Journal: Nature Communications
Article Title: Organoids transplantation attenuates intestinal ischemia/reperfusion injury in mice through L-Malic acid-mediated M2 macrophage polarization
doi: 10.1038/s41467-023-42502-0
Figure Lengend Snippet: A H & E staining of small intestinal tissue from mice 36 h after induction of intestinal I/R and quantification of the small intestinal pathology score (n = 3 in SOCS2 –/– mice/group, n = 4 in WT mice/group). Represent significant p value using two-way ANOVA followed by the Tukey test. 0.0241 (WT, I/R + Vehicle vs I/R + MA), 0.0020 (I/R + MA, WT vs SOCS2 –/– ). B Quantification of the area of Occludin, ZO-1 immunohistochemical staining (n = 3 in SOCS2 –/– mice/group, n = 4 in WT mice/group). Represent significant p value using two-way ANOVA followed by the Tukey test. For Occludin, 0.0231 (WT, I/R + Vehicle vs I/R + MA), 0.0019 (I/R + MA, WT vs SOCS2 –/– ); for ZO-1, < 0.0001 (WT, I/R + Vehicle vs I/R + MA), < 0.0001 (I/R + MA, WT vs SOCS2 –/– ). C qRT-PCR analysis of Occludin and ZO-1 mRNA in small intestinal tissues from mice 36 h after intestinal I/R (n = 3 in SOCS2 –/– mice/group, n = 4 in WT mice/group). Represent significant p value using two-way ANOVA followed by the Tukey test. For Occludin, 0.0056 (WT, I/R + Vehicle vs I/R + MA), 0.0005 (I/R + MA, WT vs SOCS2 –/– ); for ZO-1, 0.0014 (WT, I/R + Vehicle vs I/R + MA), 0.0007 (I/R + MA, WT vs SOCS2 –/– ). D ELISA detection of IL-6, IL-1β and IL-10 production in mice 36 h after intestinal I/R (n = 3 in SOCS2 –/– mice/group, n = 4 in WT mice/group). Represent significant p value using two-way ANOVA followed by the Tukey test. For IL-6, 0.046 (WT, I/R + Vehicle vs I/R + MA), 0.0046 (I/R + MA, WT vs SOCS2 –/– ); for IL-1β, 0.0159 (WT, I/R + Vehicle vs I/R + MA), 0.0052 (I/R + MA, WT vs SOCS2 –/– ); for IL-10, 0.0005 (WT, I/R + Vehicle vs I/R + MA), 0.0003 (I/R + MA, WT vs SOCS2 –/– ). E Frequency of Ly6C + MHCII - , Ly6C + MHCII + , and Ly6C - MHCII + subsets among CD45 + CD11b + cells in the small intestine of different groups (n = 3 in SOCS2 –/– mice/group, n = 4 in WT mice/group). Represent significant p value using two-way ANOVA followed by the Tukey test for Ly6C + MHCII - and Ly6C + MHCII + cells. For Ly6C + MHCII - cells, 0.0169 (WT, I/R + Vehicle vs I/R + MA), 0.0221 (I/R + MA, WT vs SOCS2 –/– ); for Ly6C + MHCII + cells, 0.0004 (WT, I/R + Vehicle vs I/R + MA), 0.0011 (I/R + MA, WT vs SOCS2 –/– ). F Quantification of CD206 + F4/80 + CD45 + CD11b + macrophages (n = 3 in SOCS2 –/– mice/group, n = 4 in WT mice/group). Represent significant p value using two-way ANOVA followed by the Tukey test. 0.0004 (WT, I/R + Vehicle vs I/R + MA), 0.0012 (I/R + MA, WT vs SOCS2 –/– ). G Representative histograms of CD206 expression in the LP of the small intestine 36 h after intestinal I/R injury. H Schematic illustration of the induction protocols for macrophage adoptive transfer. I H & E staining of small intestinal tissue from mice 36 h after induction of macrophages adoptive transfer and quantification of the small intestinal pathology score (n = 3 for recipient SOCS2 -/- mice adoptive transfer of the macrophages from MA-administrated WT mice group, n = 4 mice for the rest groups). Represent significant p value using two-way ANOVA followed by the Tukey test. 0.0163 (WT, Adoptive transfer of WT macrophages vs Adoptive transfer of SOCS2 –/– macrophages), 0.0258 (SOCS2 –/– , Adoptive transfer of WT macrophages vs Adoptive transfer of SOCS2 –/– macrophages). J Quantification of the area of Occludin, ZO-1 immunohistochemical staining (n = 3 for recipient SOCS2 -/- mice adoptive transfer of the macrophages from MA-administrated WT mice group, n = 4 mice for the rest groups). Represent significant p value using two-way ANOVA followed by the Tukey test. For Occludin, 0.0003 (WT, Adoptive transfer of WT macrophages vs Adoptive transfer of SOCS2 –/– macrophages), 0.0023 (SOCS2 –/– , Adoptive transfer of WT macrophages vs Adoptive transfer of SOCS2 –/– macrophages); for ZO-1, 0.0018 (WT, Adoptive transfer of WT macrophages vs Adoptive transfer of SOCS2 –/– macrophages), 0.0033 (SOCS2 –/– , Adoptive transfer of WT macrophages vs Adoptive transfer of SOCS2 –/– macrophages). K ELISA detection of IL-6, IL-1β and IL-10 production in mice 36 h after macrophages adoptive transfer (n = 3 for recipient SOCS2 -/- mice adoptive transfer of the macrophages from MA-administrated WT mice group, n = 4 mice for the rest groups). Represent significant p value using two-way ANOVA followed by the Tukey test. For IL-6, 0.0288 (WT, Adoptive transfer of WT macrophages vs Adoptive transfer of SOCS2 –/– macrophages), 0.0226 (SOCS2 –/– , Adoptive transfer of WT macrophages vs Adoptive transfer of SOCS2 –/– macrophages); for IL-1β, 0.0012 (WT, Adoptive transfer of WT macrophages vs Adoptive transfer of SOCS2 –/– macrophages), 0.0004 (SOCS2 –/– , Adoptive transfer of WT macrophages vs Adoptive transfer of SOCS2 –/– macrophages); for IL-10, 0.0022 (WT, Adoptive transfer of WT macrophages vs Adoptive transfer of SOCS2 –/– macrophages), 0.0154 (SOCS2 –/– , Adoptive transfer of WT macrophages vs Adoptive transfer of SOCS2 –/– macrophages). Scale bar, 200 μm. The statistical tests employed included: two-way ANOVA followed by the Tukey test for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Each dot represents data from a single sample ([ A – F ], [ I – K ]). Bar graphs represent mean ± SD. Source data are provided as a Source Data file.
Article Snippet: Small intestinal macrophages were cultured in 10% fetal bovine serum (FBS) and stained with APC-Cy7–conjugated anti-CD45 (I3/2.3, A15395, Thermo Fisher Scientific), BV510-conjugated anti-mouse CD11b antibody (clone M1/70, 101263, Biolegend), FITC-conjugated mouse F4/80 (clone BM8, 123108, Biolegend), and sorted.
Techniques: Staining, Immunohistochemistry, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Adoptive Transfer Assay
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![A Flow cytometry analysis of neutrophils <t>(CD45</t> + CD11b + Ly6G + ), monocytes (CD45 + CD11b + Ly6C + ), macrophages (CD45 + CD11b + F4/80 + ), and T cells (CD45 + CD11b - CD3 + ) in the lamina propria (LP) of the small intestine (n = 5 mice for sham group with organoids transplanted, control group at 6 h, transplanted group at 6 h, control group at 12 h, transplanted group at 48 h, n = 6 mice for transplanted group at 12 h and control group at 48 h, n = 4 mice for the rest groups). Neutrophils: represent significant p value using two-tailed student’s from left to right: 0.0270, 0.0293, 0.0406; monocytes: represent significant * p value = 0.0191 using two-tailed student’s t test at 6 h, represent significant * p value = 0.0173 using two-tailed Mann–Whitney test at 12 h, represent significant * p value = 0.0345 using two-tailed student’s t test at 36 h; macrophages: represent significant * p value = 0.0297 using two-tailed student’s t test. B Expression of Ly6C and MHCII by live CD45 + CD11b + small intestinal LP cells in organoid-transplanted and control mice 36 h after intestinal I/R and transplantation. C Expression of CX3CR1 and CD206 in the indicated cell subsets from organoid-transplanted mice. D Frequency of Ly6C + MHCII - , Ly6C + MHCII + , and Ly6C - MHCII + subsets among CD45 + CD11b + cells in the small intestine of different groups (n = 6 mice/group). Ly6C + MHCII - cells: represent significant ** p value = 0.0022 using two-tailed Mann–Whitney test; Ly6C + MHCII + cells: represent significant *** p value = 0.0008 using two-tailed student’s t test; Ly6C - MHCII + cells: represent significant ** p value = 0.0084 using two-tailed student’s t test. E Representative flow cytometric analysis of CD206 + cells in the small intestine LP 36 h after intestinal I/R injury. F Quantification of CD206 + F4/80 + CD45 + CD11b + macrophages (n = 6 mice/group). Represent significant **** p value <0.0001 using two-tailed student’s t test. G Representative histograms of CD206 expression. H Quantification of CD206 mean fluorescence intensity (MFI) in the LP of the small intestine 36 h after intestinal I/R injury (n = 6 mice/group). Represent significant *** p value = 0.0001 using two-tailed student’s t test. I qRT-PCR analysis of CD206 mRNA in F4/80 + macrophages (n = 6 mice/group). Represent significant *** p value = 0.0008 using two-tailed student’s t test. J Quantification of IL-10 + F4/80 + CD45 + CD11b + macrophages (n = 6 mice/group). Represent significant ** p value = 0.0022 using two-tailed Mann–Whitney test. K Representative histograms of IL-10 expression. L Quantification of IL-10 MFI in the small intestine LP 36 h after intestinal I/R injury (n = 6 mice/group). Represent significant ** p value = 0.0022 using two-tailed Mann–Whitney test. M qRT-PCR analysis of IL-10 mRNA expression in F4/80 + macrophages (n = 6 mice/group). Represent significant ** p value = 0.0022 using two-tailed Mann–Whitney test. The statistical tests employed included: two-tailed student’s t test and Mann–Whitney tests. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Each dot represents data from a single mouse ([ A ], [ D ], [ F ], [ H – J ] and [ L , M ]). Bar graphs represent mean ± SD. Source data are provided as a Source Data file.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_0233/pmc10600233/pmc10600233__41467_2023_42502_Fig2_HTML.jpg)
![A LP macrophages were analyzed using flow cytometry for F4/80 + CD45 + CD11b + cells in mice treated with clodronate liposomes or PBS liposomes 36 h after intestinal I/R injury and transplantation. Representative images and quantification of the macrophage population (n = 5 mice/group). Represent significant p value using two-tailed Mann–Whitney test. 0.0079 (PBS + Lipo, Ctrl vs Org trans), 0.0079 (Org trans, PBS + Lipo vs Clodronate + Lipo). B Hematoxylin and eosin (H & E) staining of small intestinal tissue from mice 36 h after induction of intestinal I/R and quantification of small intestinal pathology score (n = 5 mice/group). Represent significant p value using two-way ANOVA followed by the Tukey test. 0.0326 (PBS + Lipo, Ctrl vs Org trans), 0.0036 (Org trans, PBS + Lipo vs Clodronate + Lipo). C ELISA detection of IL-6 and IL-1β, and IL-10 production in mice 36 h after intestinal I/R and transplantation (n = 5 mice/group). Represent significant p value using two-tailed Mann–Whitney test for IL-6. 0.0079 (PBS + Lipo, Ctrl vs Org trans), 0.0079 (Org trans, PBS + Lipo vs Clodronate + Lipo). Represent significant p value using two-way ANOVA followed by the Tukey test for IL-1β. 0.0334 (PBS + Lipo, Ctrl vs Org trans), 0.0021 (Org trans, PBS + Lipo vs Clodronate + Lipo). Represent significant p value using two-tailed Mann–Whitney test for IL-10. 0.0079 (PBS + Lipo, Ctrl vs Org trans), 0.0079 (Org trans, PBS + Lipo vs Clodronate + Lipo). D qRT-PCR analysis of Occludin and ZO-1 mRNA in the small intestinal tissue of mice that underwent intestinal I/R and transplantation for 36 h (n = 5 mice/group). Represent significant p value using two-way ANOVA followed by the Tukey test for Occludin and ZO-1. For Occludin, 0.0011 (PBS + Lipo, Ctrl vs Org trans), 0.0004 (Org trans, PBS + Lipo vs Clodronate + Lipo). For ZO-1, <0.0001 (PBS + Lipo, Ctrl vs Org trans), < 0.0001 (Org trans, PBS + Lipo vs Clodronate + Lipo). E Representative immunohistochemistry images of Occludin staining and quantification of the Occludin staining area (n = 5 mice/group). Represent significant p value using two-way ANOVA followed by the Tukey test. 0.0255 (PBS + Lipo, Ctrl vs Org trans), 0.0042 (Org trans, PBS + Lipo vs Clodronate + Lipo). F Representative immunohistochemistry images of ZO-1 and quantification of the ZO-1 staining area (n = 5 mice/group). Represent significant p value using two-way ANOVA followed by the Tukey test. 0.0040 (PBS + Lipo, Ctrl vs Org trans), 0.0022 (Org trans, PBS + Lipo vs Clodronate + Lipo). G Expression of Ly6C and MHCII by live CD45 + CD11b + small intestinal LP cells in mice 36 h after intestinal I/R and transplantation. H Frequency of Ly6C + MHCII - , Ly6C + MHCII + , and Ly6C - MHCII + subsets among CD45 + CD11b + cells in the small intestine of different groups (n = 5 mice/group). Represent significant p value using two-tailed Mann–Whitney test for Ly6C + MHCII - , Ly6C + MHCII + , and Ly6C - MHCII + cells. For Ly6C + MHCII - cells, 0.0079 (PBS + Lipo, Ctrl vs Org trans), 0.0079 (Org trans, PBS + Lipo vs Clodronate + Lipo). For Ly6C + MHCII + cells, 0.0079 (Org trans, PBS + Lipo vs Clodronate + Lipo). For Ly6C - MHCII + cells, 0.0159 (PBS + Lipo, Ctrl vs Org trans), 0.0079 (Org trans, PBS + Lipo vs Clodronate + Lipo). I Quantification of CD206 + F4/80 + CD45 + CD11b + macrophages ( n = 5 mice/group). Represent significant p value using two-tailed Mann–Whitney test. 0.0159 (PBS + Lipo, Ctrl vs Org trans), 0.0079 (Org trans, PBS + Lipo vs Clodronate + Lipo). J Representative histograms of CD206 expression. K Quantification of CD206 MFI in the LP of the small intestine 36 h after intestinal I/R injury (n = 5 mice/group). Represent significant p value using two-tailed Mann–Whitney test. 0.0079 (PBS + Lipo, Ctrl vs Org trans), 0.0079 (Org trans, PBS + Lipo vs Clodronate + Lipo). Scale bar, 100 μm. The statistical tests employed included: two-way ANOVA followed by the Tukey test for multiple comparisons and Mann–Whitney tests. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Each dot represents data from a single mouse ([ A – F ], [ H , I ], and [ K ]). Bar graphs represent mean ± SD. Source data are provided as a Source Data file.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_0233/pmc10600233/pmc10600233__41467_2023_42502_Fig3_HTML.jpg)
![A H & E staining of small intestinal tissue from mice 36 h after induction of intestinal I/R and quantification of the small intestinal pathology score (n = 5 mice for transplanted groups, n = 6 mice for the rest groups). Represent significant p value using two-way ANOVA followed by the Tukey test. 0.0167 (Vehicle, Ctrl vs Org trans), 0.0172 (Ctrl, Vehicle vs Malic acid). B qRT-PCR analysis of Occludin and ZO-1 mRNA in small intestinal tissues from mice 36 h after intestinal I/R (n = 5 mice for transplanted groups, n = 6 mice for the rest groups). Represent significant p value using two-tailed Mann–Whitney test for Occludin. 0.0043 (Vehicle, Ctrl vs Org trans), 0.0043 (Ctrl, Vehicle vs Malic acid). Represent significant p value using two-way ANOVA followed by the Tukey test for ZO-1. 0.0105 (Vehicle, Ctrl vs Org trans), 0.0259 (Ctrl, Vehicle vs Malic acid). C Representative immunohistochemistry images of Occludin expression and quantification of the area of Occludin immunohistochemical staining (n = 5 mice for transplanted groups, n = 6 mice for the rest groups). Represent significant p value using two-tailed Mann–Whitney test. 0.0043 (Vehicle, Ctrl vs Org trans), 0.0022 (Ctrl, Vehicle vs Malic acid). D Representative immunohistochemistry images of ZO-1 and quantification of the area of ZO-1 staining (n = 5 mice for transplanted groups, n = 6 mice for the rest groups). Represent significant p value using two-tailed Mann–Whitney test. 0.0043 (Vehicle, Ctrl vs Org trans), 0.0022 (Ctrl, Vehicle vs Malic acid). E ELISA detection of IL-6 and IL-1β production in mice 36 h after intestinal I/R (n = 5 mice for transplanted groups, n = 6 mice for the rest groups). Represent significant p value using two-way ANOVA followed by the Tukey test. For IL-6, 0.0297 (Vehicle, Ctrl vs Org trans), 0.0190 (Ctrl, Vehicle vs Malic acid). For IL-1β, < 0.0001 (Vehicle, Ctrl vs Org trans), 0.0002 (Ctrl, Vehicle vs Malic acid). F Quantification of CD206 + F4/80 + CD45 + CD11b + macrophages (n = 5 mice for transplanted groups, n = 6 mice for the rest groups). Represent significant p value using two-tailed Mann–Whitney test. 0.0043 (Vehicle, Ctrl vs Org trans), 0.0022 (Ctrl, Vehicle vs Malic acid). G Representative histograms of CD206 expression. H Quantification of CD206 MFI in the LP of the small intestine 36 h after intestinal I/R injury (n = 5 mice for transplanted groups, n = 6 mice for the rest groups). Represent significant p value using two-way ANOVA followed by the Tukey test. 0.0056 (Vehicle, Ctrl vs Org trans), 0.0012 (Ctrl, Vehicle vs Malic acid). I Representative histograms of IL-10 expression. J Quantification of IL-10 + F4/80 + CD45 + CD11b + macrophages (n = 5 mice for transplanted groups, n = 6 mice for the rest groups). Represent significant p value using two-way ANOVA followed by the Tukey test. 0.0083 (Vehicle, Ctrl vs Org trans), 0.0085 (Ctrl, Vehicle vs Malic acid). K Quantification of IL-10 MFI in the LP of the small intestine 36 h after intestinal I/R (n = 5 mice for transplanted groups, n = 6 mice for the rest groups). Represent significant p value using two-tailed Mann–Whitney test. 0.0022 (Ctrl, Vehicle vs Malic acid). L Serum concentration of IL-10 in mice 36 h after intestinal I/R (n = 5 mice for transplanted groups, n = 6 mice for the rest groups). Represent significant p value using two-way ANOVA followed by the Tukey test. 0.0247 (Vehicle, Ctrl vs Org trans), 0.0013 (Ctrl, Vehicle vs Malic acid). M Genetic profiling of BMDMs stimulated with or without MA (n = 4 biological replicates for vehicle group, n = 6 biological replicates for MA group). Represent significant p value using two-tailed student’s from left to right: 0.0108, 0.0096, 0.0015. N Representative images of CD206 expression and quantification of CD206 + F4/80 + CD11b + in BMDMs stimulated with or without MA supplementation (n = 3 biological replicates/group). Represent significant * p value = 0.0266 using two-tailed student’s t test. O Representative images of immunostaining of CD206 (green) and DAPI (blue) in BMDMs stimulated with or without MA supplementation. Scale bar, 100 μm. The statistical tests employed included: two-way ANOVA followed by the Tukey test for multiple comparisons, two-tailed student’s t test and Mann–Whitney tests. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Each dot represents data from a single sample ([ A – F ], [ H ], and [ J – N ]). Bar graphs represent mean ± SD. Source data are provided as a Source Data file.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_0233/pmc10600233/pmc10600233__41467_2023_42502_Fig5_HTML.jpg)
![A Gene ontology (GO) pathway enrichment analyses of the upregulated DEGs between two groups. The size of the node represents the number of DEGs, and the color of the node represents the corresponding P value. B Scatter plots comparing the DEGs between the MA administrated and vehicle groups. Genes were monitored according to their expression levels (log10 intensity). Red and green dots represented upregulated and downregulated genes, respectively. C Genetic profiling of SOCS family genes in BMDMs administrated with or without MA. D Representative histograms and quantification of CD206 expression in BMDMs depleted of SOCS2 using siRNA and co-cultured with or without MA (n = 3 biological replicates for siNC administrated vehicle group, n = 4 biological replicates for the rest groups). Represent significant p value using two-way ANOVA followed by the Tukey test. 0.0034 (siNC, Vehicle vs MA), 0.0007 (MA, siNC vs siSOCS2). E Representative images of immunostaining of CD206 (green) and DAPI (blue) in BMDMs depleted of SOCS2 using siRNA and co-cultured with or without MA. F Representative histograms of IL-10 expression and quantification of IL-10 + MFI (n = 3 biological replicates/group). Represent significant p value using two-way ANOVA followed by the Tukey test. 0.0335 (siNC, Vehicle vs MA), 0.0072 (MA, siNC vs siSOCS2). G Quantification of IL-10 + F4/80 + CD45 + CD11b + macrophages in BMDMs depleted of SOCS2 using siRNA and co-cultured with or without MA (n = 3 biological replicates/group). Represent significant p value using two-way ANOVA followed by the Tukey test. 0.0353 (siNC, Vehicle vs MA), 0.0066 (MA, siNC vs siSOCS2). H ELISA detection of IL-10 concentration released by BMDMs depleted of SOCS2 using siRNA and co-cultured with or without MA (n = 3 biological replicates/group). Represent significant p value using two-way ANOVA followed by the Tukey test. 0.0020 (siNC, Vehicle vs MA), 0.0001 (MA, siNC vs siSOCS2). Scale bar, 100 μm. The statistical tests employed included: two-way ANOVA followed by the Tukey test for multiple comparisons and two-tailed student’s t test. * P < 0.05, ** P < 0.01, *** P < 0.001. Each dot represents data from a single sample ([ D ], [ F – H ]). Bar graphs represent mean ± SD. Source data are provided as a Source Data file.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_0233/pmc10600233/pmc10600233__41467_2023_42502_Fig6_HTML.jpg)
![A H & E staining of small intestinal tissue from mice 36 h after induction of intestinal I/R and quantification of the small intestinal pathology score (n = 3 in SOCS2 –/– mice/group, n = 4 in WT mice/group). Represent significant p value using two-way ANOVA followed by the Tukey test. 0.0241 (WT, I/R + Vehicle vs I/R + MA), 0.0020 (I/R + MA, WT vs SOCS2 –/– ). B Quantification of the area of Occludin, ZO-1 immunohistochemical staining (n = 3 in SOCS2 –/– mice/group, n = 4 in WT mice/group). Represent significant p value using two-way ANOVA followed by the Tukey test. For Occludin, 0.0231 (WT, I/R + Vehicle vs I/R + MA), 0.0019 (I/R + MA, WT vs SOCS2 –/– ); for ZO-1, < 0.0001 (WT, I/R + Vehicle vs I/R + MA), < 0.0001 (I/R + MA, WT vs SOCS2 –/– ). C qRT-PCR analysis of Occludin and ZO-1 mRNA in small intestinal tissues from mice 36 h after intestinal I/R (n = 3 in SOCS2 –/– mice/group, n = 4 in WT mice/group). Represent significant p value using two-way ANOVA followed by the Tukey test. For Occludin, 0.0056 (WT, I/R + Vehicle vs I/R + MA), 0.0005 (I/R + MA, WT vs SOCS2 –/– ); for ZO-1, 0.0014 (WT, I/R + Vehicle vs I/R + MA), 0.0007 (I/R + MA, WT vs SOCS2 –/– ). D ELISA detection of IL-6, IL-1β and IL-10 production in mice 36 h after intestinal I/R (n = 3 in SOCS2 –/– mice/group, n = 4 in WT mice/group). Represent significant p value using two-way ANOVA followed by the Tukey test. For IL-6, 0.046 (WT, I/R + Vehicle vs I/R + MA), 0.0046 (I/R + MA, WT vs SOCS2 –/– ); for IL-1β, 0.0159 (WT, I/R + Vehicle vs I/R + MA), 0.0052 (I/R + MA, WT vs SOCS2 –/– ); for IL-10, 0.0005 (WT, I/R + Vehicle vs I/R + MA), 0.0003 (I/R + MA, WT vs SOCS2 –/– ). E Frequency of Ly6C + MHCII - , Ly6C + MHCII + , and Ly6C - MHCII + subsets among CD45 + CD11b + cells in the small intestine of different groups (n = 3 in SOCS2 –/– mice/group, n = 4 in WT mice/group). Represent significant p value using two-way ANOVA followed by the Tukey test for Ly6C + MHCII - and Ly6C + MHCII + cells. For Ly6C + MHCII - cells, 0.0169 (WT, I/R + Vehicle vs I/R + MA), 0.0221 (I/R + MA, WT vs SOCS2 –/– ); for Ly6C + MHCII + cells, 0.0004 (WT, I/R + Vehicle vs I/R + MA), 0.0011 (I/R + MA, WT vs SOCS2 –/– ). F Quantification of CD206 + F4/80 + CD45 + CD11b + macrophages (n = 3 in SOCS2 –/– mice/group, n = 4 in WT mice/group). Represent significant p value using two-way ANOVA followed by the Tukey test. 0.0004 (WT, I/R + Vehicle vs I/R + MA), 0.0012 (I/R + MA, WT vs SOCS2 –/– ). G Representative histograms of CD206 expression in the LP of the small intestine 36 h after intestinal I/R injury. H Schematic illustration of the induction protocols for macrophage adoptive transfer. I H & E staining of small intestinal tissue from mice 36 h after induction of macrophages adoptive transfer and quantification of the small intestinal pathology score (n = 3 for recipient SOCS2 -/- mice adoptive transfer of the macrophages from MA-administrated WT mice group, n = 4 mice for the rest groups). Represent significant p value using two-way ANOVA followed by the Tukey test. 0.0163 (WT, Adoptive transfer of WT macrophages vs Adoptive transfer of SOCS2 –/– macrophages), 0.0258 (SOCS2 –/– , Adoptive transfer of WT macrophages vs Adoptive transfer of SOCS2 –/– macrophages). J Quantification of the area of Occludin, ZO-1 immunohistochemical staining (n = 3 for recipient SOCS2 -/- mice adoptive transfer of the macrophages from MA-administrated WT mice group, n = 4 mice for the rest groups). Represent significant p value using two-way ANOVA followed by the Tukey test. For Occludin, 0.0003 (WT, Adoptive transfer of WT macrophages vs Adoptive transfer of SOCS2 –/– macrophages), 0.0023 (SOCS2 –/– , Adoptive transfer of WT macrophages vs Adoptive transfer of SOCS2 –/– macrophages); for ZO-1, 0.0018 (WT, Adoptive transfer of WT macrophages vs Adoptive transfer of SOCS2 –/– macrophages), 0.0033 (SOCS2 –/– , Adoptive transfer of WT macrophages vs Adoptive transfer of SOCS2 –/– macrophages). K ELISA detection of IL-6, IL-1β and IL-10 production in mice 36 h after macrophages adoptive transfer (n = 3 for recipient SOCS2 -/- mice adoptive transfer of the macrophages from MA-administrated WT mice group, n = 4 mice for the rest groups). Represent significant p value using two-way ANOVA followed by the Tukey test. For IL-6, 0.0288 (WT, Adoptive transfer of WT macrophages vs Adoptive transfer of SOCS2 –/– macrophages), 0.0226 (SOCS2 –/– , Adoptive transfer of WT macrophages vs Adoptive transfer of SOCS2 –/– macrophages); for IL-1β, 0.0012 (WT, Adoptive transfer of WT macrophages vs Adoptive transfer of SOCS2 –/– macrophages), 0.0004 (SOCS2 –/– , Adoptive transfer of WT macrophages vs Adoptive transfer of SOCS2 –/– macrophages); for IL-10, 0.0022 (WT, Adoptive transfer of WT macrophages vs Adoptive transfer of SOCS2 –/– macrophages), 0.0154 (SOCS2 –/– , Adoptive transfer of WT macrophages vs Adoptive transfer of SOCS2 –/– macrophages). Scale bar, 200 μm. The statistical tests employed included: two-way ANOVA followed by the Tukey test for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Each dot represents data from a single sample ([ A – F ], [ I – K ]). Bar graphs represent mean ± SD. Source data are provided as a Source Data file.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_0233/pmc10600233/pmc10600233__41467_2023_42502_Fig7_HTML.jpg)