Abstract
Inflammatory bowel disease lacks a long-lasting and broadly effective therapy. Here, by taking advantage of the anti-infection and anti-inflammatory properties of natural antibodies against the small-molecule epitope phosphorylcholine (PC), we show in multiple mouse models of colitis that immunization of the animals with self-assembling supramolecular peptide nanofibres bearing PC epitopes induced sustained levels of anti-PC antibodies that were both protective and therapeutic. The strength and type of immune responses elicited by the nanofibres could be controlled through the relative valency of PC epitopes and exogenous T-cell epitopes on the nanofibres and via the addition of the adjuvant CpG. The nanomaterial-assisted induction of the production of therapeutic antibodies may represent a durable therapy for inflammatory bowel disease.
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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 author on reasonable request. Source data for the figures are provided with this paper.
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Acknowledgements
We thank Y. Wu, B. Cossette and S. Shetty for helpful advice; the staff of the Duke Surgery Substrate Services Core and Research Support, especially Histology Core members P. Newell and C. Leonard. Timeline images in Figs. 4–6, 8 and Supplementary Figs. 8 and 19 were drawn using Biorender.com. This work was supported by the National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health (NIH/NIBIB, R01 EB009701) and National Institute of Allergy and Infectious Diseases of the National Institutes of Health (NIH/NIAID, R01 AI172151), both awarded to J.H.C. E.J.C was supported by the National Science Foundation Graduate Research Fellowship Program (DGE-1644868). MALDI–TOF was conducted on an instrument supported by the North Carolina Biotechnology Center (2017-IDG-1018). Transmission electron microscopy was performed on an instrument at the Duke University Shared Materials Instrumentation Facility (SMIF), a member of the North Carolina Research Triangle Nanotechnology Network (RTNN), which is supported by the National Science Foundation (ECCS-2025064) as part of the National Nanotechnology Coordinated Infrastructure (NNCI).
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E.J.C. designed and performed experiments, analysed and interpreted data, and wrote and edited the manuscript. E.F.R., H.F.H., M.E.W. and N.L.V. performed experiments, analysed data and edited the manuscript. A.R.A. and T.S. conceptualized confocal tissue section quantification and analysis. L.P.H. designed experiments, analysed and interpreted data, and wrote and edited the manuscript. J.H.C. designed experiments, analysed and interpreted data, wrote and edited the manuscript, and supervised the research.
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E.J.C. and J.H.C. are listed as inventors on a pending patent application (PCT/US2023/019876) for the described technology. The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Intraperitoneal immunization with PC-Q11 and CpG adjuvant increases antibody and T-cell responses.
Mice were immunized either i.p. or s.c. at weeks 0, 2 and 4 with PC-Q11 co-assembled with PADRE-Q11 and with or without CpG adjuvant. a, Serum total anti-PC IgG responses showed that i.p. immunization raised the greatest responses and that these responses were vastly improved with the addition of CpG. b, Serum anti-PC IgM responses showed significantly greater IgM for mice immunized i.p. with CpG. c, Week 5 ELISpot of splenocytes restimulated with PADRE peptide showed heightened T-cell responses after immunizations containing CpG with biasing towards IFNγ for i.p. immunized mice. SFC = spot forming cell. AUC = area under the curve. Mean + or ± s.e.m. are shown. Statistical significance determined by two-way RM ANOVA with Tukey’s multiple comparison test for a and b, two-way ANOVA with Šídák’s multiple comparisons test for c. n = 5 mice.
Extended Data Fig. 2 Additional cysteines in nanofibres do not improve anti-PC antibody responses.
Negative-stained TEMs of (a) PC-Q11 (1 mM PC-Q11 co-assembled with 0.2 mM Cys4-Q11, 0.05 mM PADRE-Q11, and 0.75 mM Q11) and (b) PCM-Q11 (0.2 mM PCM-Q11 co-assembled with 0.2 mM Cys4-Q11, 0.05 mM PADRE-Q11, and 1.55 mM Q11) both co-assembled with 10% Cys4-Q11 showed that the addition of excess free cysteines did not inhibit nanofiber formation. c, Mice were immunized i.p. at weeks 0, 2 and 4 with PC-Q11 or PCM-Q11 co-assembled with PADRE-Q11 and with or without Cys4-Q11. Serum total anti-PC IgG responses indicated that the addition of Cys4-Q11 did not significantly enhance anti-PC IgG antibody levels for PC-Q11 or PCM-Q11. Mean + s.e.m. are shown. AUC = area under the curve. ns = not significant via two-way RM ANOVA with Tukey’s multiple comparison test. n = 5 mice.
Extended Data Fig. 3 Increasing PCM-Q11 epitope content 2.5x does not increase anti-PC antibody responses.
Mice were immunized i.p. at weeks 0, 2, 4 and 9 with 10% PCM-Q11 as used in other experiments (0.2 mM PCM-Q11, 1.75 mM Q11, and 0.05 mM PADRE-Q11) or 25% PCM-Q11 with 2.5x the amount of PC epitope (0.5 mM PCM-Q11, 1.45 mM Q11, and 0.05 mM PADRE-Q11) with CpG adjuvant. Serum total anti-PC IgG responses showed that both 10% PCM-Q11 and 25% PCM-Q11 raised robust anti-PC IgG responses that were not statistically different from each other via two-way RM ANOVA. AUC = area under the curve. Mean ± s.e.m. are shown. n = 5 mice.
Extended Data Fig. 4 Administration of an anti-CD4 monoclonal antibody depletes the CD4+ T-cell population.
a, Timeline of CD4+ T-cell depletion experiment. Mice were injected i.p. with 200 μg of either anti-CD4 or isotype control antibody on days -3, 1, 3, 7, 11, 15 and 19 and immunized i.p. on day 0 and 14 with PC-Q11 or PCM-Q11 co-assembled with PADRE-Q11 and CpG adjuvant. b,c, Representative flow plots verifying CD4+ T-cell depletion in unimmunized mice at (b) day 0 and (c) day 22 in the spleen, draining lymph nodes (axial, brachial, and inguinal), and mesenteric lymph nodes. d,e, Representative histograms of the CD4+ population in naïve or depleted mice at (d) day 0 and (e) day 22. n = 2 mice per group per timepoint.
Extended Data Fig. 5 Bacteria cultured from the spleen and serum FITC-dextran levels were not significantly altered after chronic colitis compared to healthy controls.
a, Spleen CFUs, an indication of bacterial spread from colon damage, were not different among groups. b, Measures of FITC-dextran in serum after oral gavage were not different among groups. Data from chronic colitis experiment 2. Mean ± s.e.m. are shown. Lack of statistical significance determined by one-way ANOVA with Tukey’s multiple comparison test. n = 10 mice.
Extended Data Fig. 6 PC immunization reduces bleeding but does not prevent histological damage in an Il10–/– colitis model.
a, Timeline of Il10-/- colitis model where mice were immunized i.p. on days -35, -21 and -7 with PCM-Q11 co-assembled with PADRE-Q11 and CpG adjuvant or PBS (disease control) followed by piroxicam administration in food fines for 7 days to trigger colitis and then 16 additional days of symptom monitoring. b,c, Robust anti-PC IgG (b) and IgM (c) antibody responses were generated in Il10-/- mice. d, IgG subclasses elicited showed the same biasing as in wild-type C57BL/6 mice immunized with PCM-Q11/CpG. e,f, There was no significant difference in weight loss (e) or fecal consistency (f) scores between piroxicam-administered groups. g, PCM-Q11/CpG immunization significantly improved bleeding scores compared to disease controls. h, Both PCM-Q11/CpG and PBS-administered mice given piroxicam developed moderate colitis levels as indicated by total histology scores. i-k, Hematoxylin and eosin-stained sections showed similar colon mucosal hyperplasia and inflammation in Il10-/- mice exposed to piroxicam, whether immunized with PCM-Q11/CpG (i) or with PBS alone (j). Mice immunized with PCM-Q11/CpG but not exposed to piroxicam had minimal to no colitis (k). Distal colon is shown for all mice. Scale bar indicates 200 µm. Mean +, - or ± s.e.m. are shown. Statistical significance determined by mixed-effects analysis with Tukey’s multiple comparison test for b,c and e-g, two-way ANOVA with Tukey’s multiple comparison test for d, and one-way ANOVA with Tukey’s multiple comparison test for h. n = 10 mice for PCM-Q11/CpG with piroxicam group or n = 9 mice for other groups.
Extended Data Fig. 7 Levels of B cells, B1a cells and plasma cells in the colons of mice after immunization, after DSS-induced colitis, and after piroxicam-triggered colitis.
a-d, CD19+ B cells isolated from the colon after: (a) immunization, (b) therapeutic DSS-induced colitis (c) chronic DSS-induced colitis and (d) Il10-/- piroxicam-triggered colitis. e-h, B220+ B cells isolated from the colon after: (e) immunization, (f) therapeutic DSS-induced colitis (g) chronic DSS-induced colitis and (h) Il10-/- piroxicam-triggered colitis. i-l, CD19+CD5+ B1a cells isolated from the colon after: (i) immunization, (j) therapeutic DSS-induced colitis (k) chronic DSS-induced colitis and (l) Il10-/- piroxicam-triggered colitis. m-p, B220+CD138+ plasma cells isolated from the colon after: (m) immunization, (n) therapeutic DSS-induced colitis (o) chronic DSS-induced colitis and (p) Il10-/- piroxicam-triggered colitis. Percentages are reported as the percent of the parent population. Mean ± s.e.m. are shown. Statistical significance determined by one-way ANOVA with Tukey’s multiple comparison test. For immunization group, n = 5 mice, for chronic DSS-induced colitis, n = 10 mice except for the PBS with DSS group where n = 9 mice, for therapeutic colitis, n = 10 mice, and for Il10-/- piroxicam-induced colitis, n = 9 mice except for the PCM-Q11/CpG with piroxicam group where n = 10 mice.
Extended Data Fig. 8 Levels of the tight-junction proteins ZO-1 and occludin in colon sections after immunization and after chronic DSS-induced colitis.
a-c, Immunization with PCM-Q11 co-assembled with PADRE-Q11 and with or without CpG adjuvant at weeks 0, 2 and 4, does not significantly alter ZO-1 or occludin levels in the colon at week 5. a, Representative images showing colon section overviews (top row, scale bar = 500 μm) and region of interest ZO-1 and occludin staining (bottom two rows, scale bar = 50 μm). b,c, ZO-1 (b) and occludin (c) levels are not significantly different in immunized mice versus healthy controls. d-f, ZO-1 and occludin levels in the colon after chronic colitis experiment 2. d, Representative images showing colon section overviews (top row, scale bar = 500 μm) and region of interest ZO-1 and occludin staining (bottom two rows, scale bar = 50 μm). e,f, ZO-1 (e) and occludin (f) levels varied slightly among groups with PCM-Q11/CpG administered mice have significantly less ZO-1 and occludin than some other groups. Mean ± s.e.m. are shown. Statistical significance determined by one-way ANOVA with Tukey’s multiple comparison test. n = 5 mice. DAPI = blue, ZO-1 = green, and occludin = red.
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Curvino, E.J., Roe, E.F., Freire Haddad, H. et al. Engaging natural antibody responses for the treatment of inflammatory bowel disease via phosphorylcholine-presenting nanofibres. Nat. Biomed. Eng (2023). https://doi.org/10.1038/s41551-023-01139-6
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DOI: https://doi.org/10.1038/s41551-023-01139-6
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