Studies on the exaggerated inflammatory response caused by streptococcus suis at systemic and central nervous system levels

Streptococcus suis serotype 2 is an important swine pathogen responsible for diverse infections, meningitis being its most striking feature. In addition, it is an emerging agent of zoonosis, which has gained worldwide attention due to important outbreaks in Asia. Understanding the pathogenesis of S. suis infections still represents a challenge. Up to present, the pro-inflammatory response due to S. suis has only been studied in vitro, and there is still a great need of appropriate experimental models for both septic shock and meningitis. In the present study, we successfully developed an in vivo model of S. suis infection in adult mice infected by the intraperitoneal route. This model served to investigate the pro-inflammatory events that take place at both the systemic and Central Nervous System (CNS) levels associated with this important pathogen. In addition, this model was useful to determine if susceptibility to S. suis infection may be influenced by the genetic background of the host.  The mouse model of S. suis infection was standardized in CD1 mice. Results showed sustained bacteremia during the 3 days post-infection (p.i.), accompanied by a quick and substantial release of different pro-inflammatory cytokines (TNF-α, IL-6, IL-12p40/p70, IFN-ɣ)  and chemokines (KC, MCP-1 and RANTES) that lead to septic shock and 20% mortality in mice. Once the hallmark of the septic phase of S. suis infection was established in CD1 mice, research continued with the objective to confirm the role of inflammation in mortality and to determine if the genetic background of the host may influence the inflammatory response toward this pathogen and the further outcome of the disease. For this, the mouse model of S. suis infection was used with two genetically different inbred mouse strains, this is, C57BL/6 (B6) and A/J mice,  which are considered as the prototype of Th1-type  and Th2-type mice, respectively. Results demonstrated a striking susceptibility to S. suis infection in A/J mice in comparison to B6 mice, with 100% mortality in the former mice strain at 20 h p.i., and 16 % mortality at 36 h p.i. for the latter. Very interestingly, and similarly to CD1 mice, bacteremia did not seem to be responsible for the death of mice, as both mice strains presented similar amounts of bacteria in blood and organs. Thus, it was postulated that the higher mortality in S. suis-infected A/J mice was due to uncontrolled septic shock. In fact, A/J mice presented very high levels of TNF-α, IL-12p40/p70, IL-1β and IFN-ɣ, that significantly exceeded those found in B6 mice. Remarkably, chemokine levels were similar between strains, suggesting their limited participation in the development of septic shock by S. suis. A greater survival of B6 mice was partially related to a better regulation of the pro-inflammatory cytokine cascade, as they showed a higher production of the anti-inflammatory cytokine IL-10 than A/J mice. The potential beneficial role of the IL-10 in mice infected with S. suis was confirmed using two approaches: the first, by blockage of the cell receptor of IL-10 (IL-10R) with an anti-mouse IL-10R monoclonal antibody (Mab) in B6 mice and the second by administrating recombinant mouse (rm)IL-10 (rmIL-10) to A/J mice. B6 mice that received the IL-10R MAb treatment before challenge with S. suis developed a clinical acute disease similar to that observed with A/J mice, with a striking and rapid increase in mortality and higher levels of TNF-α in comparison to those of infected mice that did not receive the treatment. Controversially, treatment with rmIL-10 significantly delayed the onset of septic shock in A/J mice infected with S. suis. These results show that survival from S. suis septic shock requires a tight regulation of pro- and anti-inflammatory mechanisms, and that the latter should be activated at the same time or soon after the onset of the pro-inflammatory response. This part of the study may represent a first step in the identification of host genes associated with resistance against S. suis.  One of the most important achievements of the mouse model of infection described in this project is the development of distinct clinical signs of neurological disease in CD1 mice from 4 days p.i. Indeed, in CD1 mice that survived sepsis due to S. suis infection, clinical signs of neurological disease and vestibular syndrome, which are quite similar to those observed in clinical cases of S. suis meningitis in both pigs and humans, were observed. Studies of the brains of infected CD1 mice using in situ hybridization combined with immunocytochemistry, demonstrated that the CNS inflammatory response began with a significant increase in the transcription of Toll-like receptor (TLR)2 and CD14 initially in the brain microvasculature and choroid plexuses, suggesting that S. suis may use these structures as portals of entry to the brain. There also was activation of NF-κB (as indicated by transcriptional activation of IκBα as a reporter system) and TNF-α, IL-1β and MCP-1, mainly in cells identified as microglia and to a lesser extent in astrocytes. These signals reached different brain structures, mainly the brain cortex, corpus callosum, hippocampus, choroid plexuses, thalamus, hypothalamus and meninges. All of these pro-inflammatory events were associated with extensive areas of inflammation and necrosis, severe demyelination and presence of antigens of S. suis inside microglia.  In vitro studies were conducted in order to better understand the interactions of S. suis and microglia. For this, mouse microglia were infected with a virulent wild type (WT) strain of S. suis. Two isogenic mutants deficient in capsule (CPS) or hemolysin production (suilysin, SLY) respectively, were also included for comparative purposes. The CPS was important for S. suis resistance to phagocytosis, and it also modulated the inflammatory response by hiding pro-inflammatory components from the bacterial cell wall. On the other hand, the absence of SLY, a potential cytotoxic factor, did not have a major impact on S. suis interactions with microglia. Studies with microglia helped to confirm previous findings in vivo in mice, as the WT S. suis strain induced the up-regulation of TLR2 and the production of several pro-inflammatory mediators, including TNF-α and MCP-1. As observed in mice, S. suis induced NF-kB translocation, which was more rapid for cells stimulated with the CPS-deficient mutant, suggesting that bacterial cell wall components are potent inducers of NF-kB. Moreover, WT S. suis promoted phosphotyrosine, PKC and different mitogen-activated protein kinase (MAPK) events. However, microglia infected with the CPS-deficient mutant showed overall stronger and more sustained phosphorylation profiles. Finally, the CPS also modulated S. suis-induced inducible nitrogen oxide synthase (iNOS) expression and further nitric oxide production in microglia, which could be related to neurotoxicity and vasodilatation in vivo.   We are confident that our results may help to more fully understand the mechanisms underlying S. suis induction of inflammation, leading to the design of more efficient anti-inflammatory strategies for sepsis and meningitis. Finally, we believe this experimental model of infection in mice could also be useful for studying the pathogenesis of infections of the CNS, due to other bacteria.

Keyphrases: Streptococcus suis, mouse, sepsis, meningitis, inflammation, cytokine, chemokine, microglia, TLR2, MAPK, Streptococcus suis, sepsis, inflammation, cytokine, microglia, TLR2, MAPK