Increased inflammation with crude E. coli LPS protects against acute leptospirosis in hamsters

Abstract

Leptospirosis is a worldwide zoonotic disease that causes acute kidney injury, liver disease, bleeding disorders, and even death. Treatment of the disease is largely dependent on the use of antibiotics, but recent studies on pathogenesis of leptospirosis have shown that immunomodulation may also be an effective treatment for this disease. Since the delay in inflammation correlates with higher pathogenicity of leptospira, we studied the effect of inducing inflammation on leptospirosis by using TLR4 activator LPS. In accordance with our hypothesis, treatment with LPS protected against leptospirosis by enhancing the inflammatory response in hamsters. The gene expression levels of TLR2, TLR4, NLRP3 and inflammatory factors were higher in LPS-treated group during leptospira infection in hamsters. Although the levels of NO and iNOS were higher in LPS-treated group than in Leptospira-infected group, the protective effect induced by LPS is iNOS-independent. Treatment with LPS induced higher anti-leptospira IgG level than infection with leptospira alone. Then, expressions of costimulatory molecules and maturation markers were analysed. The data showed that treatment with LPS enhanced the expression of CD40, CD80 and CD86. Our results indicate that increased inflammation induced by LPS derived from Escherichia coli (E. coli) protects against leptospirosis in hamsters.

Keywords: LPS; Leptospirosis; hamster; inflammation; treatment.

Figure 1.

Treatment with LPS protects against leptospirosis with the improving inflammation levels in hamsters. (A–C) The animal experiment was repeated three times. Survival curves of hamsters in the infected control group (n = 4) and the group treatment with LPS (n = 4). *P < 0.05 versus infected controls as determined by a Kaplan-Meier log rank test. (D) A single dose of LPS also contributed to the survival of hamsters. Survival differences between study groups (n = 4) were compared using the log-rank test. *P < 0.05 vs. the Lep group. (E) Histopathology of kidneys, livers, and lungs of hamsters in the infected control group and the LPS-treated group. Magnification, ×400. Samples were collected at 2 d p.i., and representative photographs are presented. (F) 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 = 3) and the LPS-treated group (n = 3) was performed by using the Wilcoxon rank sum test. *P < 0.05. (G) MPO activity in kidneys, livers and lungs of hamsters in infected controls and LPS-treated group at 2 d p.i.. Data are presented as mean ± SEM and analysed by t-test. *P < 0.05. (H) Leptospiral burdens in the kidneys, livers, and lungs of hamsters in the infected control group (n = 3) and the LPS-treated group (n = 3) was determined by qPCR. Samples were collected at 4 d p.i.. The results are presented as numbers of genome equivalents per microgram of tissue DNA, and the differences were compared by t-test. *P < 0.05. (I) Weight changes in control group (n = 4) and LPS-treated group (n = 4). The differences were compared by t-test. *P < 0.05.

Figure 2.

Gene expressions of TLR2, TLR4, NLRP3 and cytokines in tissues after injection of leptospires. Samples were collected from infected controls (n = 3) and LPS-treated group (n = 3) at 2 d p.i.. TLR2 (A), TLR4 (B), NLRP3 (C), IL-10 (D), TNF-α (E) and IL-1β (F) mRNA levels were quantified by RT-qPCR. The results were normalized to the expression level of the housekeeping gene GAPDH. The levels of these genes in different tissues from three healthy individuals were given a value of 1.0. Different mRNA expression levels between the infected controls and LPS-treated group were compared by t-test. *P < 0.05.

Figure 3.

Treatment with LPS increases the levels of NO and iNOS during leptospira infection in hamsters. (A) Gene expression of iNOS in organs was detected by RT-qPCR at 2 d p.i.. The results were normalized to the expression level of the housekeeping gene GAPDH. The levels of iNOS in different tissues from three healthy individuals were given a value of 1.0. Different mRNA expression levels between the infected controls and LPS-treated group were compared by t-test. *P < 0.05. (B) The expression of NO was quantified by detecting the concentration of nitrate/nitrite. Samples were collected at 2 d p.i. from different groups. Data are presented as mean ± SEM. *P < 0.05 versus infected controls as determined by a t-test. (C) The protective effect induced by LPS is iNOS-independent and the dosage of LPS on hamster is suitable. Survival differences between study groups (n = 8) were compared using the log-rank test. *P < 0.05 vs. the Lep group.

Figure 4.

Treatment with LPS improves the anti-leptospira IgG level. The serum and spleens of hamsters in different groups were collected at 4 d p.i.. (A) Titres of anti-leptospira IgG was measured in the serum of different groups and analysed by the t-test. *P < 0.05. (B) Anti-Leptospira IgG quantification in sera from infected controls and LPS-treated group. Data are expressed as the OD at 450 nm measured in sera (n = 3 per group) at the dilution 1/400, and the differences were compared by t-test. *, P < 0.05. (C–D) Anti-Leptospira IgG1 and IgG2/3 quantification in sera from infected controls and LPS-treated group. Data are expressed as the OD at 450 nm measured in sera (n = 3 per group) at the dilution 1/400, and the differences were compared by t-test. *P < 0.05. (E) Western blot about anti-Leptospira IgG from different groups.

Figure 5.

Treatment with LPS improves the expressions of maturation markers. (A–D) Gene expressions of CD40, CD80, CD86 and MHCII in organs were detected by RT-qPCR. The results were normalized to the expression level of the housekeeping gene GAPDH. The levels of these genes in different tissues from three healthy individuals were given a value of 1.0. Different mRNA expression levels between the infected controls and LPS-treated group were compared by t-test. *P < 0.05.