IL-33 protects from recurrent C. difficile infection by restoration of humoral immunity

Abstract

Clostridioides difficile infection (CDI) recurs in 1 of 5 patients. Monoclonal antibodies targeting the virulence factor TcdB reduce disease recurrence, suggesting that an inadequate anti-TcdB response to CDI leads to recurrence. In patients with CDI, we discovered that IL-33 measured at diagnosis predicts future recurrence, leading us to test the role of IL-33 signaling in the induction of humoral immunity during CDI. Using a mouse recurrence model, IL-33 was demonstrated to be integral for anti-TcdB antibody production. IL-33 acted via ST2+ ILC2 cells, facilitating germinal center T follicular helper (GC-Tfh) cell generation of antibodies. IL-33 protection from reinfection was antibody-dependent, as μMT KO mice and mice treated with anti-CD20 mAb were not protected. These findings demonstrate the critical role of IL-33 in generating humoral immunity to prevent recurrent CDI.

Keywords: Adaptive immunity; Immunology; Infectious disease.

Figure 1. IL-33 increases toxin-specific antibody in mice after first infection with C. difficile.

IL-33 (0.75 μg) was administered i.p. on days –4 to 0 to C57BL/6J mice (A–H) and/or ST2^−/− mice (I–M). Mice were infected with C. difficile strain R20291 (A–E and G–M) or VPI 10463 (F). On day 15 after infection, antibodies were measured in plasma and cecal content. (A) Schematic diagram showing infection and treatment timeline; (B) survival curves; (C) weight loss; (D) clinical scores; (E) plasma toxin B–specific IgG from mice infected with C. difficile strain R20291; (F) plasma toxin B–specific IgG from mice infected with C. difficile strain VPI 10463; (G) plasma toxin B–specific IgM from mice infected with C. difficile strain R20291; (H) cecal content toxin B–specific IgA from mice infected with C. difficile strain R20291. (I–M) WT versus ST2^−/− mice infected with C. difficile strain R20291: (I) Schematic diagram showing infection and treatment timeline; (J) plasma IgG; (K) cecal IgG; (L) cecal IgA; and (M) plasma IgM. (B) Comparison made by log-rank test (n = 30). (C and D) Comparison made by 2-tailed Student’s t test (C and D n = 30). E (n = 16), F (n = 11), G (n = 25), H (n = 18), J (n = 14), K (n = 7), L (n = 7), M (n = 7), A 2-tailed t test for normally distributed data and a Mann-Whitney test for nonnormally distributed data were used. *P < 0.05, **P < 0.01, and ***P < 0.001. The error bar indicates SEM.

Figure 2. IL-33 protects from a second C. difficile infection.

IL-33 (0.75 μg) was administered i.p. on days –4 to 0 and WT mice were infected on day 0 and again on day 60 with C. difficile strain R20291. (A) Experimental design for second infection; (B) second infection weight loss (n = 13); (C) clinical scores (n = 13); (D) FITC-dextran gut permeability test (n = 13); (E) epithelial damage scoring (n = 13); (F) representative H&E stain of the colon. Scale bars: 174.2 μm (left) and 145.6 μm (right). (B and C) Comparison made by 2-tailed Student’s t test. (D) Mann-Whitney test for nonnormally distributed data was used (E) Šídák’s multiple comparisons test was used to determine the statistical significance between groups. *P < 0.05, **P < 0.01, and ***P < 0.001. The error bar indicates SEM in B, D, and E, and in D indicates the median with interquartile range.

Figure 3. IL-33 protection from a second C. difficile infection is antibody dependent.

WT (n = 15) and μMT-KO (n = 15) mice were administered IL-33 (0.75 μg) i.p. on days –4 to 0 and mice infected on day 0 and reinfected on day 60 with C. difficile strain R20291. (A) Experimental design; (B) first infection weight loss; (C) clinical scores. WT and μMT-KO mice were reinfected with C. difficile R20291 60 days after the first infection. Reinfection (D) weight loss; (E) Stool C. difficile toxin A and B measured by ELISA kit (Techlab); (F) FITC-dextran gut permeability assay; (G) H&E stain; (H) epithelial damage scoring. Scale bars: 267.3 μm (left) and 231.2 μm (right). (B–D) Comparison made by 2-tailed Student’s t test (D n=18). (E) a 2-tailed t test was used and the error bar indicates SEM. (F) a Mann-Whitney test was used and the error bar indicates the median with interquartile range. (H) Šídák’s multiple comparisons test was used. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 4. Antibody-deficient mice (anti-CD20 treated) lost IL-33–mediated protection from a second C. difficile infection.

Anti-CD20 was administered to deplete B cells on days –7, 3, 18, 33, 48, and 63, and mice infected on day 0 and infected for the 2nd time on day 60 with C. difficile strain R20291. (A) Experimental design; (B) survival curve; (C) first infection weight loss; (D) first infection clinical scores; (E) reinfection weight loss; (F) reinfection clinical scores (G) epithelial damage scoring; (H) H&E stain. Scale bars: 118.7 μm (left) and 191.4 μm (right). (I) Toxin B–specific plasma IgG; (J) IgA; (K) cecal IgG; and (L) cecal IgA measured on day 10 after second infection. MLN and colon were harvested on day 10 after reinfection. Colonic (M and N) B cells (CD45⁺ CD3^– CD19⁺); (O and P) TH2 cells (CD45⁺CD3⁺ CD4⁺ GATA3⁺); (Q and R) neutrophils; (S–U) Treg (CD45⁺CD3⁺ CD4⁺ FOXP3⁺), and TH17 (CD45⁺CD3⁺ CD4⁺ RORγt⁺) cells; and (V and W) MLN B cells. (B) Comparison made by log-rank test (n = 26 in both groups). (C–F) Comparison made by 2-tailed Student’s t-test (C and D n = 26, E and F n = 15). (G) Šídák’s multiple comparisons test was used. (I–M, P, Q, T, U, and V) A 2-tailed t test for normally distributed data and a Mann-Whitney test for nonnormally distributed data were used. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. The error bar indicates SEM.

Figure 5. IL-33 increased mesenteric lymph node ILC2 and decreased TH1 cells during first C. difficile infection.

IL-33 (0.75 μg) was administered i.p. on days –4 to 0 and mice infected on day 0. (A and B) ILC populations on day 0, prior to infection, and (C and D) at 2 daysafter infection. TH1, TH2 populations (E–H) before infection, day 0; (I–L) day 2; and (M–P) day 6 after infection. A 2-tailed t test for normally distributed data and a Mann-Whitney test (B, F, and H n = 10, D, J, and L n = 20) for nonnormally distributed data were used. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. The error bar indicates SEM.

Figure 6. IL-33 increased mesenteric lymph node GC-TFH, Tregs, and decreased TH17 cells during a primary C. difficile infection.

IL-33 (0.75 μg) was administered i.p. on days –4 to 0 and mice infected on day 0. Mesenteric lymph nodes were harvested to analyze T helper cells before infection, and again on day 2 and day 6 after first infection. (A–C) TH17 cells (CD45⁺ CD3⁺ CD4⁺ RORgt⁺) and Treg cells (CD45⁺ CD3⁺ CD4⁺ FOXP3⁺) on day 0 prior before infection (n = 10); (D–F) on day 2 (n = 20); (G–I) on day 6 after infection (n = 24). (J–M) TFH cells were defined as germinal center (GC) TFH (CD45⁺ CD3⁺ CD4⁺ CD44⁺ PD1 high CXCR5⁺) and non-GC TFH cells (CD45⁺ CD3⁺ CD4⁺ CD44⁺ PD1-low CXCR5⁺) by flow cytometry. (J and K) TFH subsets on day 0 prior to infection (n = 10); (L and M) TFH subsets on day 6 after infection(n = 28). A 2-tailed t test for normally distributed data and a Mann-Whitney test for nonnormally distributed data were used. *P < 0.05, **P < 0.01, and ***P < 0.001. The error bar indicates SEM.

Figure 7. ILC2s mediated production of toxin B–specific antibodies, protecting against CDI reinfection.

ST2⁺ ILC2s (from uninfected IL-33 treated mice) were ex-vivo expanded, purified by flow-sorting, and adoptively transferred into ST2-KO mice. (A and B) Mice were pretreated with antibiotics and injected with 0.75 μg per dose per mouse of IL-33 in the gut 1 day after the adoptive transfer of 3 × 10⁵ ILC2s (n = 12) or saline (n = 12) per mouse. At day 16 after primary infection, plasma toxin B–specific (B) IgG was measured in plasma. (C–G) Mice were rechallenged with C. difficile on day 60. On day 70 (10 days after the second infection), (C–E), ILC2 was measured in the colon and MLN. (F and G) GC-TFH measured in the MLN (H) Day 70 representative epithelial damage (H&E) of treatment groups and (I) assessed by blinded scoring of infected tissue. Scale bars: 184.4 μm (left) and 114.3 μm (right). Depletion of ILC2 decreased toxin-specific antibodies in CDI; IL-33 (0.75 μg) was administered intraperitoneally from days –4 to 0 to R26-DTR^Nmur1 mice (n = 9) or littermate control R26-DTR mice (n = 9) (in which ILC2s lack DTR). Mice were then given intraperitoneal (i.p.) injections with diphtheria toxin (DT) on days –12, –9, –6, 1, and 8 after infection and infection was done with C. difficile strain R20291. (J) Experimental design; On day 15 after infection, ILC2 abundance and anti-TcdB antibodies were measured. (K) Density plot to show the depletion of ILC2s from the colon (L) Toxin-specific antibodies were measured in plasma and cecal content. A 2-tailed t test for normally distributed data and a Mann-Whitney test for nonnormally distributed data were used. (J) Šídák’s multiple comparisons test was used. *P < 0.05, **P < 0.01, and ***P < 0.001. The error bar indicates SEM.

Figure 8. IL-33 is a biomarker for recurrent C. difficile infection in humans.

(A) Plasma IL-33 was measured in healthy controls (n = 17), patients with uncomplicated CDI (n = 39), and recurrent CDI (n = 12) (excluding 5 patients who died) within 8 weeks of diagnosis. (B) Recurrence-free survival among the patients with C. difficile infection (1A), grouped by serum IL-33 quartile (Wilcoxon P = 0.002). (C) IHC staining of IL-33 from colon tissue biopsies of patients without or with recurrence. Scale bars: 20 μm. The Kaplan–Meier method was used to measure recurrence-free survival curves and to evaluate the effects of IL-33 on risk for recurrent C. difficile infection. To account for competing risk against recurrent infection, death within the 8-week follow-up period was treated as a censoring event.*P < 0.05 and ***P < 0.001.