A balanced IL-1β activity is required for host response to Citrobacter rodentium infection

Abstract

Microbial sensing plays essential roles in the innate immune response to pathogens. In particular, NLRP3 forms a multiprotein inflammasome complex responsible for the maturation of interleukin (IL)-1β. Our aim was to delineate the role of the NLRP3 inflammasome in macrophages, and the contribution of IL-1β to the host defense against Citrobacter rodentium acute infection in mice. Nlrp3(-/-) and background C57BL/6 (WT) mice were infected by orogastric gavage, received IL-1β (0.5 µg/mouse; ip) on 0, 2, and 4 days post-infection (DPI), and assessed on 6 and 10 DPI. Infected Nlrp3(-/-) mice developed severe colitis; IL-1β treatments reduced colonization, abrogated dissemination of bacteria to mesenteric lymph nodes, and protected epithelial integrity of infected Nlrp3(-/-) mice. In contrast, IL-1β treatments of WT mice had an opposite effect with increased penetration of bacteria and barrier disruption. Microscopy showed reduced damage in Nlrp3(-/-) mice, and increased severity of disease in WT mice with IL-1β treatments, in particular on 10 DPI. Secretion of some pro-inflammatory plasma cytokines was dissipated in Nlrp3(-/-) compared to WT mice. IL-1β treatments elevated macrophage infiltration into infected crypts in Nlrp3(-/-) mice, suggesting that IL-1β may improve macrophage function, as exogenous administration of IL-1β increased phagocytosis of C. rodentium by peritoneal Nlrp3(-/-) macrophages in vitro. As well, the exogenous administration of IL-1β to WT peritoneal macrophages damaged the epithelial barrier of C. rodentium-infected polarized CMT-93 cells. Treatment of Nlrp3(-/-) mice with IL-1β seems to confer protection against C. rodentium infection by reducing colonization, protecting epithelial integrity, and improving macrophage activity, while extraneous IL-1β appeared to be detrimental to WT mice. Together, these findings highlight the importance of balanced cytokine responses as IL-1β improved bacterial clearance in Nlrp3(-/-) mice but increased tissue damage when given to WT mice.

Figure 1. IL-1β treatments reduce intestinal colonization and dissemination of C. rodentium in Nlrp3^−/− mice.

Infected mice were given repeated IL-1β treatments on 0, 2, and 4 DPI. C. rodentium colonization of the (A) cecum, (B) colon, and (C) dissemination to MLN were assessed on 6 and 10 DPI by plating homogenates on MacConkey agar plates in serial dilutions. IL-1β treatments of Nlrp3^−/− mice significantly lowered cecum colonization on 6 DPI, and colon colonization and dissemination to MLN on 10 DPI. Values were Log₁₀ transformed and data presented as mean ± SE. One asterisk: P<0.05, two asterisks: P<0.01.

Figure 2. IL-1β treatments modulate C. rodentium penetration and epithelial integrity.

Biotin was administered into the lumen of the distal colon at 6 or 10 DPI. Cryosections were stained and viewed under fluorescent microscopy for C. rodentium infiltration (in red; indicated by arrowheads in the enlarged image of the inset highlighted by boxes in the left panels) and biotin penetration (green; indicated by asterisks). Uninfected IL-1β-treated mice retained biotin to the apical surface. In non-treated mice at 6 DPI, the bacterial infiltration and biotin penetration was increased. IL-1β treatments at 6 DPI did not seem to have a major effect on infiltration or epithelial integrity. In non-treated mice at 10 DPI, the bacterial infiltration and epithelial barrier disruption were lowered in WT and elevated in Nlrp3^−/− mice, relative to 6 DPI. In IL-1β-treated mice at 10 DPI, the bacterial infiltration and barrier disruption were elevated in WT and lowered in Nlrp3^−/− mice. Bacterial colonization followed the same trend as biotin penetration. Magnification X400, bar 50 µm.

Figure 3. Colitis is modulated by IL-1β treatments.

(A) IL-1β treatment of infected mice reduced WT and Nlrp3^−/− body weights at 2 DPI compared to untreated mice, but weights later recovered (error bars not included for clarity); (B) Crypt lengths were measured using the ZEN software on Zeiss-acquired micrographs by an individual blinded to the identity of the slides. Crypt lengths increased significantly within 6 DPI in Nlrp3^−/− mice compared to WT mice. IL-1β treatments increased the crypt lengths at 6 DPI in WT mice compared to untreated mice. Data represents mean per crypt ± SE; (C) Histological scores of colitis were assessed in a blinded fashion and graded as described previously . Scoring revealed elevated severity of disease in IL-1β-treated WT mice at 10 DPI, while IL-1β-treated Nlrp3^−/− mice had reduction in damage. Data represents mean ± SE; (D) H&E-stained colonic sections of untreated and treated mice. Increase in crypt lengths, loss of goblet cells, and cellular damage can be noted in infected mice (further details in Figure S2). Hematoxylin and eosin staining, magnification X200, bar 100 µm. One asterisk: P<0.05, three asterisks: P<0.001.

Figure 4. IL-1β treatments modulate the systemic cytokine response.

IL-6 (A), IL-12p70 (B), TNF-α (C), IL-1β (D), KC/gro (E), and IFN-γ (F) were measured using ELISA-based assays. Generally, secretion of pro-inflammatory cytokines was dissipated in Nlrp3^−/− mice at 10 DPI compared to WT mice. One asterisk: P<0.05, two asterisks: P<0.01, three asterisks: P<0.001; ND, not detected.

Figure 5. IL-1β treatments augment macrophage colonic infiltration.

Macrophage infiltration was assessed by staining cryosections of distal colons. An increase in the number of macrophages (red; indicated by arrowhead in higher magnification panels on the left) in close proximity of C. rodentium (green) at 10 DPI in Nlrp3^−/− mice is noted. With IL-1β treatments, the macrophages in crypts and mucosal lining appeared increased and bacterial infiltration into the crypts reduced. Bar 50 µm.

Figure 6. IL-1β enhances C. rodentium phagocytosis, while overcompensation of IL-1β augments epithelial barrier damage in vitro.

(A) Exogenous IL-1β induced C. rodentium phagocytosis (arrowhead) by peritoneal macrophages in vitro, especially in Nlrp3^−/− macrophages. Magnification X630, bar 50 µm; (B) Quantification showed an increase of intracellular C. rodentium in IL-1β treated Nlrp3^−/− macrophages but no effect on WT macrophages. Data represents mean ± SE. One asterisk P<0.05; (C) Western blot analysis of mature IL-1β in the supernatant of peritoneal macrophages and pro-IL-1β in the cellular component; (D) The epithelial integrity of C. rodentium-infected CMT-93 cells assessed by ECIS, deteriorated in presence WT macrophages and even more with the addition of IL-1β. Data represents the mean of two independent experiments.