Exposure to live saprophytic Leptospira before challenge with a pathogenic serovar prevents severe leptospirosis and promotes kidney homeostasis

Abstract

Previous studies demonstrated that Leptospira biflexa, a saprophytic species, triggers innate immune responses in the host during early infection. This raised the question of whether these responses could suppress a subsequent challenge with pathogenic Leptospira. We inoculated C3H/HeJ mice with a single or a double dose of L. biflexa before challenge with a pathogenic serovar, Leptospira interrogans serovar Copenhageni FioCruz (LIC). Pre-challenge exposure to L. biflexa did not prevent LIC dissemination and colonization of the kidney. However, it rescued weight loss and mouse survival thereby mitigating disease severity. Unexpectedly, there was correlation between rescue of overall health (weight gain, higher survival, lower kidney fibrosis marker ColA1) and higher shedding of LIC in urine. This stood in contrast to the L. biflexa unexposed LIC challenged control. Immune responses were dominated by increased frequency of effector T helper (CD4+) cells in spleen, as well as significant increases in serologic IgG2a. Our findings suggest that exposure to live saprophytic Leptospira primes the host to develop Th1 biased immune responses that prevent severe disease induced by a subsequent challenge with a pathogenic species. Thus, mice exposed to live saprophytic Leptospira before facing a pathogenic serovar may withstand infection with far better outcomes. Furthermore, a status of homeostasis may have been reached after kidney colonization that helps LIC complete its enzootic cycle.

Keywords: L. interrogans; Leptospira; commensalism; immunology; infectious disease; inflammation; kidney; leptospirosis; microbiology; mouse; vaccine.

Figure 1.. Weight loss, kidney colonization, shedding in urine, and survival to challenge with L. interrogans following a single exposure to L. biflexa.

Male C3H/HeJ mice were inoculated once with 10⁸ L. biflexa (LB) at 6 weeks and they were challenged with 10⁸ L. interrogans serovar Copenhageni FioCruz (LIC) at 8 weeks. (A) Experimental layout; (B) body weight measurements (%) acquired for 15 days post challenge with LIC; (C) mouse survival within the 15 days post challenge with LIC; (D) 16S rRNA qPCR quantification of live LIC in urine; (E) 16S rRNA qPCR quantification of Leptospira burden in kidney tissue harvested on d15 post challenge with LIC and (F) 16S rRNA qPCR from kidney EMJH cultures containing live Leptospira previously observed by dark-field microscopy (DFM). DFM positive culture from the total data is represented in numbers under the graph. Statistical analysis was performed by ordinary one-way ANOVA followed by Tukey’s multiple comparison correction between challenged groups and their respective controls, *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001, N=6–8 mice per group. Data represents two independent experiments.

Figure 1—figure supplement 1.. qPCR to quantify L. interrogans load in blood of mice using 16S rRNA Leptospira-specific primers and probes from the single L. biflexa exposure experiment.

Data represents two experiments.

Figure 2.. Weight loss, kidney colonization, shedding in urine, and survival to challenge with L. interrogans following a double exposure to L. biflexa.

Male C3H/HeJ mice were inoculated twice with 10⁸ L. biflexa at 6 and 8 weeks, and at 10 weeks they were challenged with 10⁸ L. interrogans ser Copenhageni FioCruz (LIC). (A) Experimental layout; (B) body weight measurements (%) acquired for 15 days post challenge with LIC; (C) mouse survival within the 15 days post challenge with LIC; (D) 16S rRNA qPCR quantification of live LIC in urine; (E) 16S rRNA qPCR quantification of Leptospira burden in kidney tissue harvested on d15 post challenge with LIC and (F) 16S rRNA qPCR from kidney EMJH cultures containing live Leptospira previously observed by dark-field microscopy (DFM). DFM positive culture from the total data is represented in numbers under the graph. Statistical analysis was performed by ordinary one-way ANOVA followed by Tukey’s multiple comparison correction between challenged groups and their respective controls, *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. N=6–8 mice per group. Data represents two independent experiments.

Figure 2—figure supplement 1.. qPCR to quantify L. interrogans load in blood of mice using 16S rRNA Leptospira-specific primers and probes from the double L. biflexa exposure experiment. Data represents two experiments.

Figure 3.. Kidney histopathology and quantification of renal fibrosis.

Representative hematoxylin and eosin (H&E)-stained kidney tissue sections from both single and double exposure studies are included in (A) and (C), respectively. The images were captured at ×40 magnification. (B) and (D) represent the mRNA expression of kidney fibrosis marker ColA1 by qPCR normalized to endogenous β-actin expression. Data was analyzed by ordinary one-way ANOVA followed by Tukey’s multiple comparison correction between challenged groups with their respective controls; *p-values are included in the graphs. Data represents one of two independent experiments.

Figure 3—figure supplement 1.. Morphometric analysis of kidney of mice from the double L. biflexa exposure experiment. Data represents one experiment.

Figure 4.. Detection of IgG1, IgG2a, and IgG3 specific to L. interrogans in serum from experimental mice.

(A) represents IgG isotypes specific to L. interrogans in 10-week serum of mice exposed once to L. biflexa before L. interrogans challenge. (B) represents IgG isotypes specific to L. interrogans in 12-week serum of mice exposed twice to L. biflexa before L. interrogans challenge. Ordinary one-way ANOVA followed by Tukey’s multiple comparison correction test was used to compare between challenged groups with their respective controls; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, and ns = not significant; N=6–8 mice per group. Data represents two independent experiments.

Figure 5.. Frequency of lymphocytes in spleen of mice subjected to a double exposure of L. biflexa before challenge with L. interrogans.

(A–E) show B cell (CD19+), T cell (CD3+), NK cell (CD49b+), helper T cell (CD4+), and cytotoxic T cell (CD8+) frequencies in groups of experimental mice. Ordinary one-way ANOVA followed by Tukey’s multiple comparison correction test was used to compare between challenged groups and their respective controls; **p<0.01, ***p<0.001, ****p<0.0001, and ns = not significant; N=3–4 mice per group. Data represents one of two independent experiments.

Figure 5—figure supplement 1.. Flow cytometry gating strategy.

The outlined gating strategy was used to immune phenotype spleen immune cells acquired in the double L. biflexa exposure experiment. Black arrows represent gating events to analyze different immune cell populations derived from their parent populations in the spleen. X and Y axis represents specific cell surface markers to distinguish a specific cell type.

Figure 6.. Frequency of T cell subsets (CD62L/CD44) in spleen of mice subjected to a double exposure of L. biflexa before challenge with L. interrogans.

(A–D) represent naïve, early effector, effector, and memory subsets of CD4+ helper T lymphocytes, respectively. (E–H) represent naive, early effector, effector, and memory subsets of CD8+ cytotoxic T lymphocytes, respectively. Ordinary one-way ANOVA followed by Tukey’s multiple comparison correction test was used to compare between challenged groups and their respective controls; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, and ns = not significant; N=3–4 mice per group. Data represents one of two independent experiments.