Toll-Like Receptor 2 Agonist Pam3CSK4 Alleviates the Pathology of Leptospirosis in Hamster

Abstract

Leptospirosis, caused by pathogenic spirochetes, is a zoonotic disease of global importance. The detailed pathogenesis of leptospirosis is still unclear, which limits the ideal treatment of leptospirosis. In this study, we analyzed the expression of Toll-like receptor 2 (TLR2) and TLR4 in target organs of both resistant mice and susceptible hamsters after Leptospira interrogans serovar Autumnalis infection. TLR2 but not TLR4 transcripts in mouse organs contrasted with delayed induction and overexpression in hamster organs. Coinjection of leptospires and the TLR2 agonist Pam3CSK4 into hamsters improved their survival rate, alleviated tissue injury, and decreased the abundance of leptospires in target organs. The production of interleukin-10 (IL-10) from tissues was enhanced in hamsters of the group coinjected with leptospires and Pam3CSK4 compared with the leptospira-injected group. Similarly, IL-10 levels in TLR2-deficient mice were lower than those in wild-type mice. A high ratio of IL-10/tumor necrosis factor alpha (TNF-α) levels was found in both infected wild-type mice and hamsters coinjected with leptospires and Pam3CSK4. Moreover, TLR2-dependent IL-10 expression was detected in peritoneal macrophages after leptospira infection. Our data demonstrate that coinjection of leptospires and Pam3CSK4 alleviates the pathology of leptospirosis in hamsters; this effect may result from the enhanced expression of TLR2-dependent IL-10.

FIG 1

Modulation of TLR2 mRNA expression in tissues after injection of leptospires. TLR2 mRNA levels in kidneys, livers, and lungs from BALB/c mice and hamsters were quantified by RT-qPCR at a series of time points. The results were normalized to the expression level of the housekeeping gene GAPDH. Bars show the levels of TLR2 (means ± standard deviations) in tissues of mice and hamsters at each time point (n = 3). The levels of TLR2 in different tissues from three healthy individuals at 0 h were given a value of 1.0. Different mRNA expression levels between the infected group and the healthy group (0 h) were compared by one-way ANOVA. *, P < 0.05.

FIG 2

Modulation of TLR4 mRNA expression in tissues after injection of leptospires. TLR4 mRNA levels in kidneys, livers, and lungs from BALB/c mice and hamsters were quantified by RT-qPCR at a series of time points. The results were normalized to the expression levels of the housekeeping gene GAPDH. Bars show levels of TLR4 (means ± standard deviations) in tissues of mice and hamsters at each time point (n = 3). The levels of TLR4 in different tissues from three healthy individuals at 0 h were given a value of 1.0. Different mRNA expression levels between the infected group and the healthy group (0 h) were compared by one-way ANOVA. *, P < 0.05.

FIG 3

Influence of TLR2 agonist Pam3CSK4 on pathology of hamsters with leptospirosis. (A) Effect of Pam3CSK4 on leptospiral growth. A total 10⁶ leptospires in 0.5 ml of EMJH medium supplemented with or without 100 μg of Pam3CSK4 were cultured at 29°C. Growth was analyzed for 4 days by using a Petroff-Hausser chamber and a dark-field microscope. Each data point exhibits the means from triplicates ± standard deviations for three independent experiments. P values of <0.05 were considered significant (*, P < 0.05). (B) Survival curves of hamsters in the infected control group (n = 6) and the group coinjected with leptospires and Pam3CSK4 (n = 6). Hamsters were injected with leptospires or coinjected with leptospires and Pam3CSK4. *, P < 0.05 versus untreated controls as determined by a by Kaplan-Meier log rank test. (C) Histopathology scores for kidneys, livers, and lungs of hamsters. The data represent the mean histopathology scores for the two groups of hamsters. Statistical analysis of the results for infected controls (n = 6) and the group coinjected with leptospires and Pam3CSK4 (n = 6) was performed by using the Wilcoxon rank sum test. *, P < 0.05. (D) Histopathology of kidneys (a and b), livers (c and d), and lungs (e and f) of hamsters in the infected control group (a, c, and e) and the group coinjected with leptospires and Pam3CSK4 (b, d, and f). Magnification, ×200. Samples were collected over a 21-day experimental period, and representative photographs are presented. (E) Leptospiral burdens in the kidneys, livers, and lungs of hamsters in the infected control group (n = 6) and the group coinjected with leptospires and Pam3CSK4 (n = 6) as determined by qPCR. Samples were collected over a 21-day experimental period. The results are presented as numbers of genome equivalents per microgram of tissue DNA, and the differences were compared by one-way ANOVA. *, P < 0.05. (F) Survival curves of hamsters treated with Pam3CSK4 6 h after infection with leptospires (n = 8) and untreated controls (n = 8). Hamsters were injected with leptospires. Six hours later, Pam3CSK4 (100 μg) was intraperitoneally injected into hamsters. *, P < 0.05 versus untreated controls as determined by a Kaplan-Meier log rank test.

FIG 4

TLR2 contributes to the control of leptospiral burden in kidney and alleviates pathology in mice. (A) Histopathology scores for the kidneys, livers, and lungs of mice. The data represent the mean histopathology scores for the two groups of mice. Statistical analysis of results for WT mice (n = 6) and TLR2^−/− mice (n = 6) was performed by using the Wilcoxon rank sum test. *, P < 0.05. (B) Histopathology of kidneys (a and b), livers (c and d), and lungs (e and f) of WT mice (a, c, and e) and TLR2^−/− mice (b, d, and f) after infection with leptospires at 4 days p.i. Magnification, ×200. Representative photographs are presented. (C) Leptospiral burdens in kidneys, livers, and lungs of WT mice (n = 6) and TLR2^−/− mice (n = 6) as determined by qPCR at 4 days p.i. The results are presented as numbers of genome equivalents per microgram of tissue DNA, and the differences were compared by one-way ANOVA. *, P < 0.05.

FIG 5

IL-10/TNF-α ratios in mice and hamsters after injection of leptospires. (A) IL-10/TNF-α ratios in hamsters in the infected control group and the group coinjected with leptospires and Pam3CSK4 at 24 h p.i. based on the data shown in Table 2. (B) IL-10/TNF-α ratios in WT and TLR2^−/− mice at 4 days p.i. based on the data shown in Table 3.

FIG 6

IL-10 and TNF-α expression levels in peritoneal macrophages of hamsters and mice. Peritoneal macrophages of hamsters were challenged with leptospires or with leptospires plus Pam3CSK4 for 24 h. Peritoneal macrophages from WT, TLR2^−/−, and TLR4^−/− mice were challenged with leptospires for 24 h, and the mRNA levels of IL-10 and TNF-α were quantified by RT-qPCR. (A and B) IL-10 (A) and TNF-α (B) mRNA levels in peritoneal macrophages of WT, TLR2^−/−, and TLR4^−/− mice. (C) IL-10 and TNF-α mRNA levels in peritoneal macrophages of hamsters. The results were normalized to the expression level of the housekeeping gene GAPDH. Bars show mean levels of cytokines ± standard deviations, and differences were analyzed by one-way ANOVA. *, P < 0.05; ns, none significant.