Viral infection. Prevention and cure of rotavirus infection via TLR5/NLRC4-mediated production of IL-22 and IL-18

Abstract

Activators of innate immunity may have the potential to combat a broad range of infectious agents. We report that treatment with bacterial flagellin prevented rotavirus (RV) infection in mice and cured chronically RV-infected mice. Protection was independent of adaptive immunity and interferon (IFN, type I and II) and required flagellin receptors Toll-like receptor 5 (TLR5) and NOD-like receptor C4 (NLRC4). Flagellin-induced activation of TLR5 on dendritic cells elicited production of the cytokine interleukin-22 (IL-22), which induced a protective gene expression program in intestinal epithelial cells. Flagellin also induced NLRC4-dependent production of IL-18 and immediate elimination of RV-infected cells. Administration of IL-22 and IL-18 to mice fully recapitulated the capacity of flagellin to prevent or eliminate RV infection and thus holds promise as a broad-spectrum antiviral agent.

Fig. 1. Flagellin protects mice from RV infection and diarrhea

(A and B) Eight-week-old female (A) C57BL/6 mice or (B) Rag1^−/− mice were orally inoculated with mouse RV, EC strain. Mice were administered 0.2 ml of phosphate-buffered saline (PBS) (vehicle) ± 20 μg of flagellin by means of intraperitoneal injection, and then every other day from 0 to 18 days after inoculation. Feces were collected daily and assayed for RV antigens by means of enzyme-linked immunosorbent assay (ELISA). Results are shown as mean ± SEM [two-way analysis of variance (ANOVA), n = 4 mice, P < 0.001) for (A) and (B). p.i., post-inoculation. (C) Seven-day-old C57BL/6 mice were orally inoculated with RV (supplementary materials). Mice were treated with PBS or flagellin (10 μg) every day from 0 to 9 days after inoculation and monitored for incidence of diarrhea daily (χ² test, n = 11 mice, *P < 0.01). Flagellin-treated mice exhibited significantly reduced duration of diarrhea and days of active diarrhea (Student’s t test, n = 11 mice, *P < 0.01). (D) Seven-day-old mice, treated as in (C), were euthanized 3 days after inoculation. Total RNA of small intestines from those mice were prepared and analyzed for RV NSP3 RNA level (Student’s t test, n = 6 mice, *P < 0.05). (E) Three-week-old Rag1^−/− mice were inoculated with mouse RV. Three weeks after inoculation, at which point a chronic infection had been established, mice were treated with PBS ± flagellin (20 μg) every 2nd day from 22 to 40 days after inoculation as indicated with red arrows. Feces were collected at indicated days and assayed for RVantigens by means of ELISA. Asterisk indicates that the difference between mice given PBS and flagellin was statistically significant. Results are shown as mean ± SEM (two-way ANOVA, n = 5 mice, P < 0.0001).

Fig. 2. Flagellin’s antiviral activity is mediated by TLR5/NLRC4 on dendritic cells

(A to D) Eight-week-old (A) Tlr5^−/−, (B) Nlrc4^−/−, (C) Tlr5/Nlrc4^−/−, and (D) MyD88^−/− mice were orally inoculated with mouse RV and administered PBS ± flagellin (20 μg) every other day from 0 to 8 days after inoculation. Feces were assayed for RV antigens by means of ELISA. Results in (A) to (D) are shown as mean ± SEM [Student’s t test, n = 5 mice, P < 0.05 on day 3 in (A) and day 5 in (B)]. Differences between PBS and flagellin groups in (C) and (D) were not significant [two-way ANOVA, n = 4 to 5 mice, P = 0.6361 for (C) and P = 0.3871 for (D)]. (E) CD11c-DTR reconstituted bone marrow chimeras were either untreated or injected with DT at 8 ng/gram body weight once a day for 2 days. Flow cytometry plots show the extent of depletion of DCs (CD45⁺/CD19⁻/MHC class II⁺/CD11c⁺ splenocytes). Mice were then studied as in (A) to (D). The difference between the PBS and flagellin groups was statistically significant in absence of DT (two-way ANOVA, n = 4 mice, P < 0.001) and nonsignificant in DT-treated group (two-way ANOVA, n = 6 to 7 mice, P = 0.3821). (F and G) Tlr5/Nlrc4^−/− mice were adoptively transferred with 4 × 10⁶ fluorescence-activated cell–sorted DCs (purity > 98.5%) from Tlr5/Nlrc4^−/− (F) or WT C57BL/6 (G) mice. Twelve hours later, the mice were studied as in (A) to (D). The difference between the PBS and flagellin groups was statistically significant in (G) (Student’s t test on days 2 to 4, n = 4 mice, P < 0.05) but not (F) (Student’s t test, n = 4 mice, P > 0.05 on all days). (H) Tlr5/Nlrc4^−/− mice were adoptively transferred with 20 million magnetic bead–sorted DCs (purity > 95.0%) from WT C57BL/6 mice, and 12 hours later, the mice were studied as in (A) to (D) (Student’s t test on days 2 to 5, n = 4 mice, P < 0.05).

Fig. 3. Flagellin-mediated protection against RV infection requires both IL-22 and IL-18

(A to H) Indicated strains of genetically modified 8-week-old mice were orally inoculated with mouse RV, EC strain. Mice were treated with PBS ± flagellin (20 μg), via intraperitoneal injection, every other day from 0 to 8 days after inoculation. Feces were collected daily and assayed for RV antigens by means of ELISA. The following strain was used in each panel: (A) p40^−/−, (B) Rag2/Il2rg^−/−, (C) WT C57BL/6 mice treated with IL-17–neutralizing mAb, (D) Il22^−/−, (E) WT C57BL/6 mice treated with IL-22–neutralizing mAb, (F) Il1r^−/−, (G) IL-18BP TG, and (H) Il18^−/− mice. Results in (A) to (H) are shown as mean ± SEM (n = 4 to 6 mice). The difference between mice given PBS and flagellin was statistically significant for (C) and (F) (two-way ANOVA, P < 0.0001) and significant at individual days of (A), (B), (D), (E), (G), and (H) [Student’s t test, P < 0.05 on day 3 in (A), days 7 and 9 of (B), day 5 of (D), day 2 of (E), days 3 and 4 in (G), and days 3 and 4 in (H)].

Fig. 4. IL-22/IL-18 treatment prevents and treats RV infection

(A) Eight-week-old C57/BL6 mice were treated with 0.2 ml PBS (vehicle), 2 μg IL-22, 1 μg IL-18, or 2 μg IL-22 plus 1 μg IL-18 by means of intraperitoneal injection and subsequently inoculated with RV. Such cytokines were administered every other day from 0 to 8 days after inoculation. Feces were assayed for RV antigens by means of ELISA. Results are shown as mean ± SEM. Difference between PBS and IL-22/IL-18 groups were statistically significant (two-way ANOVA, n = 4 mice, P < 0.0001). (B) Rag1^−/− mice, chronically infected with RV, were treated with PBS, 10 μg IL-22, 1 μg IL-18, or both on days 24 and 26 after inoculation (indicated with red arrows). Difference between PBS and IL-22/IL-18 groups was statistically significant (two-way ANOVA, n = 4 mice, P < 0.0001). (C) Seven-day-old C57BL/6 mice were orally inoculated with RV. Mice were administered 50 μl PBS (vehicle) or 1 μg IL-22 plus 0.2 μg IL-18 immediately before inoculation, and 1 to 9 days after inoculation, and monitored for incidence of diarrhea daily (χ² test, n = 5 to 6 mice, *P < 0.05), duration and active days of diarrhea (Student’s t test, n = 5 to 6 mice, *P < 0.001). (D to F) Chronically RV-infected Rag1^−/− mice were treated with one injection of PBS, PBS containing 10 μg IL-22, 1 μg IL-18, or 10 μg IL-22 plus 1 μg IL-18. (D) Venn diagram representation of significant changes in intestinal epithelial gene expression 3 hours after cytokine treatment. (E) Intestinal levels of RV genomes and replication rates as reflected by NSP3 RNA levels and +/− RV strand ratios at 3 hours (Student’s t test, n = 4 mice, *P < 0.001 for RV genome, *P < 0.05 for RV RNA +/− strand ratio). (F) RV genomes levels at indicated time (Student’s t test, n = 4 mice, *P < 0.0001).