Leptospiral Hemolysins Induce Proinflammatory Cytokines through Toll-Like Receptor 2-and 4-Mediated JNK and NF-κB Signaling Pathways

Abstract

BackgroundInfection with pathogenic Leptospira species causes serious systemic inflammation in patients. Although a few leptospiral proinflammatory molecules have been identified, Leptospira likely encodes other unidentified strong inflammation stimulators. The pathogenic L. interrogans genome encodes numerous putative hemolysin genes. Since hemolysins from other bacteria can cause inflammatory reactions, we hypothesized that leptospiral hemolysins may function as proinflammatory stimulators that contribute to the strong inflammation associated with Leptospira infection.Methodology/Principal FindingsWe first used cytokine protein microarrays for systematic analysis of serum cytokine profiles in leptospirosis patients and leptospire-infected mice. We found that IL-1β, IL-6 and TNF-α were the main proinflammatory cytokines in the sera of both the patients and the mice. We then analyzed eight putative hemolysins in L. interrogans strain Lai. The results showed that five of them, Sph1, Sph2, Sph3, HlpA and TlyA were secreted and had hemolytic activity. More importantly, these five hemolysins induced the strong production of IL-1β, IL-6 and TNF-α in human and mouse macrophages (although a bit lower in the latter). Furthermore, blockade of TLR2 or TLR4 with either antibodies or inhibitors of the NF-κB or JNK signaling pathways significantly reduced the production of hemolysin-induced IL-1β, IL-6 and TNF-α. Macrophages isolated from TLR2-, TLR4-or double TLR2-and 4-deficient mice also confirmed that the leptospiral hemolysins that induce proinflammatory cytokines are both TLR2-and TLR4-dependent.Conclusions/SignificanceOur findings demonstrate that L. interrogans secretes many hemolysins that function as powerful inducers of proinflammatory cytokines through both TLR2-and TLR4-dependent JNK and NF-κB pathways.

Specific CD4+T-Cell Reactivity and Cytokine Release in Different Clinical Presentations of Leptospirosis

“…Elevated cytokine production in severe disease compared to mild disease is consistent with our data, since patients with severe disease revealed more prominent production of all cytokines tested. It has been shown that extensive release of proinflammatory mediators such as TNF-␣, IL-1␤, and IL-6 causes pathological inflammatory disorders, tissue injury, and organ failure (25). Consistent with our findings, there seems to be a dominant role of TNF-␣ as a mediator of severe disease.…”

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

“…Previous studies have shown that TLR4 is vital for the control of the leptospiral burden in vivo, whereas both TLR2 and TLR4 control the leptospiral burden in the kidney, and tissue differences in TLR signaling may exist (8,9). In vitro, many hemolysins of Leptospira induce proinflammatory cytokines through both the TLR2-and TLR4-dependent c-Jun N-terminal kinase (JNK) and nuclear factor B (NF-B) pathways (10), and leptospiral membrane proteins stimulate proinflammatory chemokines by TLR2 in renal proximal tubule cells (11). All those studies indicate that the TLRs, particularly TLR2 and TLR4, can play a crucial role in the development of leptospirosis.…”

Decrease in antithrombin III and prothrombin serum levels contribute to coagulation disorders during leptospirosis

“…Similar to sepsis, human leptospirosis was reported to be associated with high plasma levels of inflammatory cytokines such as TNF-a, IL-6, IL-8, IL-1b and IL-12 (Matsui et al, 2011;Wang et al, 2012;Reis et al, 2013). Indeed, the production of inflammatory cytokines such as TNF-a by CD4+ T-cells after stimulation with antigens of Leptospira was highest in patients with severe disease (Volz et al, 2015).…”

“…Nevertheless, some of these proteins are secreted and induced pro-inflammatory cytokines (Wang et al, 2012). Furthermore, lipopolysaccharide (Werts et al, 2001), peptidoglycans (Cinco et al, 1996), glycoproteins (Diament et al, 2002), lipoproteins (Yang et al, 2006a), and outer membrane proteins (OMPs) (Yang et al, 2006a) are capable of promoting host immune response and cytokine secretion.…”