P38α De ciency in Macrophages Ameliorates Murine Experimental Colitis by Regulating In ammation and Immune Process

Wei Chen Shanghai Jiao Tong University A liated Sixth People Hospital Rui Liang Shanghai Jiao Tong University A liated Sixth People Hospital Youcai Yi Shanghai Jiao Tong University A liated Sixth People Hospital Xiaoyu Chen Shanghai Shanghai Jiao Tong University A liated Sixth People Hospital Huining Fan Shanghai Jiao Tong University A liated Sixth People Hospital Jinshui Zhu (  zhujs1803@163.com ) Shanghai Jiao Tong University A liated Sixth People Hospital Jing Zhang Shanghai Jiao Tong University A liated Sixth People Hospital


Introduction
In ammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), is characterized by chronic and recurrent in ammatory and immune in ltration in gastrointestinal tract 1 .
Increasing evidence unveils that the pathogenesis of IBD is associated with multiple factors such as genetic polymorphisms, environmental factors, and immunological disorders 2,3 . A better understanding of the molecular pathogenesis of IBD is critical to develop promising therapeutic strategies for this disease.
The core of the in ammatory signaling is mediated by reversible phosphorylation by protein kinases, of which mitogen-activated protein kinase (MAPK) signaling acts in linking in ammatory stimuli to cellular responses 4 . The p38 MAPKs are a family of serine/threonine protein kinases that consist of four isoforms p38α, p38β, p38γ and p38δ 5 , of which p38α is the most commonly expressed in human and mouse tissues 6 and controls in ammatory response and cancer cell proliferation 5,7 . Hepatocyte-derived p38α is a pivotal negative regulator in maintaining hepatic gluconeogenesis 8 . P38α appears to have dual roles in certain cancers. It suppresses colitis-associated tumor initiation, but contributes to the proliferation and survival of colon cancer 9 . P38α deletion in myeloid cells improves experimental colitis, but its de ciency in intestinal epithelial cells (IECs) increases the susceptibility to colitis 10 .
Emerging evidence indicates that macrophage-speci c p38α accelerates acute liver injury 11 and steatohepatitis progression through inducing pro-in ammatory cytokine secretion and M1 polarization 12 .
However, the effects of macrophage-derived p38α on IBD pathogenesis remain undocumented. In the present study, p38α was co-localized with CD68 in the cytoplasm and their co-expression displayed an increased level in colitis tissues. We then used macrophage-speci c p38α knockout mice (p38α ΔMφ ) and WT mice to construct DSS-induced experimental colitis and found that p38α de ciency in macrophages ameliorated murine experimental colitis by inhibiting cytokine release, promoting colonic mucosa repair and regulating immune process.

Materials And Methods
Tissue Sample 10 cases of colitis and 5 normal colon tissues were purchased from Xi'an Best Biotechnology Co., Ltd.

Animals And Treatments
C57BL/6 male p38α ΔMφ mice and wild type (WT) littermates (6-8 weeks of age) were provided by Medical College of Xiamen University and housed in the SPF standard laboratory conditions. As previously described 13 , p38α ΔMφ mice (n = 5) and WT mice (n = 6) were administrated by the addition of 3% DSS (36-50 kDa, MP Biomedicals, USA) in the drinking water for 7 consecutive days. In another cohort, male C57BL/6 mice (6-8 weeks old) were intraperitoneally injected with p38 MAPK inhibitors or vehicle (DMSO) for 7 consecutive days. According to previous literature 12 , the injection doses of p38 MAPK inhibitors SB203580 and BIRB796 were 50 mg/kg/day and 15 mg/kg/day, respectively. Mouse body weight, colon length, clinical phenotype and pathological scoring were assessed.

Evaluation Of Dss-induced Colitis
The mice were observed every morning. Weight loss, fecal consistency and intestinal bleeding were recorded in accordance with the previous report 13 . The DAI value was evaluated based on the above Page 4/21 records.

Rna Sequencing And Bioinformatic Analysis
Brie y, total RNA was extracted from the colon tissues of the WT and p38α ΔMφ groups. The quantitative and quality control of total RNA were detected by NanoDrop ND-1000. 1-2 µg RNA was extracted from each sample to generate the RNA library, which were sequenced on an Illumina Hiseq 4000 platform after cluster generation. Data were analyzed as follows: sequencing quality control, sequencing data comparison, quantitative analysis of differentially-expressed genes, and GO and KEGG enrichment analysis.
Quantitative Real-time Pcr (Qrt-pcr) Trizol reagent (Invitrogen, USA) was used to isolate total RNA from colon tissues Complementary DNA (cDNA) is obtained by RNA reverse transcription. The reaction procedure is based on our previous reports 17 . The 2 −ΔΔCt method was used to calculate the gene expression level. GAPDH was used as the internal control. All reactions were performed more than twice. All primers were listed in Table S1.

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Western blot analysis The protein levels of Claudin-2, OAS2 and OAS3 in colon tissues in each group were detected by Western blotting. In brie y, total proteins from colon tissues were obtained with RIPA lysis buffer (Beyotime, Shanghai, China) and protease inhibitor (Servicebio, China). After the determination of protein concentration, the protein was denatured by boiling water for 15 minutes. The primary antibodies claudin-2 (AF0128, A nity Biosciences, USA), OAS2 (DF12680, A nity Biosciences, USA), OAS3 (ER1914-27, HuaBio, China), and anti-β-actin (AF7018, A nity Biosciences, USA) were thinned and incubated overnight at 4°C. We added HRP-labeled goat anti-rabbit secondary antibodies in a 1:3000 dilution, and incubated them at room temperature for 30 minutes.
Wash the membranes three times with PBST. The immunoreactive bands were detected with ECL Luminescent Solution (Servicebio, China).

Tdt-mediated Dutp Nick-end Labeling (Tunel) Assay
Apoptosis of IECs was detected by TUNEL kit (Servicebio, China). In short, colon tissue sections were cultured in TUNEL reaction mixture. Then, it was stained with 4', 6-diamino-2-phenylindole (DAPI). After that, all samples were observed under the uorescence inverted microscope. Each sample was observed in three random elds.

Statistical analysis
Statistical analyses were performed with SPSS 20.0 and GraphPad Prism 8.0.1. All data were displayed as the mean ± SEM. The differences between groups were analyzed by One-way ANOVA. P < 0.05 means statistical difference.

Results
The expression of macrophage-derived p38α is increased in human colitis tissues The protein levels of phosphorylated p38α (p-p38α) in colitis and normal tissues were detected by IHC analysis, which indicated that, p-p38α presented no differential expression between colitis and normal tissues (P = 0.1363, Fig. 1A, B). Further investigations showed that p38α was co-localized with CD68 in the cytoplasm of colitis tissue cells (Fig. 1C) and they harbored a positive correlation in colitis tissues (R 2 = 0.5015, Fig. 1D). FISH analysis also con rmed that p38α or CD68 possessed no differential expression between colitis and normal tissues, but the co-expression level of p38α and CD68 were signi cantly higher than those in normal tissues (P = 0.0312, Fig. 1E).

Macrophage-speci c Knockout Of P38α Attenuated Murine Experimental Colitis
To understand how macrophage-derived p38α leads to the severe colitis, we used p38α ΔMφ and WT mice to establish DSS-induced experimental colitis. The body weight changes, stool consistency, intestinal bleeding and colon length were observed. The results showed that, with or without DSS intervention, the body weight was increased, but DAI was decreased in p38α ΔMφ group as compared with the WT group ( Fig. 2A, B). Likewise, after exposure to DSS treatment, the colon length was raised in p38α ΔMφ group as compared with the WT group (Fig. 2C, D). Histological examination of colon sections showed that p38α ΔMφ mice administrated by DSS displayed a lower histopathological scoring in colon tissues accompanied by less neutrophil in ltration, submucosal edema, and loss of crypt and goblet cell as compared with WT mice (Fig. 2E, F).
Macrophage-speci c knockout of p38α inhibits pro-in ammatory cytokine secretion in experimental colitis To clarify the effects of macrophage-derived p38α on proin ammatory cytokine production in experimental colitis, we investigated the levels of TNF-α, IL-1β and IL-6 in p38α ΔMφ and WT groups.
Previous studies have indicated that the secretion of cytokines TNF-α, IL-1β and IL-6 was associated with the pathogenesis of IBD 19,20 . IHC analysis indicated that the expression levels of TNF-α, IL-1β and IL-6 in experimental colitis in p38α ΔMφ group were signi cantly lower than those in the WT group ( Fig. 3A-C).

Macrophage-speci c knockout of p38α promotes intestinal barrier repair in experimental colitis
Intestinal mucosal integrity can be altered during active colitis, leading to mucosal metabolism disorder and aggravated colon in ammation 21 . To understand how p38α in macrophages affects intestinal barrier function, we tested the tissue levels of mucosal barrier-related markers such as occludin, ZO-1, claudin-1, claudin-2, muc-1 and muc-2 in experimental colitis in p38α ΔMφ and WT groups. qRT-PCR and IHC analysis displayed that occludin, ZO-1, claudin-1 and muc-1 harbored no differential expression ( Supplementary Fig. S1), but claudin-2 and muc-2 were upregulated in p38α ΔMφ group as compared with the WT group (Fig. 4A, B). The similar results were further validated by Western blot and IF analyses in p38α ΔMφ and WT groups. (Fig. 4C, D).
Macrophage-speci c knockout of p38α regulates the proliferation and apoptosis of IECs in experimental colitis Accumulating data show that p38α is related to cell proliferation in IBD 22 . To determine the effect of p38α in macrophages on the proliferation of IECs, ki-67 proliferation index and cell apoptosis in experimental colitis were measured by IHC and TUNEL, which indicated that ki-67 proliferation index of IECs was increased (Fig. 5A), whereas cell apoptosis was decreased in p38α ΔMφ group as compared with the WT group (Fig. 5B).
Macrophage-speci c knockout of p38α enhances immune system process in experimental colitis To further dissect the underlying mechanisms of macrophage-derived p38α in experimental colitis, we performed a RNA-seq analysis in experimental colitis between p38α ΔMφ and WT groups. Heatmaps and volcano analysis revealed that 53 differentially-expressed genes were identi ed between these two groups (Fig. 6A, B). The results of GO analysis revealed that these differential genes were enriched in multiple biological processes, of which immune system process ranked the second place and aroused our attention (Fig. 6C, D). The related genes were shown in Fig. 6E. We then validated the expression of these immune system process-related genes between p38α ΔMφ and WT groups by qRT-PCR analysis. The results indicated that the mRNA levels of Lgals9, Rtp4, Ddx60, Nlrp1b, Hsh2d, Oas2 and Oas3 were upregulated in p38α ΔMφ group compared with the WT group (Fig. 6F). The protein levels of Oas2 and Oas3 were further validated by IHC analysis (Fig. 6G).

The P38 Mapk Inhibitor Improves Dss-induced Experimental Colitis
To assess the effects of targeting p38 MAPK on DSS-induced colitis, we used p38 MAPK inhibitors SB203580 and BIRB796 to treat DSS-induced colitis. It was found that the mice in SB203580 group gained an increasement of weight and decline of DAI scoring compared with the DSS group (Fig. 7A, B). Meanwhile, the colon length was increased in SB203580 group as compared with the DSS group (Fig. 7C,  D). Histopathological examination demonstrated that SB203580 could inhibit a higher colon pathological scoring in experimental colitis, accompanied by less neutrophil in ltration, loss of crypt and goblet cell, and submucosal edema as compared to with DSS group (Fig. 7E, F).

Discussion
It has been reported that macrophage-derived p38α promotes liver injury 11 and steatohepatitis progression 12 . Herein, we estimated the role of macrophage-derived p38α in DSS-induced experimental colitis. We found that the expression of macrophage-derived p38α was elevated in colitis tissues, and macrophage-speci c knockout of p38α improved the clinical symptoms, DAI and histopathological scoring. Our ndings indicated that macrophage-derived p38α might be a promising target for colitis.
Accumulating data show that the activation of p38 MAPK signal is involved in intestinal in ammation and responsible for overactive immune system in IBD [23][24][25] . Inhibition of p38 MAPK can powerfully reduce the production of pro-in ammatory cytokines and reduce intestinal in ammation 26 . Macrophages are considered as the gatekeepers of intestinal immune homeostasis and their dysregulation leads to the chronic in ammation in IBD 27 . In our study, we found that macrophage-derived knockout of p38α improved experimental colitis by reducing the secretion of pro-in ammatory cytokines TNF-α, IL-1β and IL-6.
Intestinal barrier composed of mucous layer and epithelial cell layer can protect the intestinal mucosa from the attack of pathogens and antigens 28 . Previous study showed that muc-2 de ciency in mice spontaneously induces the colitis 28 . In addition, IECs are interconnected by tight junctions (TJs), which regulate paracellular permeability [25][26] . The members of claudin family are the principal constituents of the TJs, of which claudin-2 favors immune-mediated colitis and targeting claudin-2 relieved its progression 29,30 . However, our results indicated that macrophage-derived knockout of p38α increased the enrichment of muc-2 and claudin-2 in experimental colitis, indicating that macrophage-derived knockout of p38α might improve experimental colitis by promoting mucosal homeostasis.
MAPKs are involved in multiple physiological processes, including cell growth, metabolism, differentiation and cell death [29][30][31] . Activation of p38 MAPK suppresses the proliferation of corneal endothelial cells, whereas the p38 MAPK inhibitor counteracts this effect 32 . P38α de ciency in neonatal muscle regulates cellular hyperproliferation and maturation 33 . Neonatal mice lacking P38α in cardiac muscle exhibit an increase in DNA synthesis and mitosis in cardiomyocytes 34 . Herein, we investigated the effects of macrophage-derived p38α on colon cell proliferation and found that Ki-67 proliferation levels were markedly increased in experimental colitis in p38α ΔMφ group as compared with the WT group.
In addition, increasing data indicate the pro-apoptotic role of p38 MAPKs in multiple cell types 35,36 . We found that macrophage-derived knockout of p38α reduced cell apoptosis of IECs in DSS-induced colitis. Our results suggested that macrophage-derived knockout of p38α might improve experimental colitis by regulating cell proliferation and apoptosis of IECs.
It has been widely accepted that the pathogenesis of IBD is associated with the imbalance of intestinal mucosal immune responses to environmental factors in genetically-predisposed individuals 37 . Innate and adaptive immune responses cause in ammatory lesions in IBD 38 . Of note, p38 MAPK acts a prominent role in innate and adaptive immune responses 39 . Our results identi ed that immune processrelated factors including Lgals9, Rtp4, Ddx60, Nlrp1b, Hsh2d, Oas2 and Oas3 were enriched in p38α ΔMφ group as compared with the WT group. Lgals9 is a β-galactoside-binding lectin that acts in physiological and pathological conditions, such as immune response 40 . It induces T cell apoptosis 41,42 and dendritic cell (DC) activation 43 . Lgals9 −/− mice harbors DSS-induced intestinal injury 44,45 . RTP4 controls IFN-I response by affecting TBK1 and IRF3 phosphorylation 46 . DDX60 acts as a ligand-speci c sentinel activated by RIG-1 to participate in innate immune response 47,48 . Nalp1b, a member of (NOD)-like receptors family recognizes pathogen-associated molecular patterns, such as lipopolysaccharide 49 .
HSH2D is a weighty signal molecule that involves in the activation of T cells 50 , leading to tumor malignancy and drug resistance in Tcell acute lymphoblastic leukaemia 51 . The OASs are a family of IFNand virus-induced proteins, consisting of OAS1, OAS2, OAS3, OASL 52 . OAS2 and OAS3 have antiviral activity 52,53 and OAS3 negatively regulates chemokines and interferon-responsive factors in macrophages 54 . Our results further validated that Lgals9, Rtp4, Ddx60, Nlrp1b, Hsh2d, Oas2 and Oas3 were upregulated in p38α ΔMφ group as compared with the WT group, suggesting that macrophagederived knockout of p38α might improve experimental colitis by regulating immune process.
Pharmacological inhibition of p38 MAPK has been used to treat various in ammatory diseases, such as rheumatoid arthritis, Alzheimer's disease and IBD 55,56 . SB203580 as a p38 MAPK inhibitor reduces the production of IFN-γ and IL-12p70 in colitis 24 . Herein, our results suggested that p38 inhibitors could signi cantly ameliorate experimental colitis. This may provide a potential theoretical basis for treatment of UC.

Conclusion
Taken together, we unveil a novel role of macrophage-derived p38α in colitis and found that macrophagederived p38α facilitates the pathogenesis of colitis by reducing in ammation response, protecting mucous barrier and regulating cell proliferation and immune process. Our work may provide a new approach for colitis treatment.

Declarations
Ethics approval and consent to participate All experiments involving animals were conducted according to the ethical policies and procedures approved by the ethics committee of Shanghai Sixth People's Hospital (2020-0015). All subjects provided written informed consent and the study protocol was approved by the ethics committee of Shanghai Sixth People's Hospital. (2020-231).

Consent for publication
All the authors agreed to publish articles in the journal.

Availability of data and material
The data underlying this article will be shared on reasonable request to the corresponding author.    The expression levels of TNF -α, IL-6 and IL-1β in colon tissues were analyzed by IHC analysis. *P <0.05, **P <0.01, ***P <0.001. Representative IF images of muc-2 and claudin-2 in WT and p38αΔMφ mice. Data are showed as means ± SEM. * P < 0.05 and ** P < 0.01. The apoptosis of IECs was detected by TUNEL. Data are showed as means ± SEM. * P < 0.05 and *** P < 0.001. major genes that related to immune system process. (F) The mRNA expression of Lgals9, Rtp4, Ddx60, Nlrp1b, Hsh2d, Oas2 and Oas3 in colonic tissues of WT and p38αΔMφ mice on day 7 calculated. Data are showed as means ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001.

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