines were probably produced locally by myeloid cells. Figure 4. Downregulation of myeloid p38α reduces IGF-1 production and signaling during intestinal inflammation and tumorigenesis Colon protein lysates obtained from mice either untreated or treated with DSS for 6 days were analyzed at the indicated times to measure IGF-1 protein levels by ELISA (n ≥ 3). Intestinal macrophages were isolated from mice treated with DSS for 6 days and analyzed at day 13. IGF-1 mRNA levels were quantified by qRT–PCR (n ≥ 5). Representative colon sections from mice either untreated or treated with DSS for 6 days were analyzed at the indicated times for Ki67 staining. Quantifications are shown in the histogram (n ≥ 4). Scale bars, 100 μm. Representative colon sections from untreated mice or colon and tumor sections from AOM/DSS-treated mice were stained for phospho-IGF1R. Quantifications are shown in the histogram (n = 4). Scale bars, 100 μm. Data information: Statistical analysis was performed by using Mann–Whitney test for the comparison of two groups or ANOVA using Bonferroni post hoc correction for multiple groups. Data are expressed as the average ± SD. Download figure Download PowerPoint Consistent with the known mitogenic properties of IGF-1, we found significant differences in cell proliferation as determined by Ki67 staining between the colons of DSS-treated WT and p38α-ΔMC in the repair phase at day 13 (Fig 4C and Appendix Fig S3C), when IGF-1 protein levels were significantly different in colon extracts (see Fig 4A above). IGF-1 binds to and induces the autophosphorylation of the IGF-1 receptor (IGF1R). To assess IGF-1 signaling activity during intestinal inflammation and tumorigenesis, we performed immunohistochemistry staining for phospho-IGF1R followed by TMarker analysis (Schuffler et al, 2013) to differentiate between different staining intensities (Appendix Fig S3D). Importantly, IGF1R phosphorylation was reduced in colon tumors from p38α-ΔMC mice compared to WT mice (Fig 4D). IGF-1 promotes intestinal inflammation and inflammation-associated tumorigenesis IGF-1 has been implicated in inflammatory processes and immune cell recruitment (Mourkioti & Rosenthal, 2005). To evaluate the role of myeloid IGF-1 in intestinal inflammation, we used mice expressing LysM-Cre and IGF-1-lox alleles (IGF-1-ΔMC). The efficiency of IGF-1 mRNA downregulation was confirmed by qRT–PCR in isolated intestinal macrophages (Fig 5A) and peritoneal macrophages (Fig EV3A). We observed that DSS-treated IGF-1-ΔMC mice showed small differences in body weight loss (Fig EV3B), but had a more significant reduction in DAI (Fig EV3C and Appendix Fig S3E) as well as less epithelial damage at day 7 compared to WT mice (Figs 5B and EV3D). Figure 5. Impaired IGF-1 signaling decreases immune cell recruitment to the inflamed colon Relative IGF-1 mRNA levels in isolated intestinal macrophages (n = 5). Epithelial damage was evaluated in H&E-stained colon sections from mice treated with DSS for 6 days and analyzed at the indicated days (n ≥ 13). The experiment was performed three times. Percentage of Ly6ChiCCR2+ cells in the bone marrow cells that were alive and CD45+CD11b+ from mice treated for 6 days with DSS and analyzed at day 7 (n ≥ 5). Representative colon sections from mice treated with DSS for 6 days and analyzed at the indicated days were stained for F4/80. Quantifications are shown in the histogram (n ≥ 4). Scale bars, 100 μm. IGF-1 mRNA levels in colons from untreated mice were determined by qRT–PCR (n ≥ 5). Epithelial damage was quantified in mice untreated or treated with either PQ401 or vehicle and then with DSS for 6 days, and analyzed at the indicated days (n ≥ 7). This experiment was performed twice. Percentage of Ly6ChiCCR2+ cells in the bone marrow cells that were alive and CD45+CD11b+ from mice treated with vehicle or PQ401 and DSS for 6 days and sacrificed at day 7 (n ≥ 4). F4/80-positive cells were quantified in colon sections from untreated mice or mice treated with either PQ401 or vehicle and then untreated or treated with DSS for 6 days, and analyzed at day 7 (n ≥ 4). Data information: Statistical analysis was performed by using Mann–Whitney test for the comparison of two groups or ANOVA using Bonferroni post hoc correction for multiple. Data are expressed as the average ± SD. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Downregulation of IGF-1 in myeloid cells reduces susceptibility to intestinal inflammation A. Relative IGF-1 mRNA levels in peritoneal macrophages (n ≥ 8). B, C. Body weight (B) and disease activity index (C) were recorded daily during DSS-induced colitis (n ≥ 31). This is a pool from three experiments, which are shown individually in Appendix Fig S3C. D. Representative images of H&E-stained sections from mice either untreated or treated with DSS for 6 days and analyzed at the indicated days. Scale bars, 100 μm E, F. Representative colon sections stained for phospho-STAT3 (E) or phospho-IGF1R (F) from mice treated with DSS for 6 days and analyzed at the indicated days. Quantifications are shown in the histograms (n ≥ 7). Scale bars, 100 μm. Data information: Statistical analysis was performed by using Mann–Whitney test for the comparison of two groups or ANOVA using Bonferroni post hoc correction for multiple groups. Data are expressed as the average ± SD. Download figure Download PowerPoint Next, we investigated the role of IGF-1 in inflammatory cell recruitment and found significantly less Ly6ChiCCR2+ inflammatory monocytes in the bone marrow of DSS-treated IGF-1-ΔMC mice compared to WT mice (Fig 5C). In agreement with this, we observed a reduced number of macrophages (F4/80+; Fig 5D), and STAT3 phosphorylation levels were also reduced in colons from DSS-treated IGF-1-ΔMC mice (Fig EV3E). Interestingly, IGF-1 mRNA levels were reduced in colons from untreated IGF-1-ΔMC mice compared to WT mice, suggesting an important contribution of the IGF-1 produced by myeloid cells in the colon (Fig 5E). In line with this idea, quantification of IGF1R phosphorylation revealed a reduction of IGF-1 signaling in colons from DSS-treated IGF-1-ΔMC mice compared to WT mice (Fig EV3F). To confirm the implication of IGF-1 signaling in DSS-induced intestinal inflammation, we used PQ401, a chemical inhibitor of IGF1R autophosphorylation. WT mice were treated daily with PQ401, starting 1 day before the DSS treatment, and were compared with WT and p38α-ΔMC mice treated with vehicle and DSS (Appendix Fig S3F). As expected, the levels of IGF1R phosphorylation were reduced in the colon of mice treated with PQ401 (Appendix Fig S3G). In agreement with our results using IGF-1-ΔMC mice, treatment of WT mice with PQ401 r" @default.
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• W2808558566 title "Myeloid p38α signaling promotes intestinal <scp>IGF</scp> ‐1 production and inflammation‐associated tumorigenesis" @default.
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