MSCs mediate long-term efficacy in a Crohn's disease model by sustained anti-inflammatory macrophage programming via efferocytosis

Abstract

Mesenchymal stem cells (MSCs) are novel therapeutics for the treatment of Crohn's disease. However, their mechanism of action is unclear, especially in disease-relevant chronic models of inflammation. Thus, we used SAMP-1/YitFc (SAMP), a chronic and spontaneous murine model of small intestinal inflammation, to study the therapeutic effects and mechanism of action of human bone marrow-derived MSCs (hMSC). hMSC dose-dependently inhibited naïve T lymphocyte proliferation via prostaglandin E₂ (PGE₂) secretion and reprogrammed macrophages to an anti-inflammatory phenotype. We found that the hMSCs promoted mucosal healing and immunologic response early after administration in SAMP when live hMSCs are present (until day 9) and resulted in a complete response characterized by mucosal, histological, immunologic, and radiological healing by day 28 when no live hMSCs are present. hMSCs mediate their effect via modulation of T cells and macrophages in the mesentery and mesenteric lymph nodes (mLN). Sc-RNAseq confirmed the anti-inflammatory phenotype of macrophages and identified macrophage efferocytosis of apoptotic hMSCs as a mechanism that explains their long-term efficacy. Taken together, our findings show that hMSCs result in healing and tissue regeneration in a chronic model of small intestinal inflammation and despite being short-lived, exert long-term effects via sustained anti-inflammatory programming of macrophages via efferocytosis.

Fig. 1. hMSC suppresses murine T cell proliferation and reprogram murine macrophages to an anti-inflammatory phenotype.

a Mesodermal differentiation potential of BM-hMSCs and b Human fibroblasts (FB). c SAMP T cell proliferation in MLR (S: SAMP splenocytes, B6: C57Bl/6 J derived irradiated splenocytes) treated with hMSC and FB for 96 hours. d PGE₂ concentration measured in mixed lymphocyte reaction (MLR) supernatants using an enzyme immunoassay. e SAMP T cell proliferation in MLR treated with hMSCs and COX inhibitor indomethacin (10 µM). f PGE₂ concentration measured in MLR supernatant using an enzyme immunoassay. g Heat Map showing the gene expression of Crohn’s RT-PCR profiler array on MLR reaction, h Relative gene expression of indicated markers and cytokine measured in total RNA extracted from murine bone marrow-derived macrophages (mBM-Mϕ). The gene expression was determined by qRT-PCR, normalized to GAPDH, and expressed as fold change (2-^ΔΔCt). i PGE₂ concentration measured in reaction supernatant from hMSCs and murine bone marrow-derived macrophages (mBM-Mϕ) co-culture using an enzyme immunoassay. Data were expressed as mean ± SD from at least two independent experiments. P < 0.05 considered significant, by 1-way ANOVA (c–f) and 2-tailed, unpaired Student’s t test (h, i).

Fig. 2. hMSC survive in the SAMP peritoneum cavity up to day 9.

a Representative images showing time course BLI of transduced hMSCs in AKR and SAMP mice on days 0, 5, and 9. b Representative time course epifluorescence images of SAMP mice showing the presence of intraperitoneally administered NIR dye IVIS680 labeled hMSCs. Images were acquired on days 0, 9, and 28. c Quantitative estimation of total radiant efficiency in mice treated with IVIS680 tagged hMSCs. Data were expressed as mean ± SD from at least 2 independent experiments. P < 0.05 considered significant, by 1-way ANOVA. d Representative ex-vivo epifluorescence image of SAMP peritoneum cavity showing the presence of IVIS680 tagged hMSCs (yellow arrow) on day 9. e Flow cytometry gating scheme to detect hMSC cell population in the peritoneal lavage of hMSC administered SAMP at day 9.

Fig. 3. hMSC treatment shows mucosal healing and early sign of immunological response on day 9.

a Schematic showing the treatment regimen of the experiment (n = 22). b Percent abnormal mucosa in PBS, hMSCs, and DEX-treated groups. c Representative comparative stereoscopic view of small intestine from DEX, hMSC, and PBS-treated groups (yellow arrow showing abnormal mucosa; blue arrow showing normal mucosa). d Flow cytometry gating strategy showing e Absolute number of CD3⁺, f CD4⁺, and g CD8⁺ cells in mesenteric lymph nodes (mLN). h Relative gene expression of cytokines from Th-1, Th-2, and Th-17 pathways was measured in total RNA extracted from mesenteric lymph node cells. Gene expression was determined by qRT-PCR, normalized to GAPDH, and expressed as fold change (2-ΔΔCt). i Schematic showing treatment regimen of the experiment (n = 14). j Percent abnormal mucosa in PBS and DEX-treated groups. k Representative comparative stereoscopic view of the small intestine in PBS and DEX treated groups (yellow arrows showing abnormal mucosa). l The inflammatory index for disease severity in SAMP mice treated with PBS and DEX. m Representative histopathology photomicrograph of ileum tissue from PBS, and DEX treated groups (Red triangle= villus distortion, black triangle=crypts hyperplasia, red arrow=immune infiltration in submucosa, black arrow= muscle hypertrophy). The scale bar represents 200 µm. Data were expressed as mean ± SD from at least two independent experiments unless specified. Each data point represents one mouse. In box and whiskers-plot, center line: median; box limits: 25-75 percentile; whiskers: min. to max. with all data points. P < 0.05, P < 0.01, P < 0.001 considered significant, by 1-way ANOVA and unpaired t-test (j, l).

Fig. 4. hMSC treatment alleviate intestinal inflammation in SAMP on day 28.

a Schematic showing the treatment regimen of the experiment (n = 38). b Percent abnormal mucosa in PBS, hMSCs and DEX-treated groups. c Representative comparative stereoscopic view of the small intestine in DEX, hMSCs, and PBS-treated groups (yellow arrows showing abnormal mucosa; blue arrow showing normal mucosa). d The Inflammatory Index for disease severity in PBS, hMSCs, and DEX-treated groups showing histologic small intestine (SI) inflammation. e Representative histopathology photomicrograph of ileum tissue from PBS, hMSCs and DEX-treated groups (Red triangle = villus distortion, black triangle = crypts hyperplasia, red arrow = immune infiltration in submucosa, black arrow = muscle hypertrophy). The scale bar represents 200 µm. f MR image for SAMP shows human CD features, including SI wall thickening, stricture, free fluid collection, enlarged mesenteric lymph node(mLN). g MR image annotation in SAMP, h Radiomic feature extraction. i Radiomics analysis involved 100 radiomic features from four different classes to quantify SI wall appearance in terms of heterogeneity and gradient responses in PBS and DEX-treated groups. j Top-ranked features (Haralick and Sobel) were selected and used to train a machine-learning Random Forest classifier to yield a radiomics-based likelihood of disease severity. k SIMPle score resulted in further enhancement of disease severity assessment and discrimination between treatment groups (n = 26). Data were expressed as mean ± SD from at least two independent experiments unless specified, each data point represents one mouse. In the Box and whiskers-plot, center line: median; box limits: 25–75 percentile; whiskers: min. to max. with all data points. P < 0.05 considered significant, by 1-way ANOVA.

Fig. 5. Sc-RNAseq was performed on single-cell suspension of mesenteric stromal vascular fraction (SVF) from SAMP mice treated with PBS and hMSC.

a–e Sc-RNAseq showing analysis on day 9 samples. a UMAP plot showing identified clusters at a resolution of 1.0. b Volcano plots showing differential gene expression in all cell types with a threshold value (FDR < 0.05). The top three significant differentially expressed genes (logFC >1.5 with FDR < 0.05) are highlighted and labelled in the plots. c Volcano plots showing differential gene expression in macrophage cluster with a threshold value (FDR < 0.05). The top 3 significant differentially expressed genes (logFC>1.5 with FDR < 0.05) (upregulated and downregulated) are labelled and highlighted in the plot. d UMAP plots of the representative macrophage markers genes (Mrc1, Cd163, H2-Ab1) showing normalized gene expression in the macrophage cluster. e Dot plots showing the average expression of selective genes expressed by the proportion of the cells in the macrophage cluster across the treatment. f–j Single-cell RNA sequencing showing analysis on day 28 samples. f UMAP plot showing identified clusters at cluster resolution 0.4. g Volcano plots show differential gene expression in all cell types with a threshold value (FDR < 0.05). Cdk8 was significant differentially expressed gene (logFC >1.5 with FDR < 0.05) in hMSC-treated mice on day 28. h Volcano plots showing differential gene expression in macrophage cluster with a threshold value (FDR < 0.05). Two genes were significantly differentially expressed (FC > 1.5 with FDR < 0.05) in hMSCs-treated mice. i UMAP plots of the representative macrophage markers genes (Mrc1, Cd163, H2-Ab1) showing normalized gene expression in the macrophage cluster. j Dot plots showing average expression of selective genes and proportion of the cells representing in the cluster across the treatment.

Fig. 6. hMSC secrete PGE₂, educates mLN, mesenteric, and peritoneum macrophages to anti-inflammatory phenotype, and suppresses T cells to alleviate small intestine inflammation in SAMP.

Relative gene expression of TNF-α and Arginase I on day 9 of the treatment, measured in the total RNA extracted from CD11b⁺ cells from (a) mesenteric lymph node and (b) mesenteric stromal vascular fraction. c, d Relative gene expression of TNF-α and Arginase I on day 28 of the treatment measured in the total RNA extracted from CD11b⁺ cells from (c) mesenteric lymph node (d) mesenteric stromal vascular fraction. Gene expression was determined by qRT-PCR, normalized to GAPDH, and expressed as fold change (2-ΔΔCt). e Arginase 1/ TNF-α ratio measured by flow cytometry in SAMP peritoneal macrophages. f, g Cell Tracker Red^TM CMPTX dye labelled hMSCs showing in-vivo phagocytosis by CD11b⁺ macrophages in peritoneal lavage (P.L.) and SVF at day 9. h Arginase 1/ TNF-α ratio measured by flow cytometry in SAMP peritoneal and SVF Cd11b⁺ macrophages (Efferocytes) phagocytosed hMSC. i Brightfield microscopic images of SAMP peritoneal macrophages co-cultured with live hMSCs (no visible apoptosis observed) and apoptotic hMSCs (white arrow showing phagosome formation in macrophage), fluorescence image showing phagocytosis of apoptotic hMSCs by SAMP peritoneal macrophages, arrows showing engulfed apoptotic bodies of hMSC. Scale bar 10 µm. j PGE₂ concentration measured in the peritoneal fluid of SAMP. k Absolute numbers of CD3, CD4, and CD8 lymphocytes measured in mLN at day 28 of the treatment. l Relative gene expression of cytokines in CD4 lymphocytes from hMSCs-treated SAMP at day 28. Data were expressed as mean ± SD from at least two independent experiments, each data point represent one mouse. P < 0.05, considered significant, by 2-tailed, unpaired Student’s t test.

Fig. 7. Schematic showing putative mechanism of hMSCs mediated immunosuppression in experimental model of Crohn’s disease.

In the early stage, live hMSC secretion of molecules like PGE₂ is the dominant mechanism. PGE₂ is released by hMSCs in the peritoneal cavity which diffuses through the fenestrated mesenteric lymphatic vessels to reach mLN, inhibit naïve T cell proliferation and educate SVF and mLN resident macrophages to anti-inflammatory phenotype with subsequent downregulation of proinflammatory cytokines, and enhance macrophages efferocytosis capacity. In the later phase, macrophages efferocytose apoptotic hMSCs, which leads to their proliferation and reprogramming to an anti-inflammatory phenotype that maintains and mediates the long-term effect.