IL-23 p19 knockout mice exhibit minimal defects in responses to primary and secondary infection with Francisella tularensis LVS.

Our laboratory's investigations into mechanisms of protective immunity against Francisella tularensis Live Vaccine Strain (LVS) have uncovered mediators important in host defense against primary infection, as well as those correlated with successful vaccination. One such potential correlate was IL-12p40, a pleiotropic cytokine that promotes Th1 T cell function as part of IL-12p70. LVS-infected IL-12p40 deficient knockout (KO) mice maintain a chronic infection, but IL-12p35 KO mice clear LVS infection; thus the role that IL-12p40 plays in immunity to LVS is independent of the IL-12p70 heterodimer. IL-12p40 can also partner with IL-23p19 to create the heterodimeric cytokine IL-23. Here, we directly tested the role of IL-23 in LVS resistance, and found IL-23 to be largely dispensable for immunity to LVS following intradermal or intranasal infection. IL-23p19 KO splenocytes were fully competent in controlling intramacrophage LVS replication in an in vitro overlay assay. Further, antibody responses in IL-23p19 KO mice were similar to those of normal wild type mice after LVS infection. IL-23p19 KO mice or normal wild type mice that survived primary LVS infection survived maximal doses of LVS secondary challenge. Thus p40 has a novel role in clearance of LVS infection that is unrelated to either IL-12 or IL-23

T cell functions and cytokine production are similar between WT mice and p19G KO mice, regardless of priming route.

<p>WT mice and p19G KO mice were infected with 10⁵ LVS ID or 10³ LVS IN. Mice were sacrificed after 8 weeks and splenocytes isolated; splenocytes were also obtained from naïve mice. Bone marrow-derived macrophages from WT mice (shown) or p19G KO mice (not shown) were infected with LVS, and then 5×10⁶ homologous splenocytes were added to triplicate wells of infected macrophages. A) Intracellular bacterial burdens were assessed immediately after infection at 0 hours (not shown) and at 72 hours after infection. B) Supernatants were harvested from cultures at 72 hours after infection, and the levels of RNI measured. Supernatants were also assayed for cytokines by sandwich ELISA, including C) IFN-γ and D) IL-17A. IL-12p40 and IL-12p70 were also measured (data not shown). Results are shown for one representative experiment of three experiments of similar design and outcome. *p value≤0.05 by Student’s <i>t</i> test.</p

Bacterial burdens in tissues are comparable between infected WT mice and p19G KO mice infected with LVS ID and IN.

<p>Groups of 3 WT mice or p19G KO mice were infected either ID with 10⁵ LVS (A), or were anesthetized and infected with 2×10³ LVS IN (B). Mice were sacrificed at the indicated time points, and lungs, livers, and spleens plated to enumerate CFU. There were no significant differences in organ burdens between WT mice and p19G KO mice for any organ at any time point. Results shown are representative of two experiments of similar design and outcome. p19M KO mice were also included in one experiment with similar results (data not shown).</p

Similar titers of serum antibodies titers in LVS-vaccinated WT and IL-23 KO mice.

a<p>Groups of 5 WT or IL-23 KO mice (p19M KO and p19G KO) were infected with 10⁵ LVS ID and sera collected at Day 42, or were anesthetized and infected with 2×10³ LVS IN and sera collected at day 35. Sera from individual mice for each group were pooled and assayed for LVS specific total IgM and IgG antibodies, as well as the IgG subclasses (IgG₁, IgG_2b, IgG_2c, and IgG₃) by ELISA. Serum titers were calculated as described in Materials and Methods. Results shown are representative of two experiments of similar design and outcome.</p><p>Similar titers of serum antibodies titers in LVS-vaccinated WT and IL-23 KO mice.</p

Similar survival of lethal challenge by LVS-vaccinated WT and IL-23 KO mice.

a<p>In experiment 1 (Exp. 1), WT C57BL/6J mice or IL-23 KO mice (either p19M KO or p19G KO mice, as indicated) were infected either with 10⁵–10⁶ ID or 10² IN LVS. One month later, when primary infection was cleared, surviving mice were given a secondary high dose challenge of 2×10⁶ IP or 8×10⁷ LVS IN. Naïve control mice treated with PBS ID or IN were challenged as indicated IN or IP, where all mice died.</p>b<p>Exp. 2, mice were given a primary vaccination of 2×10² or 2×10³ IN and challenged with 2×10⁶ IP. The average time to death (TTD) of those mice that died within a group is also shown, as well as the range of the days of death. Groups where all the mice succumbed on the same day are given a range of (N/A). Data are representative of three experiments of similar design and outcome.</p><p>Similar survival of lethal challenge by LVS-vaccinated WT and IL-23 KO mice.</p

C57BL/6J and IL-23 deficient mice exhibit comparable survival patterns following IN or ID LVS infection.

<p>Groups of 5 to 10 WT mice and p19M KO mice were infected IN with LVS doses ranging from 10¹–10⁶ CFU (doses 10¹ and 10⁶ not shown for clarity). For ID infections, groups of 5 WT mice and p19M KO were infected ID with LVS doses ranging from 10⁴–10⁶. The calculated LD₅₀ for IN and ID infections are shown (inset table). No mice from any ID-infected group succumbed to infection. Results shown are representative of three experiments of similar design and outcome. p19G KO mice were also included in some experiments with similar results (data not shown).</p