Research ArticleIMMUNOLOGY

Immunomodulatory nanogels overcome restricted immunity in a murine model of gut microbiome–mediated metabolic syndrome

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Science Advances  27 Mar 2019:
Vol. 5, no. 3, eaav9788
DOI: 10.1126/sciadv.aav9788
  • Fig. 1 PLGA nanovaccines manifest limited humoral immune response in male TLR5−/−mice.

    (A) Left: Images of fat pad mass for WT, TLR5−/−, and WT mice on a high fat diet (HFD). Photo credit: J.D.G., Cornell University. Middle and right: Mouse fat pad masses as a function of age and gender. Statistics was performed using a one-way analysis of variance (ANOVA) with Tukey’s post hoc correction (n = 8 to 16). (B and C) Comparison of leptin (B) and insulin (C) levels in TLR5−/− and WT male mice. Statistics was performed using an unpaired, two-tailed t test (n = 8 TLR5−/− and n = 11 WT). (D) Comparison of inflammatory markers in TLR5−/− and WT male mice. Statistics was performed using an unpaired, two-tailed t test (n = 10 each). (E) Timeline for vaccination, germinal center immune response, and gating strategy. The scatterplot presents the percentage of GL7+FAS+CD19+ germinal center B cells in the lymph node of male mice 10 days after booster vaccination with either soluble NP-OVA (4-hydroxy-3-nitrophenylacetyl hapten conjugated to ovalbumin) antigen or PLGA nanovaccines formulated with NP-OVA. PBS, phosphate-buffered saline; FAS, fatty acid synthase. (F) Gating strategy for CD138+ plasma cells. The scatterplot presents the percentage of CD138+ plasma cells in the lymph node after booster vaccination. (G) The scatterplot presents the antigen-specific antibodies in the serum of male mice after immunization. (H and I) Scatterplots present the major histocompatibility complex class II (MHCII) expression on CD11c+ dendritic cells (H) and the percentage of CD3+ T cells (I) in the lymph node after booster vaccination. In (E) to (I), statistics was performed using a one-way ANOVA with Tukey’s post hoc correction (n = 4 except for the soluble NP-OVA WT mice with n = 3). (J) The scatterplot compares the percentage of germinal center B cells in the lymph node of TLR5−/− to WT female mice. Statistics was performed using an unpaired, two-tailed t test (n = 3). In all studies, *P < 0.05 and **P < 0.01. ns denotes nonsignificant differences. MFI, mean fluorescence intensity.

  • Fig. 2 TLR5−/−phenotype in male mice leads to reduced nanoparticle trafficking from the injection site to lymphoid tissue.

    (A) IVIS images demonstrating distribution of nanoparticles at day 0, day 2, and day 4 after injection for TLR5−/− and WT male mice. Images are representative of a cohort of four mice. (B) Quantification of signal at the injection site on days 2 and 4, normalized to day 0 (mean ± SEM). Comparisons at each day were performed using an unpaired, two-tailed t test. (C) Fluorescent images of organs at day 2 and day 6 after injection in male mice. Total counts represent the sum of all counts for all pixels inside the region of interest. (D) Quantification of IVIS signal in lymph node at day 2 and day 6 (n = 4). Statistics was performed using an unpaired, two-tailed t test. (E) Left: Gating strategy for flow cytometry. Top left is an unstained control; bottom left represents a stained sample. Right: Scatterplots represent percent FITC+CD169+ macrophage, FITC+CD11c+ dendritic cells, and FITC+F4/80 macrophages in the lymph node on day 2. Comparisons were made with an unpaired, two-tailed t test. (F) Percent CD19+ B cells, CD11c+ dendritic cells, and CD3+ T cells at the injection site on day 6 after immunization. Comparisons were performed using an unpaired, two-tailed t test. For all flow cytometry data, readouts are presented as means ± SEM. For trafficking studies, n = 3 for both groups on day 2. n = 4 for TLR5−/− group on day 6 and n = 5 for WT group on day 6. In all studies, *P < 0.05, **P < 0.01, and ***P < 0.001. ns denotes nonsignificant differences.

  • Fig. 3 PLGA nanovaccine immunization alters gut microbiome composition.

    (A and B) Differentially abundant microbial phyla in stool samples from WT (n = 3) versus TLR5−/− (n = 3) male mice, before and after booster doses. (C) Differentially abundant microbial phyla in the stool samples from male (n = 3) versus female (n = 3) TLR5−/− mice, after booster doses. Statistical analysis was performed using paired two-tailed t test. In all studies, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. ns denotes nonsignificant differences.

  • Fig. 4 Antibiotic-mediated dysbiosis of gut microbiome alters adaptive immune response in male WT mice.

    (A) Timeline of antibiotic administration and immunization. Antibiotics were continued from weaning through the end of vaccination. (B) Weight and fat pad mass of mice fed on antibiotics and TLR5−/− mice presented as means ± SEM. Comparisons were made by t test [n = 12 for WT, n = 7 for WT (antibiotics), and n = 16 for TLR5−/−]. (C) Differentially abundant microbial phyla in stool samples from WT (n = 3) versus WT antibiotic-treated (n = 7) male mice before immunization. (D) Percentage of GL7+FAS+CD19+ germinal center B cells in the lymph node and spleen of antibiotic-treated male mice after booster vaccination with either soluble NP-OVA or nanovaccine-formulated NP-OVA. Statistics was performed using an unpaired, two-tailed t test, with populations presented as means ± SEM (n = 3). (E) Scatterplot presents the antigen-specific antibodies in the serum of mice after immunization with nanovaccine and soluble formulation (n = 3). Statistics was performed using an unpaired, two-tailed t test, with populations presented as means ± SEM (n = 3). (F) Inflammation marker levels in the serum of WT and antibiotic-fed WT mice. Statistics was performed using an unpaired, two-tailed t test, with cytokines presented as means ± SEM (n = 7). In all studies, *P < 0.05, **P < 0.01, ***P < 0.001. ns denotes nonsignificant differences.

  • Fig. 5 Pyr-pHEMA nanogels rescue the antigen-specific germinal center response in male TLR5−/−mice and improve particle trafficking.

    (A) Chemical structure of pyridine functionalized pHEMA and schematic of nanogel interaction with immune cells. (B) Contour plot of diffusion-ordered spectroscopy (DOSY) NMR spectrum of Pyr-pHEMA in DMSO-d6 at 25°C confirming that the backbone, linker, and pyridine have experimentally indistinguishable diffusion properties. ppm, parts per million. (C) Solubility analysis of Pyr-pHEMA and pHEMA as compared to water. Photo credit: M.J.M., Cornell University. (D) Turbidity analysis of Pyr-pHEMA and pHEMA as compared to water using absorbance of solution at 570 nm (n = 5 per formulation). All groups were compared by one-way ANOVA with Tukey’s post hoc test and presented as a box-whisker plot. (E) Loading ratio of Pyr-pHEMA to protein at various soluble protein loading concentrations (n = 5 per formulation, presented as means ± SEM). Comparisons were made by an unpaired, two-tailed t test (*P < 0.05) between protein loading concentrations. (F) Protein release of Pyr-pHEMA in in vitro conditions. All groups were compared by two-way ANOVA with Bonferroni’s correction, and data are presented as means ± SEM (n = 4; ***P < 0.001). (G to J) Pyr-pHEMA nanogel induced germinal center B cell population (G), CD138+ population (H), antigen-specific antibody levels in the serum (I), and CD11c+ dendritic cell population (J) in immunized male TLR5−/− mice. Comparisons were made by an unpaired, two-tailed t test, and all readouts were expressed as means ± SEM (n = 5 WT and n = 5 TLR5−/−). In all studies, *P < 0.05, **P < 0.01, and ***P < 0.001. ns denotes nonsignificant differences. (K and L) Pyr-pHEMA nanogel trafficking and expression of MHCII on CD169 macrophages (K) and CD11c+ dendritic cells (L) in the lymphoid tissues were compared to soluble antigen. Comparisons were made by an unpaired, two-tailed t test, and all data were presented as means ± SEM (n = 5 nanogel and n = 5 soluble formulation). (M) Fold change in GL7+FAS+CD19+ germinal center B cell population between Pyr-pHEMA nanogel, PLGA nanovaccines, and PLGA nanovaccines adjuvanted with alum. All particles were loaded with NP-OVA antigen. All groups were compared by one-way ANOVA with Tukey’s post hoc test, and data are presented as means ± SEM (n = 4). In this study, *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 6 Immunomodulatory effects of Pyr-pHEMA are mediated through TLR2.

    (A) TLR stimulatory activity of Pyr-pHEMA nanogels and controls in HEK293 cells transfected with TLR2, TLR4, and TLR9. HEKs endogenously express TLR5. Luciferase activity indicates activation of a TLR. Statistics was performed using a two-way ANOVA with Bonferroni’s correction (n = 5). (B) Schematic presenting the proposed hypothesis that Pyr-pHEMA nanogels function through TLR2. (C) MFI of MHCII and CD80 on bone marrow–derived dendritic cells that are exposed to Pyr-pHEMA nanogels. Statistics was performed using a one-way ANOVA with Tukey’s post hoc correction (n = 3), with all data presented as means ± SEM (n = 3). (D) Fold change in CD19+ B cell and GL7+FAS+CD19+ germinal center B cell population between nanogel and soluble formulations. All nanogel groups were compared by an unpaired, two-tailed t test with their soluble counterparts, and data are presented as means ± SEM (n = 5 WT nanogel, n = 4 WT soluble, n = 6 TLR2−/− nanogel, n = 5 TLR2−/− soluble, n = 4 TLR4−/− nanogel, and n = 3 TLR4−/− soluble). In all the studies, *P < 0.05, **P < 0.01, and ***P < 0.001. ns denotes nonsignificant differences.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/3/eaav9788/DC1

    Fig. S1. Characterization of WT and TLR5−/− mice (related to Fig. 1).

    Fig. S2. Pyr-pHEMA nanogels are equivalent in size to PLGA nanoparticle vaccines (related to Fig. 1 and Fig. 5).

    Fig. S3. Knockout of the TLR5 receptor results in lower germinal center formation in mice immunized with a PLGA nanovaccine (related to Fig. 1).

    Fig. S4. PLGA nanoparticle trafficking from the injection site to lymphoid tissue on day 6 and accumulation in the liver and kidneys at days 2 and 6 after injection (related to Fig. 2).

    Fig. S5. Expression of CD86 activation marker (related to Fig. 2).

    Fig. S6. Injection site analysis (related to Fig. 2).

    Fig. S7. Cell populations in the spleen and lymph node of immunized antibiotic-fed mice (related to Fig. 4).

    Fig. S8. Immunological characterization of Pyr-pHEMA nanogels (related to Fig. 5).

    Fig. S9. Pyr-pHEMA nanogels do not differentially accumulate in tissue after 6 days relative to soluble formulation (related to Fig. 5).

    Fig. S10. Immunomodulatory effects of Pyr-pHEMA are mediated through TLR2 (related to Fig. 6).

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Characterization of WT and TLR5−/− mice (related to Fig. 1).
    • Fig. S2. Pyr-pHEMA nanogels are equivalent in size to PLGA nanoparticle vaccines (related to Fig. 1 and Fig. 5).
    • Fig. S3. Knockout of the TLR5 receptor results in lower germinal center formation in mice immunized with a PLGA nanovaccine (related to Fig. 1).
    • Fig. S4. PLGA nanoparticle trafficking from the injection site to lymphoid tissue on day 6 and accumulation in the liver and kidneys at days 2 and 6 after injection (related to Fig. 2).
    • Fig. S5. Expression of CD86 activation marker (related to Fig. 2).
    • Fig. S6. Injection site analysis (related to Fig. 2).
    • Fig. S7. Cell populations in the spleen and lymph node of immunized antibiotic-fed mice (related to Fig. 4).
    • Fig. S8. Immunological characterization of Pyr-pHEMA nanogels (related to Fig. 5).
    • Fig. S9. Pyr-pHEMA nanogels do not differentially accumulate in tissue after 6 days relative to soluble formulation (related to Fig. 5).
    • Fig. S10. Immunomodulatory effects of Pyr-pHEMA are mediated through TLR2 (related to Fig. 6).

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