Differential cytokine gene expression according to outcome in a hamster model of leptospirosis

Abstract

Background: Parameters predicting the evolution of leptospirosis would be useful for clinicians, as well as to better understand severe leptospirosis, but are scarce and rarely validated. Because severe leptospirosis includes septic shock, similarities with predictors evidenced for sepsis and septic shock were studied in a hamster model.

Methodology/principal findings: Using an LD50 model of leptospirosis in hamsters, we first determined that 3 days post-infection was a time-point that allowed studying the regulation of immune gene expression and represented the onset of the clinical signs of the disease. In the absence of tools to assess serum concentrations of immune effectors in hamsters, we determined mRNA levels of various immune genes, especially cytokines, together with leptospiraemia at this particular time-point. We found differential expression of both pro- and anti-inflammatory mediators, with significantly higher expression levels of tumor necrosis factor alpha, interleukin 1alpha, cyclo-oxygenase 2 and interleukin 10 genes in nonsurvivors compared to survivors. Higher leptospiraemia was also observed in nonsurvivors. Lastly, we demonstrated the relevance of these results by comparing their respective expression levels using a LD100 model or an isogenic high-passage nonvirulent variant.

Conclusions/significance: Up-regulated gene expression of both pro- and anti-inflammatory immune effectors in hamsters with fatal outcome in an LD50 model of leptospirosis, together with a higher Leptospira burden, suggest that these gene expression levels could be predictors of adverse outcome in leptospirosis.

Figure 1. Preliminary experiments results leading to the selection of day 3 post-infection (as shown by the red lines) as an appropriate study time point.

A. Time course of mortalities in the LD50 infection model (n = 70, green line) and in the animals sampled for the gene expression studies (n = 36, blue line). B. Relative normalized gene expression levels (see text) time courses (y-axes use different scales for different gene targets).

Figure 2. Mean expression levels of differentially expressed genes (relative normalized expression, see text).

Expression levels are shown at day 3 post-infection in the LD50 Leptospira infection (according to the outcome) and after the injection of a high dose of a virulent or the high-passage variant (A) and Leptospira burdens (B) in infected hamsters. Error bars indicate 95% confidence intervals.

Figure 3. IFNγ, TGFβ and IL-6 gene expression levels at day 3 post Leptospira infection (relative normalized expression, see text).

Error bars indicate 95% confidence intervals. The insert shows the high variability observed in IL-6 gene expression levels.