Title: Myeloid cells protect intestinal epithelial barrier integrity through the angiogenin/plexin‐B2 axis
Abstract: Article8 June 2020free access Source DataTransparent process Myeloid cells protect intestinal epithelial barrier integrity through the angiogenin/plexin-B2 axis Rongpan Bai Institute of Environmental Medicine, and Cancer Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, Hangzhou, China Search for more papers by this author Desen Sun Institute of Environmental Medicine, and Cancer Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Muxiong Chen Institute of Environmental Medicine, and Cancer Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Xiaoliang Shi Institute of Environmental Medicine, and Cancer Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Liang Luo Department of Gastroenterology, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Zhengrong Yao Institute of Environmental Medicine, and Cancer Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Yaxin Liu Institute of Environmental Medicine, and Cancer Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Xiaolong Ge Department of General Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Xiangwei Gao orcid.org/0000-0002-8358-6320 Institute of Environmental Medicine, and Cancer Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Guo-fu Hu Molecular Oncology Research Institute, Tufts Medical Center, Boston, MA, USA Search for more papers by this author Wei Zhou Department of General Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Jinghao Sheng Corresponding Author [email protected] orcid.org/0000-0002-1207-7449 Institute of Environmental Medicine, and Cancer Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, Hangzhou, China Program in Molecular and Cellular Biology, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Zhengping Xu Corresponding Author [email protected] orcid.org/0000-0003-0922-736X Institute of Environmental Medicine, and Cancer Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, Hangzhou, China Program in Molecular and Cellular Biology, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Rongpan Bai Institute of Environmental Medicine, and Cancer Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, Hangzhou, China Search for more papers by this author Desen Sun Institute of Environmental Medicine, and Cancer Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Muxiong Chen Institute of Environmental Medicine, and Cancer Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Xiaoliang Shi Institute of Environmental Medicine, and Cancer Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Liang Luo Department of Gastroenterology, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Zhengrong Yao Institute of Environmental Medicine, and Cancer Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Yaxin Liu Institute of Environmental Medicine, and Cancer Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Xiaolong Ge Department of General Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Xiangwei Gao orcid.org/0000-0002-8358-6320 Institute of Environmental Medicine, and Cancer Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Guo-fu Hu Molecular Oncology Research Institute, Tufts Medical Center, Boston, MA, USA Search for more papers by this author Wei Zhou Department of General Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Jinghao Sheng Corresponding Author [email protected] orcid.org/0000-0002-1207-7449 Institute of Environmental Medicine, and Cancer Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, Hangzhou, China Program in Molecular and Cellular Biology, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Zhengping Xu Corresponding Author [email protected] orcid.org/0000-0003-0922-736X Institute of Environmental Medicine, and Cancer Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, Hangzhou, China Program in Molecular and Cellular Biology, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Author Information Rongpan Bai1,2,‡, Desen Sun1,‡, Muxiong Chen1,‡, Xiaoliang Shi1, Liang Luo3, Zhengrong Yao1, Yaxin Liu1, Xiaolong Ge4, Xiangwei Gao1, Guo-fu Hu5, Wei Zhou4, Jinghao Sheng *,1,2,6 and Zhengping Xu *,1,2,6 1Institute of Environmental Medicine, and Cancer Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China 2Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, Hangzhou, China 3Department of Gastroenterology, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China 4Department of General Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China 5Molecular Oncology Research Institute, Tufts Medical Center, Boston, MA, USA 6Program in Molecular and Cellular Biology, Zhejiang University School of Medicine, Hangzhou, China ‡These authors contributed equally to this work. *Corresponding author. Tel: +86 571 88208164; Fax: +86 571 88208164; E-mail: [email protected] *Corresponding author. Tel: +86 571 88208008; Fax: +86 571 88208164; E-mail: [email protected] EMBO J (2020)39:e103325https://doi.org/10.15252/embj.2019103325 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 Communication between myeloid cells and epithelium plays critical role in maintaining intestinal epithelial barrier integrity. Myeloid cells interact with intestinal epithelial cells (IECs) by producing various mediators; however, the molecules mediating their crosstalk remain incompletely understood. Here, we report that deficiency of angiogenin (Ang) in mouse myeloid cells caused impairment of epithelial barrier integrity, leading to high susceptibility to DSS-induced colitis. Mechanistically, myeloid cell-derived angiogenin promoted IEC survival and proliferation through plexin-B2-mediated production of tRNA-derived stress-induced small RNA (tiRNA) and transcription of ribosomal RNA (rRNA), respectively. Moreover, treatment with recombinant angiogenin significantly attenuated the severity of experimental colitis. In human samples, the expression of angiogenin was significantly down-regulated in patients with inflammatory bowel disease (IBD). Collectively, we identified, for the first time to our knowledge, a novel mediator of myeloid cell-IEC crosstalk in maintaining epithelial barrier integrity, suggesting that angiogenin may serve as a new preventive agent and therapeutic target for IBD. Synopsis Angiogenin is a secreted RNase with distinct roles in tRNA-derived stress-induced small RNA (tiRNA) generation and ribosomal RNA (rRNA) transcription Here, stroma-derived angiogenin is shown to maintain intestinal epithelial barrier integrity via regulation of tRNA and rRNA metabolism. Reduced angiogenin expression leads to increased intestinal inflammation and is linked to inflammatory bowel disease. Myeloid cell-derived angiogenin regulates intestinal barrier integrity. Angiogenin acts through its receptor plexin-B2 in intestinal epithelial cells to promote cell survival and proliferation. Angiogenin promotes tiRNA and rRNA synthesis in intestinal epithelium. Recombinant angiogenin protects against intestinal inflammation in mice. Introduction Inflammatory bowel disease (IBD), typically including Crohn's disease (CD) and ulcerative colitis (UC), is characterized with epithelial barrier integrity impairment and intestinal inflammation (Torres et al, 2017; Ungaro et al, 2017). Epithelial barrier damage leads to an excessive exposure of mucosal tissue to microbial antigens, followed by leukocyte recruitment, soluble mediator release, and ultimately mucosal inflammation (Peterson & Artis, 2014; Martini et al, 2017). A key event in epithelial barrier maintenance is to balance survival and growth of IECs, which is orchestrated by both epithelium itself and recruited immune cells in mucosal tissue (Negroni et al, 2015; Luissint et al, 2016). Mucosal innate myeloid leukocytes, such as neutrophils, monocytes, and macrophages, have been found to reside near the epithelium and interact with IECs via mediators in normal condition and during inflammation (Bain & Mowat, 2014; Grainger et al, 2017). Therefore, understanding the crosstalk between myeloid cells and IECs will provide valuable insight into dysregulation of intestinal homeostasis and IBD pathogenesis. Several pro-inflammatory mediators, such as IFN-γ and TNF-α, are known to induce IEC apoptosis (Marini et al, 2003; Nava et al, 2010). By contrast, other mediators, such as IL-6, promote IEC activation and survival (Grivennikov et al, 2009). However, therapeutic approaches designed to augment IEC functions by targeting these molecules have limited effectiveness, mainly due to the resultant immune imbalance in the intestine. For example, aside from enhancing IEC proliferation, IL-27 also exerts a pro-inflammatory effect by inducing T-cell activation and TH1-type cytokine production, limiting its direct application in IBD patients (Diegelmann et al, 2012). IL-6 can stimulate proliferation and survival of IECs, but simultaneously activate other target cells, including antigen-presenting cells and T cells (Atreya et al, 2000; Grivennikov et al, 2009). Therefore, it would be beneficial to identify novel myeloid cell-derived factors that improve epithelial barrier integrity in IBD with more specific functions and less inflammatory side-effects. Angiogenin (ANG), a secretory vertebrate-specific ribonuclease, enhances cell growth and survival by regulating intracellular RNA processing (Sheng & Xu, 2016). The secreted ANG is endocytosed in target cells via its receptor plexin-B2 (PLXNB2) and undergoes context-specific subcellular localization (Goncalves, Silberstein et al, 2016; Yu et al, 2017). Under growth conditions, ANG accumulates in the nucleus where it regulates rRNA transcription to promote cell proliferation (Moroianu & Riordan, 1994; Xu et al, 2002). Upon stress stimulation, ANG translocates to cytosol where it cleaves tRNAs to produce tRNA-derived stress-induced small RNAs (tiRNAs), resulting in reprogramming of protein translation and inhibition of apoptosome formation, thereby promoting cell survival (Fu et al, 2009; Ivanov et al, 2011; Goncalves et al, 2016; Li et al, 2018b). This protein may play a role in IBD pathogenesis. Two studies reported that serum ANG is significantly elevated in IBD patients (Koutroubakis et al, 2004; Oikonomou et al, 2011), while a third one found the opposite result (Magro et al, 2004). To explore the function of ANG in IBD, we first established their correlation in IBD patient samples and DSS-induced experimental colitis and then uncovered that ANG mediates a crosstalk between myeloid cells and IECs to maintain epithelial barrier integrity. Results ANG is down-regulated in IBD patient samples To establish the correlation of ANG with IBD progression, we first collected colonic tissue samples from IBD patients and measured ANG expression level. The results showed that ANG mRNA was significantly down-regulated in IBD patients (including UC and CD) compared to normal controls (Fig 1A). Western blotting and immunohistochemical (IHC) staining confirmed that ANG protein was reduced in IBD samples (Fig 1B–D). We further evaluated ANG expression in mild and severe IBD patients with IHC staining (Fig 1E) and found that ANG levels were significantly decreased in severe colitis samples compared to those in the mild ones (Fig 1F and G). These data indicate that ANG expression is down-regulated in IBD tissue and inversely correlated with IBD progression. Figure 1. ANG is down-regulated in IBD patient samples A. mRNA expression of ANG in colonic tissues from normal controls (n = 25) and UC (n = 27) or CD (n = 46) patients. B. Representative Western blotting of ANG in colonic tissues from normal controls and UC or CD patients. C. Representative images showing ANG immunohistochemical (IHC) staining in normal control and UC or CD sample. D. Corresponding statistical analysis of ANG expression score in normal controls (n = 10) and UC (n = 10) or CD (n = 15) patients. E. Representative images showing hematoxylin–eosin (HE) staining and ANG IHC staining in mild and severe IBD samples. F, G. Statistical analyses of ANG expression scores in mild (n = 18) and severe (n = 19) UC (F) or mild (n = 20) and severe (n = 20) CD (G) patients. Data information: Scale bar, 100 μm; data are shown as violin plot (A) or mean ± SEM (D, F, and G); Kruskal–Wallis with Dunn's multiple comparisons test (A and D) and Mann–Whitney test (F and G) are used to determine statistical significance (**P < 0.01, ***P < 0.001). UC: ulcerative colitis; CD: Crohn's disease. Source data are available online for this figure. Source Data for Figure 1 [embj2019103325-sup-0009-SDataFig1.zip] Download figure Download PowerPoint Ang-deficient mice are hyper-susceptible to DSS-induced experimental colitis To validate the relationship between ANG and IBD, we established a colitis mouse model with DSS induction. Following 2.5% DSS treatment, Ang gradually declined in inflammatory colon tissue (Fig 2A), suggesting a potential functional role of ANG in colitis development. Although Ang−/− mice did not display any aberrant inflammation or overt colon phenotype at baseline, strikingly, their survival rate was significantly diminished compared to WT littermates after challenging with 3.5% DSS (Fig 2B). To carefully monitor disease progression, both mice were administered with 2.5% DSS for 7 days. Compared to their WT counterparts, Ang−/− mice exhibited obvious daily weight loss (Fig 2C), increased disease activity index (DAI) (Fig 2D), shorter colon length (Fig 2E), and enhanced intestinal permeability (Fig 2F) on day 9 when they were sacrificed. Consistent with these findings, hematoxylin and eosin (HE) staining showed a dramatically aggravated colonic histopathology, including inflammatory infiltrate and ulceration formation (Fig 2G and H). Meanwhile, Ang deficiency led to a significant increase in mRNAs and secretion of pro-inflammatory cytokines in mucosal tissue (Fig 2I and J). These results reveal that ANG attenuates DSS-induced colitis in mice. Combined with the down-regulation of ANG in inflamed patient colon, our data suggest that ANG acts as a protective factor in the development of inflammation. Figure 2. Ang deficiency enhances susceptibility to DSS-induced colitis in mice A. Ang mRNA expression in colonic tissue from 2.5% DSS-treated WT mice at indicated time point (n = 4 mice/time point). B. Kaplan–Meier curve of 3.5% DSS-treated Ang-deficient mice (Ang−/−, n = 14) or littermate controls (WT, n = 13). C, D. (C) Body weight loss and (D) disease activity index (DAI) of WT and Ang−/− mice with (n = 9) or without (n = 6) 2.5% DSS treatment. E–H. (E) Colon length, (F) serum FITC-dextran level, (G) representative HE staining image, and (H) histopathological score of colonic section from the WT and Ang−/− mice on day 9 with (n = 9) or without (n = 6) 2.5% DSS treatment. I. Quantitative mRNA expression of cytokine genes in colonic tissue from the WT and Ang−/− mice on day 0 (n = 3) or day 9 (n = 9) during 2.5% DSS treatment. J. Soluble cytokine level in supernatant of cultured colonic tissue isolated from WT and Ang−/− mice on day 0 (n = 3) or day 9 (n = 7) during 2.5% DSS treatment. Data information: Scale bar, 50 μm; data are shown as mean ± SEM; ANOVA with Dunnett's multiple comparisons test (A), log-rank test for equality of survivor function (B), Mann–Whitney test (D, F, H, I, and J), and two-tailed unpaired Student's t-test (C and E) are used to determine statistical significance (*P < 0.05, ** P < 0.01, ***P < 0.001). Ctrl: control; DSS: dextran sulfate sodium; FITC-dextran: fluorescein isothiocyanate-labeled dextran. Source data are available online for this figure. Source Data for Figure 2 [embj2019103325-sup-0010-SDataFig2.xlsx] Download figure Download PowerPoint Deficiency of Ang in myeloid cells accounts for the aggravated colitis Hematopoietic stem and progenitor cells contribute to inflammation. Previous work has shown that ANG differentially regulates the fate of both cells (Goncalves et al, 2016), prompting us to ask whether the increased colitis observed in Ang−/− mice was due to the dysregulation of these two cell types. Therefore, we established reconstituted mouse models with either WT or Ang-null hematopoietic cells by transferring WT (CD45.2) or Ang−/− (CD45.2) bone marrow-derived cells to WT (CD45.1) recipients (WT→WT and Ang−/−→WT) and subjected them to DSS treatment. Similar to those observed in Ang−/− mice, the mice reconstituted with Ang-deficient hematopoietic cells (Ang−/−→WT) exhibited exacerbated weight loss (Fig 3A), increased DAI score (Fig 3B), shorter colon length (Fig 3C), enhanced intestinal permeability (Fig 3D), more severe histological score (Fig 3E and F), and increased expression of cytokines in the colonic tissue (Fig EV1A), suggesting that hematopoietic cell-derived ANG protects the mice against colitis progression. To further discriminate the relative contribution of primitive and differentiated hematopoietic cells, we assessed bone marrow reconstitution capacity. Consistent with a previous finding (Goncalves et al, 2016), Ang deficiency in hematopoietic cells did not change the differentiation capability of primitive cells, as reflected by similar percentage of myeloid cells, T cells, and B cells in WT recipients’ peripheral blood (Fig EV1B), suggesting that it is the ANG derived from differentiated immune cells responsible for the increased colitis. On the other hand, it is known that intestinal epithelial cells can also express and secrete ANG. To evaluate the contribution of epithelium-derived ANG to colitis, we reconstituted Ang−/− mice with WT hematopoietic cells (WT→Ang−/−) or conditionally knocked out Ang in epithelial cells (i.e., Villincre;Angfl/fl mice) and then subjected them to colitis induction. The data clearly showed that there were no differences between (WT→WT) and (WT→Ang−/−) (Fig 3A–F) or between WT and Villincre;Angfl/fl mice (Fig EV2A–C), further supporting that hematopoietic rather than epithelial cell-derived ANG protects mice from intestine inflammation. Figure 3. Deficiency of Ang in myeloid cells accounts for the aggravated colitis A, B. (A) Body weight loss and (B) DAI of WT→WT, Ang−/−→WT and WT→Ang−/− chimera mice with (n = 7) or without (n = 5) 2.5% DSS treatment. C–F. (C) Colon length, and (D) serum FITC-dextran level, (E) representative HE staining image, and (F) histopathological score of colonic section from these chimera colitis mice on day 8 with (n = 7) or without (n = 5) 2.5% DSS treatment. G. Ang mRNA expression in mouse lymphoid and myeloid cells; data represent two independent experiments (n = 2). H, I. (H) Body weight loss and (I) DAI of Angfl/fl and LyzMcre;Angfl/fl mice with (n = 7) or without (n = 3) 2.5% DSS treatment. J–M. (J) Colon length, (K) serum FITC-dextran level, (L) representative HE staining image, and (M) histopathological score of colonic section from the Angfl/fl and LyzMcre;Angfl/fl mice on day 8 with (n = 7) or without (n = 3) 2.5% DSS treatment. Data information: Scale bar, 50 μm; data are shown as mean ± SEM; Kruskal–Wallis with Dunn's multiple comparisons test (A, B and F), ANOVA with Dunnett's multiple comparisons test (C and D), and two-tailed unpaired Student's t-test (H, I, J, K, and M) are used to determine statistical significance (*P < 0.05, **P < 0.01, ***P < 0.001). Source data are available online for this figure. Source Data for Figure 3 [embj2019103325-sup-0011-SDataFig3.xlsx] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Colonic cytokine production and multilineage reconstitution in chimera mice A. Quantitative mRNA expression of cytokine genes in colonic tissue from WT→WT and Ang−/−→WT chimera mice on day 0 (n = 3) or day 8 (n = 7-8) during DSS treatment. B. Multilineage reconstitution analysis in chimera mice (n = 5). Data information: Data are shown as mean ± SEM, Mann–Whitney test, or two-tailed unpaired Student's t-test is used to determine statistical significance (*P < 0.05). Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Myeloid-derived ANG plays a non-cell-autonomous role in attenuating DSS-induced colitis A, B. (A) Body weight loss and (B) DAI of Angfl/fl and Villincre;Angfl/fl mice with (n = 8) or without (n = 5) 2.5% DSS treatment. C. Colon length of the Angfl/fl and Villincre;Angfl/fl mice on day 8 with (n = 8) or without (n = 5) 2.5% DSS treatment. D. Ang mRNA expression in colonic myeloid cells from 2.5% DSS-treated WT mice at indicated time point. (n = 3–4/time point). E. Kaplan–Meier curve of WT (n = 21) or Ang−/− (n = 19) mice in LPS-induced endotoxin shock model. F. Kaplan–Meier curves of WT (n = 12) or Ang−/− (n = 12) mice in Listeria monocytogenes (LM)-induced sepsis model. G, H. mRNA expression of cytokine genes in BMDMs from WT or Ang−/− mice administrated with (G) LPS or (H) synthetic RNA duplex poly(I:C) at indicated time point (n = 2–3/time point per group). I, J. (I) Representative plot of flow cytometry analysis and (J) the percentages of macrophage and dendritic cell subsets in cLP of WT or Ang−/− mice (n = 6) in steady state. K, L. The percentages of CD45+CD11b+ (K), CD11b+IL6+, and CD11b+TNFα+ (L) subsets in LPS-stimulated cLP of Angfl/fl or LyzMcre;Angfl/fl mice (n = 4) with DSS treatment (day 0 and 4). M. MFI of CD11b+IL6+ and CD11b+TNFα+ in LPS-stimulated cLP of Angfl/fl or LyzMcre;Angfl/fl mice with DSS treatment (day 0 and 4) (n = 4). Data information: Data are shown as mean ± SEM; ANOVA with Dunnett's multiple comparisons test (D) is used to determine statistical significance (***P < 0.001); BMDM: bone marrow-derived macrophage; MFI: mean fluorescence intensity; LPS: lipopolysaccharide. Source data are available online for this figure. Download figure Download PowerPoint To further identify the target cell types, we measured Ang expression levels in differentiated immune cells and found that it was highly expressed in myeloid cells, especially in monocytes and macrophages (Fig 3G), and was gradually reduced in colonic myeloid cells following DSS treatment (Fig EV2D). To directly determine the role of myeloid cell-derived ANG in colitis mitigation, mice (LysMcre;Angfl/fl) with conditional Ang knockout in myeloid cells were generated and then administrated with DSS. These mice exhibited exacerbated colitis compared to controls (Fig 3H–M). Taken together, these data reveal that Ang expression in myeloid cells is critical for protecting mice against DSS-induced colitis. Myeloid-derived ANG plays a non-cell-autonomous role in colitis attenuation Myeloid cells play key role in innate immune response. To explore whether ANG participates in this process, we established lipopolysaccharide (LPS)-triggered endotoxin shock and Listeria monocytogenes-induced sepsis models and found that there were no significant differences in survival rate between WT and Ang−/− mice in both models (Fig EV2E and F). On the other hand, in vitro stimulation of bone marrow-derived macrophages (BMDM) from both mice with LPS or synthetic RNA duplex poly(I:C) induced a similar extent of pro-inflammatory cytokine production (Fig EV2G and H). These data indicate that Ang deficiency in myeloid cells has no significant influence on systemic innate immune response. To determine whether ANG affects mononuclear phagocytes (MPs) composition in the colonic lamina propria (cLP), we did phenotypic analysis with flow cytometry. The data showed that there were no differences in the frequencies of CX3CR1hiCD11b+ macrophages, CD103+CD11b− dendritic cells (DCs), and CD103+CD11b+ DCs between WT and Ang−/− mice in the steady state (Fig EV2I and J), and 4-day DSS treatment induced a comparable degree of myeloid cells infiltration into cLP in Angfl/fl and LysMCre;Angfl/fl mice (Fig EV2K). Furthermore, these mice exhibited a similar alteration in both the frequency and the mean fluorescence intensity (MFI, indicating the production of cytokines) of IL-6+CD11b+ and TNF-α+CD11b+ cells in cLP upon LPS stimulation (Fig EV2L and M), suggesting Ang has no influence on the activation of colonic myeloid cells under both steady and inflammatory states. Taken together, although peculiarly expressed in the myeloid cells, Ang is dispensable for the inflammatory cytokine production in systemic innate immune response and the composition and activation of myeloid cells in local mucosal immune system, indicating that myeloid-derived ANG plays a non-cell-autonomous role in the attenuation of DSS-induced colitis. ANG-PLXNB2 axis is essential for the colitis attenuation PLXNB2 was recently found to be a functional receptor to the secretory ANG protein (Yu et al, 2017), giving rise to the possibility that myeloid cell-derived ANG may mitigate colitis in a paracrine manner via PLXNB2. To determine the potential ANG-targeting cells in colonic tissue, we evaluated the expression of ANG and PLXNB2 in mouse colon and found that ANG was mainly expressed in cLP cells, whereas PLXNB2 was highly expressed in IECs (Fig 4A–C). Similar results were also observed in human colonic tissue (Fig EV3A and B). These data suggest a potential crosstalk between myeloid cells and IECs via the ANG-PLXNB2 axis. Figure 4. ANG-PLXNB2 axis is essential for mitigating DSS-induced colitis in mice A, B. (A) Quantitative mRNA expression and (B) Western blotting of ANG and PLXNB2 in isolated IECs or colonic laminar propria (cLP) cells from WT mice (n = 9). C. Representative images showing immunofluorescence staining of ANG, PLXNB2, and macrophage surface marker F4/80 in frozen colonic section from WT mice. D. Representative images showing immunofluorescence staining of GFP and PLXNB2 in frozen colonic section from WT mice infected with adeno-associated virus (AAV9). E. PLXNB2 expression in IECs from the AAV9-infected WT mice (n = 3). F, G. (F) Body weight loss and (G) DAI of the AA9-infected WT mice with (n = 7) or without (n = 5) 2.5% DSS treatment. H–K. (H) Colon length, (I) serum FITC-dextran level, (J) representative HE staining image, and (K) histopathological score of colonic section from the AAV9-infected WT mice on day 8 with (n = 7) or without (n = 5) 2.5% DSS treatment. Scale bar, 50 μm; data are shown as the mean ± SEM, Mann–Whitney test (A, G, and I), and two-tailed unpaired Student's t-test (E, F, H, and K) are used to determine statistical significance (*P < 0.05, **P < 0.01; ***P < 0.001). Source data are available online for this figure. Source Data for Figure 4 [embj2019103325-sup-0012-SDataFig4.zip] Download figure Download Power