The preventable efficacy of β-glucan against leptospirosis

Abstract

Leptospirosis, caused by pathogenic Leptospira species, has emerged as an important neglected zoonotic disease. Few studies have reported the preventable effects of immunoregulators, except for antibiotics, against leptospirosis. Generally, immunostimulatory agents are considered effective for enhancing innate immune responses. Many studies have found that beta-glucan (β-glucan) could be a potent and valuable immunostimulant for improving immune responses and controlling diseases. In this study, we investigated the preventable role of β-glucan against Leptospira infection in hamsters. First, β-glucan was administered 24 h prior to, during and after infection. The results showed that β-glucan increased the survival rate to 100%, alleviated tissue injury, and decreased leptospire loads in target organs. Additionally, we found using quantitative real-time PCR that application of β-glucan significantly enhanced the expression of Toll-like receptor (TLR) 2, interleukin (IL)-1β and iNOS at 2 dpi (days post infection) and reduced the increase of TLR2, IL-1β and iNOS induced by Leptospira at 5 dpi. Furthermore, to induce memory immunity, β-glucan was administered 5 days prior to infection. β-Glucan also significantly increased the survival rates and ameliorated pathological damage to organs. Moreover, we demonstrated that β-glucan-trained macrophages exhibited elevated expression of proinflammatory cytokines (IL-1β and IL-6) in vitro, indicating that β-glucan induces an enhanced inflammatory response against Leptospira infection. These results indicate that administration of β-glucan and other immunostimulants could be potential valuable options for the control of Leptospira infection.

Fig 1. Influence of β-glucan on the pathology of hamsters with leptospirosis.

(A) Effect of β-glucan on Leptospira growth. A total of 10⁷ leptospires in 1 ml of 0.9% saline supplemented with or without 5 mg or 10 mg of β-glucan 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 represents the means ± standard deviations from 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 = 8), the β-glucan control group (n = 8) and the group coinjected with leptospires and β-glucan (n = 8). Hamsters were stimulated with β-glucan 1 day prior to leptospira challenge and on day 0 and day 1 during leptospira challenge. *, P < 0.05 versus untreated controls, as determined by Kaplan-Meier analysis with a log-rank test. (C) Histopathology of the 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 β-glucan (b, d, and f). Magnification, 100×. Samples were collected on the 4^th day after infected leptospira, and representative images are shown. (D) Histopathology scores for kidneys, livers, and lungs of hamsters. The data are presented as the mean histopathology scores for the two groups of hamsters. Statistical analysis of the results for the infected control group (n = 4) and the group coinjected with leptospires and β-glucan was performed by using the Wilcoxon rank sum test. *, P < 0.05. Magnification, × 200. (E) Leptospiral burdens in the kidneys, livers, and lungs of hamsters in the infected control group and the group coinjected with leptospires and β-glucan, as determined by qPCR. Samples were collected on the 4^th day after infected leptospira. The results are presented as the number of genomic equivalents per microgram of tissue DNA, and the differences were compared by one-way ANOVA. *, P < 0.05.

Fig 2. Modulation of TLR2, TLR4, IL-1β and iNOS mRNA expression in tissues after injection of leptospires.

The TLR2, TLR4, IL-1β and iNOS mRNA levels in the kidneys, livers, and lungs of hamsters at 2 dpi and 5 dpi were quantified by RT-qPCR. The results were normalized to the expression level of the housekeeping gene GAPDH. The bars show the levels of TLR2, TLR4, IL-1β and iNOS (means ± standard deviations) in each tissue of hamsters (n = 3). The levels of TLR2, TLR4, IL-1β and iNOS in different tissues from three healthy individuals at 0 h were assigned 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. Effect of β-glucan-induced trained immunity on leptospirosis.

(A) Survival curves of hamsters in the infected control group (n = 8), the β-glucan control group (n = 8) and the group stimulated with β-glucan and leptospires (n = 8). Hamsters were stimulated with β-glucan 5 days prior to leptospira challenge. *, P < 0.05 versus untreated controls, as determined by Kaplan-Meier analysis with a log-rank test. (B) Histopathology scores for the kidneys, livers, and lungs of hamsters. The data are presented as the mean histopathology scores for the two groups of hamsters. Statistical analysis of the results for the infected control group (n = 8) and the group stimulated with leptospires and β-glucan (n = 8) was performed by using the Wilcoxon rank sum test. *, P < 0.05. (C) Histopathology of the 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 stimulated with leptospires and β-glucan (b, d, and f). Magnification, 100×. Samples were collected over the 21-day experimental period, and representative images are shown. (D) The expression of proinflammatory factors in hamster peritoneal macrophages trained by β-glucan. Hamster peritoneal macrophages were stimulated with β-glucan 5 days prior to leptospira challenge. The levels of IL-1β, IL-6 and IL-10 at 1 dpi were measured by qRT-PCR. The results were normalized to the expression level of the housekeeping gene GAPDH. The bars show the levels of IL-1β, IL-6 and IL-10 (means ± standard deviations) in hamster peritoneal macrophages. The levels of IL-1β, IL-6 and IL-10 in blank control peritoneal macrophages were assigned a value of 1.0. Different mRNA expression levels between the infected group and the group stimulated with β-glucan and leptospires were compared using one-way ANOVA. *, P < 0.05.