TLR-7 activation enhances IL-22-mediated colonization resistance against vancomycin-resistant enterococcus

Abstract

Antibiotic administration can disrupt the intestinal microbiota and down-regulate innate immune defenses, compromising colonization resistance against orally acquired bacterial pathogens. Vancomycin-resistant Enterococcus faecium (VRE), a major cause of antibiotic-resistant infections in hospitalized patients, thrives in the intestine when colonization resistance is compromised, achieving extremely high densities that can lead to bloodstream invasion and sepsis. Viral infections, by mechanisms that remain incompletely defined, can stimulate resistance against invading bacterial pathogens. We report that murine norovirus infection correlates with reduced density of VRE in the intestinal tract of mice with antibiotic-induced loss of colonization resistance. Resiquimod (R848), a synthetic ligand for Toll-like receptor 7 (TLR-7) that stimulates antiviral innate immune defenses, restores expression of the antimicrobial peptide Reg3γ and reestablishes colonization resistance against VRE in antibiotic-treated mice. Orally administered R848 triggers TLR-7 on CD11c(+) dendritic cells, inducing interleukin-23 (IL-23) expression followed by a burst of IL-22 secretion by innate lymphoid cells, leading to Reg3γ expression and restoration of colonization resistance against VRE. Our findings reveal that an orally bioavailable TLR-7 ligand that stimulates innate antiviral immune pathways in the intestine restores colonization resistance against a highly antibiotic-resistant bacterial pathogen.

Fig. 1. Norovirus infection inversely correlates with VRE colonization in ampicillin-treated mice

(A) Schematic for antibiotic treatment, norovirus (MNV-CW3) infection, and VRE infection. Blue box represents the time mice are receiving ampicillin water. (B) Comparison of detectable viral copies of MNV-CW3 in ileal tissue versus VRE CFU in the ileal content. L.o.D., limit of detection. Red line represents log-log nonlinear regression fit for Amp + CW3 (n = 19). (C to E) Cells isolated from the PPs or mLNs of non–ampicillin-treated, VRE-infected mice (No Amp), ampicillin-treated, VRE-inoculated mice (Amp), and ampicillin-treated, MNV-CW3/VRE-coinfected (Amp + CW3) mice were incubated in medium in the presence of brefeldin A (BFA) for 3 hours and assessed for IL-22 production. Flow cytometric plots gated on live, CD45⁺, non-T non-B, Gr-1^neg CD90⁺ CD127⁺ cells. Frequency (C) and number (D) of IL-22⁺ ILCs in the PPs. Individual mice pooled into treatment groups. Data are sum of four independent experiments (n = 7 to 12 pooled samples per group). (E) Number of IL-22⁺ ILCs in the mLNs. Data are sum of three independent experiments (n = 6 to 10 mice per group). (F) Reg3g gene expression quantified by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and displayed as fold increase over No Amp mice and normalized to Hprt. Data are sum of four independent experiments (n = 15 to 18 mice per group) (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, t test). Data are means ± SEM.

Fig. 2. R848 restores Reg3γ expression and colonization resistance against VRE

(A to C) Mice were treated with ampicillin (Amp) or ABX in their drinking water for 7 days and administered daily for 3 days (days 4 to 6 of antibiotic administration) (A) R848 (50 µg) orally or flagellin (15 µg) intraperitoneally, (B) R848 (50 µg) or CpG (100 µg) orally, or (C) R848, R837, Gardiquimod, or CL907 (50 µg) orally. Protein extracts from the ileum were analyzed by Western blotting using Reg3γ-specific antiserum. Data are representative of two independent experiments (n = 3 mice per group). (D) Schematic for antibiotic treatment, R848 treatment, and VRE infection. Blue box represents the time mice are receiving ampicillin water. (E) VRE CFUs in the ileal content of non–ampicillin-treated, (No Amp), ampicillin-treated (Amp), and ampicillin + R848–treated (Amp + R848) mice 24 hours after VRE infection. Data are sum of five independent experiments (***P ≤ 0.0001, Mann-Whitney test; n = 17 to 20). (F) Comparison of Reg3g expression in the ileum of Amp + R848–treated mice versus VRE CFU in the ileal content. Reg3g expression relative to Amp mice and normalized to Hprt. Black line represents best-fit regression line for Amp + R848 mice. (G) Bacterial fluorescence in situ hybridization (FISH) staining of ileal sections from No Amp, Amp, and Amp + R848 mice. L, lumen; blue, Hoechst; red, universal 16S bacterial probe; green, VRE-specific probe.

Fig. 3. R848 rapidly induces IL-22 production from ILCs and Reg3g in the ileum

A single dose of R848 (50 µg) was administered orally to C57BL/6 mice. (A) Ifnb, Il23a, Il12b, Il1b, Il22, and Reg3g gene expression was assessed at 0.5, 3, and 6 hours after R848 treatment. Gene expression was quantified by qRT-PCR, normalized to Hprt, and displayed as fold increase over PBS-treated mice (3 hours after PBS treatment). Data are sum of two independent experiments (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, Mann-Whitney test; n = 3 to 7). (B to E) Single-cell suspensions isolated from the (B and C) mLNs or (D and E) ileum Lp cells of R848- or PBS-treated mice were incubated in medium in the presence of BFA and assessed for IL-22 production. Frequency (B) and number (C) of IL-22⁺ ILCs in the mLNs. Frequency (D) and number (E) of IL-22⁺ ILCs in the ileum Lp. FACS plots gated on live, CD45⁺, non-T non-B, Gr-1^neg CD90⁺ CD127⁺ cells. Data are representative of two independent experiments (*P ≤ 0.05, Mann-Whitney test; n = 3). b.d., below detection; SI Lp, small intestinal lamina propia. Data are means ± SEM.

Fig. 4. R848 signals through TLR-7 but does not directly act on ILCs to drive IL-22 production

Mice were treated with ampicillin for 3 days and administered PBS or R848 (50 µg) daily orally. (A and B) Single-cell suspensions isolated from the mLNs of C57BL/6 or Tlr7^−/− mice 3 hours after final PBS or R848 treatment were incubated in medium in the presence of BFA for 3 hours and assessed for IL-22 production. Frequency (A) and number (B) of IL-22⁺ ILCs. FACS plots gated on live, CD45⁺, non-T non-B, CD11b^neg, NK1.1^neg CD90⁺ cells (*P ≤ 0.05, Mann-Whitney test; n = 3). (C) Il22 gene expression in the ileum of C57BL/6 or Tlr7^−/− mice as quantified by qRT-PCR, normalized to Hprt, and displayed as fold increase over PBS-treated C57BL/6 mice (P = 0.1, Mann-Whitney test; n = 3). (D) Single-cell suspensions isolated from the mLNs of C57BL/6 or Tlr7^−/− mice 3 hours after final PBS or R848 treatment were incubated in medium supplemented with recombinant IL-23 (rIL-23; 40 ng/ml) in the presence of BFA for 3 hours and assessed for IL-22 production. (E and F) Cells isolated from the mLNs of C57BL/6, Tlr7^−/−/B6, Il22^−/−/B6, and Tlr7^−/−/Il22^−/− mixed BMC mice 3 hours after final PBS or R848 treatment were incubated in medium in the presence of BFA for 3 hours and assessed for IL-22 production. Frequency (E) and number (F) of IL-22⁺ ILCs. FACS plots gated on donor-derived, live, CD45⁺, non-T non-B, Gr-1^neg NK1.1^neg CD90⁺ cells [n = 3; multiple-comparison two-way analysis of variance (ANOVA) test]. Data are means ± SEM.

Fig. 5. R848-mediated IL-22 production is independent of type I IFN and IL-1 signaling but dependent on IL-23

(A and B) C57BL/6 and Ifnar^−/− mice were administered PBS or R848 (50 µg) orally and sacrificed 90 min later. Number of IL-22⁺ ILCs in the (A) mLNs and (B) ileum after ex vivo incubation in medium in the presence of BFA for 3 hours. Data are representative of two independent experiments (n = 3 to 5; Mann-Whitney test). (C to I) C57BL/6, Il1r1^−/−, or Il23a^−/− BMC mice were treated with ampicillin for 3 days and administered PBS or R848 (50 µg) daily orally. Number of donor-derived IL-22⁺ ILCs in the (C) mLNs and (D) ileum of C57BL/6 or Il1r1^−/− BMC mice after ex vivo incubation in medium in the presence of BFA for 3 hours (n = 3 to 4; Mann-Whitney test). Frequency (E) and number (F) of donorderived IL-22⁺ ILCs in the mLNs of C57BL/6 and Il23a BMC mice after ex vivo incubation in medium in the presence of BFA for 3 hours. FACS plots gated on donor-derived, live, CD45⁺, non-T non-B, CD11b^neg CD90⁺ CD127⁺ cells. (G) Number of donor-derived IL-22⁺ ILCs in the ileum of PBS- or R848-treated C57BL/6 and Il23a BMC mice. Il22 (H) and Reg3g (I) gene expression in the ileum of PBS- or R848-treated C57BL/6 and Il23a^−/− BMC mice as quantified by qRT-PCR and displayed as fold increase over PBS-treated C57BL/6 BMC mice and normalized to Hprt. Data are representative of three independent experiments (*P ≤ 0.05, **P ≤ 0.01, Mann-Whitney test; n = 3 to 6). Data are means ± SEM.

Fig. 6. R848 signals via TLR-7–expressing CD11c⁺ dendritic cells to induce IL-22, which is necessary to reestablish colonization resistance against VRE

(A and B) C57BL/6, Tlr7^−/−/B6, and Tlr7^−/−/CD11c.DTR mixed BMC mice were treated with ampicillin for 3 days, treated with diphtheria toxin, and administered PBS or R848 (50 µg) daily orally. Frequency (A) and number (B) of IL-22⁺ ILCs in the ileum of R848-treated C57BL/6, Tlr7^−/−/B6, and Tlr7^−/−/CD11c.DTR mixed BMC mice after ex vivo incubation in medium in the presence of BFA for 3 hours. FACS plots gated on live, CD45⁺, non-T non-B, Gr-1^neg CD90⁺ CD127⁺ cells. Data are representative of two independent experiments (*P ≤ 0.05, **P ≤ 0.01, Mann-Whitney test; n = 3). (C) Il22 gene expression in the ileum of Tlr7^−/−/B6 and Tlr7^−/−/CD11c.DTR mixed BMC mice as quantified by qRT-PCR and displayed as fold increase over PBS-treated B6 BMC mice and normalized to Hprt. Data are representative of two independent experiments (*P ≤ 0.05, **P ≤ 0.01, Mann-Whitney test; n = 3). Data are means ± SEM. (D) ABX-treated or untreated C57BL/6 or Il22^−/− BMC mice were administered PBS or R848 (50 µg) daily orally. Protein extracts from the ileum were analyzed by Western blotting using the Reg3γ-specific antiserum. Data are representative of two independent experiments. Each lane is for a representative mouse from the indicated group. (E) C57BL/6 and Il22^−/− were treated with ampicillin for 3 days and administered PBS or R848 (50 µg) daily orally. Mice were inoculated with VRE, and CFUs in the ileal content were assessed 24 hours after infection. Data are sum of three independent experiments (**P ≤ 0.01, Mann-Whitney test; n = 10 to 14).