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An injectable subcutaneous colon-specific immune niche for the treatment of ulcerative colitis

Nature Biomedical Engineering (2023)Cite this article

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Abstract

As a chronic autoinflammatory condition, ulcerative colitis is often managed via systemic immunosuppressants. Here we show, in three mouse models of established ulcerative colitis, that a subcutaneously injected colon-specific immunosuppressive niche consisting of colon epithelial cells, decellularized colon extracellular matrix and nanofibres functionalized with programmed death-ligand 1, CD86, a peptide mimic of transforming growth factor-beta 1, and the immunosuppressive small-molecule leflunomide, induced intestinal immunotolerance and reduced inflammation in the animals’ lower gastrointestinal tract. The bioengineered colon-specific niche triggered autoreactive T cell anergy and polarized pro-inflammatory macrophages via multiple immunosuppressive pathways, and prevented the infiltration of immune cells into the colon’s lamina propria, promoting the recovery of epithelial damage. The bioengineered niche also prevented colitis-associated colorectal cancer and eliminated immune-related colitis triggered by kinase inhibitors and immune checkpoint blockade.

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Fig. 1: Bioengineering and characterization of the immunosuppressive combinational colon-specific immune niche.
Fig. 2: Combinational immunosuppressive NFs and colon ECM effectively inhibit antigen-specific CD8+ T cell activation and promote classically activated M1 macrophages differentiation into alternatively activated M2 macrophages in vitro.
Fig. 3: Combinational colon-specific immune niche effectively ameliorated colitis and promoted recovery in established mouse ulcerative colitis models.
Fig. 4: Mechanistic insight: combinational colon-specific immune niche ameliorated DSS-induced colitis by inhibiting colon-specific CD8+ T cell activation and polarizing classically activated M1 macrophages into alternatively activated M2.
Fig. 5: Combinational colon-specific immune niche effectively ameliorated colitis-associated colorectal cancer and phosphatidylinositol 3-kinase inhibition-associated colitis in cell signalling pathway-targeted cancer treatment.
Fig. 6: Subcutaneously administered combinational colon-specific immune niche and intraperitoneally administered combinational NFs are equally effective in ameliorating immune-related colitis without affecting the anti-cancer activities of ICB.

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Data availability

The main data supporting the results in this study are available within the paper and its Supplementary Information. The raw and analysed datasets generated during the study are available for research purposes from the corresponding authors on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank the UTSW Animal Resource Center, UTSW ARC Diagnostic Laboratory, UTSW ARC Veterinary Services, UTSW Electron Microscopy Facility, UTSW Quantitative Light Microscopy Core, UTSW Flow Cytometry Core, UTSW Histo Pathology Core, UTSW Proteomics Core, UTSW Microarray and Immune Phenotyping Core, UTSW Microbiome Research Lab., UTSW Preclinical Radiation Core Facility and UTSW Whole Brain Microscopy Facility at the UTSW, UT Austin Texas Materials Institute, University of North Carolina at Chapel Hill Pathology Services Core, Akina, Inc. (West Lafayette, IN), RayBiotech Life, Inc. (Corners, GA), IDEXX BioAnalytics (Columbia, MI) and iHisto, Inc. (Salem, MA) for their assistance with the procedures in this article. UTSW Electron Microscopy Facility is supported by the NIH grant 1S10OD021685-01A1. UTSW Quantitative Light Microscopy Core is supported by the NIH grant 1S10OD021684-01. UT Austin Texas Materials Institute is supported by the National Science Foundation Major Research Instrument Grant CBET-1624659. Pathology Services Core at the University of North Carolina-Chapel Hill is supported in part by an NCI Centre Core Support Grant 5P30CA016080-42. A.Z.W. was supported by the NIH grants R01GM130590 and R01EB025651.

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Authors and Affiliations

  1. Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Kin Man Au & Andrew Z. Wang

  2. Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Justin E. Wilson & Jenny P.-Y. Ting

  3. Department of Microbiology, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Justin E. Wilson & Jenny P.-Y. Ting

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  1. Kin Man Au
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Contributions

K.M.A., A.Z.W., J.P.-Y.T. and J.E.W. conceived and designed the experiments. K.M.A. conceived the experiments. K.M.A. and A.Z.W. analysed the data. K.M.A. and A.Z.W. co-wrote the paper. All authors discussed the results and edited the manuscript at all stages.

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Correspondence to Kin Man Au or Andrew Z. Wang.

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Extended data

Extended Data Fig. 1 Combinational immunosuppressive nanofibers effectively inhibit antigen-specific CD8+ T cell activation in vitro.

a, T cell inhibition (exhaustion) markers and proliferation indices of CD8+ T cells cultured with different functionalized NFs in the presence of anti-CD3/anti-CD28-functionalized T cell activation beads (Dynabeads) at a 1:1 ratio for 48 h, as quantified through fluorescence-activated cell sorting. The proliferation study was performed on CFSE-labelled CD8+ T cells. (n = 4). b, T cell activation and exhaustion markers on naïve and trinitrophenol-sensitized CD8+ T cells (in splenocytes) after co-culture with trinitrophenol-modified colon epithelial cells in the presence or absence of combinational immunosuppressive molecules or combinational immunosuppressive nanofibers (48 h). (n = 4). c, ELISpot assay of IFN-gamma in splenocytes after culture with unmodified or trinitrophenol-modified colon epithelial cells in the presence or absence of combinational immunosuppressive molecules or combinational immunosuppressive nanofibers (n = 4). d, Cytotoxicity of splenocytes against colon epithelial cells in the presence or absence of combinational immunosuppressive molecules or combinational immunosuppressive nanofibers (n = 7). Combinational nanofibers, the combination of PD-L1/CD86-functionalized nanofibers, leflunomide-encapsulated nanofibers and TGF-β1 mimetic peptide-encapsulated nanofibers; combinational immunosuppressive molecules, the combination of PD-L1/CD86-functionalized nanofibers, leflunomide-encapsulated nanofibers and TGF-β1 mimetic peptide-encapsulated nanofibers. Data are presented as the mean ± s.e.m. All P values were analysed using two-way ANOVA with Tukey’s HSD multiple comparisons post-hoc test.

Source data

Extended Data Fig. 2 Therapeutic treatment with immune checkpoint molecule-bioengineered colon-specific immune niches prevents colitis-associated weight loss, and colon shortening and reduces immune cell infiltration into the lamina propria.

a, Bodyweight changes throughout the study. b, Digital photographs of colons preserved at the study endpoint, that is, 7 days after therapeutic treatments. c, Representative H&E-stained section of each colon preserved at the study endpoint after different treatments. The inserted label in each image represents the corresponding colon epithelial damage score and inflammatory cell infiltration score. P values were analysed using one-way ANOVA (a) with Tukey’s HSD multiple comparisons post-hoc test.

Source data

Extended Data Fig. 3 Treatment with subcutaneously inoculated colon-specific immune niches or intraperitoneally administered combination of immunosuppressive molecule-functionalized nanofibers inhibits the production of colitis-associated myeloperoxidase and proinflammatory cytokines in the colon.

a-c, Colonic myeloperoxidase activity (a), pro-inflammatory cytokine (b) and pro-regulatory cytokine (c) expression in the preserved colon specimens at the study endpoint (5 days after different therapeutic treatments) after different therapeutic treatments (n = 5). d, Representative immunofluorescence images of colons preserved from dextran sodium sulfate-induced colitis mice, captured 5 days after different therapeutic treatments, reveal lamina propria-infiltrated IFN-gamma+ CD8+ T cells. Data are presented as the mean ± s.e.m. All P values were analysed using one-way ANOVA with Tukey’s HSD multiple comparisons post-hoc test.

Source data

Extended Data Fig. 4 Therapeutic treatment with non-immune checkpoint molecule-functionalized colon-specific immune niches and colon-specific immune niches prevents colitis-associated colon shortening and reduces immune cell infiltration into the lamina propria.

a, Bodyweight changes throughout the study. b, Digital photographs of colons preserved at the study endpoint, that is, 7 days after different therapeutic treatments. c, Representative H&E-stained section of each colon preserved at the study endpoint after different treatments. The inserted label in each image represents the corresponding colon epithelial damage score and inflammatory cell infiltration score. P values were analysed using one-way ANOVA (a) with Tukey’s HSD multiple comparisons post-hoc test.

Source data

Extended Data Fig. 5 Colon-specific immune niches effectively ameliorate DSS-induced colitis in male C57BL/6 mice.

a-d, Evaluation of the therapeutic treatment efficiency of combinational colon-specific immune niches in the dextran sodium sulfate-induced-induced colitis mouse model. Therapeutic treatment schedule (a): colitis mice received a single subcutaneous treatment with the combo immune niche at day 6. b-c, Bodyweight change (b) and disease activity index score (c) were monitored for up to 12 days. d, Digital images and length of colon preserved at the study endpoint. e, H&E-stained sections of each colon preserved at the study endpoint and their disease scores after therapeutic treatment with the combo immune niche. The inserted label in each image represents the corresponding colon epithelial damage score and inflammatory cell infiltration score. Data are presented as the mean ± standard error of the mean (s.e.m.). All P values were analysed using one-way (d,e) or two-way (b,c) ANOVA with Tukey’s HSD multiple comparisons post-hoc test.

Source data

Extended Data Fig. 6 The combination of colon epithelial cells and combinational immunosuppressive nanofibers, or colon extracellular matrix alone are not as effective as combinational colon-specific immune niche (the combination of colon epithelial cells, colon extracellular matrix, combinational immunosuppressive nanofibers) in relieving the colitis symptoms in a dextran sodium sulfate-induced colitis model.

Bodyweight changes (a,c,e) and DAI scores (b,d,f) after different control colitis treatments with different control colon-specific immune niches. Statistical analysis was performed 4 days after different colitis treatments. (n = 6 or 7) Data are presented as the mean ± s.e.m. All P values were analysed using two-way ANOVA with Tukey’s HSD multiple comparisons post-hoc test.

Source data

Extended Data Fig. 7 The combination of colon epithelial cells and combinational immunosuppressive nanofibers, or colon extracellular matrix alone are not as effective as colon-specific immune niches (the combination of colon epithelial cells, colon extracellular matrix, combinational immunosuppressive nanofibers) in preventing inflammation-associated colon shortening, colitis-induced colon epithelial damage and immune cell infiltration into the colon in the dextran sodium sulfate colitis model.

a, Digital photographs and the lengths of colons preserved at the study endpoint after different colitis treatments. b, Representative H&E-stained sections of each colon and colon damage scores after different colitis treatments. The inserted label in each image represents the corresponding colon epithelial damage score and inflammatory cell infiltration score. (n = 6 or 7) All P values were analysed using one-way (a) or two-way (b) ANOVA with Tukey’s HSD multiple comparisons post-hoc test.

Source data

Extended Data Fig. 8 Pancreas-specific combinational immune niche cannot relieve colitis symptoms in dextran sodium sulfate colitis mice.

a, Analysis of growth factors and cytokines present in decellularized colon extracellular matrix and decellularized mouse pancreas via sandwich-type antibody microarray assay. (n = 4 biologically independent samples) b-c, Bodyweight change (b) and disease activity index score (c) after colitis treatment with combinational colon-specific immune niche or combinational pancreas-specific immune niche. d, Digital photographs of colons preserved at the study endpoint (5 days after therapeutic treatment), and their lengths (d). (n = 7 or 8) All P values were analysed using one-way (d) or two-way (a,b,c) ANOVA with Tukey’s HSD multiple comparisons post-hoc test.

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Extended Data Fig. 9 Intraperitoneal-administered combinational immunosuppressive nanofibers are as effective as subcutaneously inoculated combinational colon-specific immune niche to treat dextran sodium sulfate-induced colitis.

a, b, Bodyweight change (a) and disease activity index score (b) after colitis treatment with subcutaneously administered combinational colon-specific immune niche or intraperitoneally-administrated combinational immunosuppressive nanofibers. c, Digital photographs of colons preserved at the study endpoint (5 days after therapeutic treatment), and their lengths. d, Representative H&E-stained sections of each colon and colon damage scores after different colitis treatments. The inserted label in each image represents the corresponding colon epithelial damage score and inflammatory cell infiltration score. (n = 8) All P values were analysed using one-way (c) or two-way (a,b,d) ANOVA with Tukey’s HSD multiple comparisons post-hoc test.

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Extended Data Fig. 10 Single therapeutic treatment with combinational colon-specific immune niches effectively ameliorates colitis symptoms in a chronic dextran sodium sulfate-induced colitis model.

a, b, Disease activity index score (a) and bodyweight (b) change after treatment(s) with combinational colon-specific immune niche. c, Digital photographs and length of colons preserved at the study endpoint (48 days after the initial colitis induction) after one or three therapeutic treatments with the combo immune niche. d, Representative H&E-stained images, colon epithelial damage score, inflammatory cell infiltration score, and colonic damage score of colons preserved at the study endpoint (48 days after the first induction of colitis) after one or three therapeutic treatments with combinational colon-specific immune niche.

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Supplementary Table 3

Proteomic analysis of native colon samples versus decellularized COL ECM samples.

Supplementary Table 4

Statistical analysis of faecal microbiome compositions after different colitis treatments.

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Au, K.M., Wilson, J.E., Ting, J.PY. et al. An injectable subcutaneous colon-specific immune niche for the treatment of ulcerative colitis. Nat. Biomed. Eng (2023). https://doi.org/10.1038/s41551-023-01136-9

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