Claude Lachance

Influence of gender and interleukin-10 deficiency on the inflammatory response during lung infection with Pseudomonas aeruginosa in mice

Cystic fibrosis females have a worse prognosis compared to male patients. Furthermore, cystic fibrosis patients infected with Pseudomonas aeruginosa have been shown to have dysregulated cytokine profiles, as higher levels of tumour necrosis factor alpha (TNF‐α), interleukin (IL)‐8, and lower levels of IL‐10 are found in the bronchoalveolar lavage fluid compared to healthy controls. The present study was aimed at investigating the importance of gender and IL‐10 in the susceptibility of C57BL/6 mice to pulmonary infection with Pseudomonas aeruginosa. We found that wildtype females were more susceptible than males to infection, as we observed greater weight loss, higher bacterial load, and inflammatory mediators in their lungs. IL‐10 knockout mice, both females and males, had higher levels of TNF‐α in the lungs compared to wildtype mice and maintained higher levels of polymorphonuclear cells and lower levels of macrophages for a longer period of time. Our results demonstrate that the number of bacteria recovered from the lungs of IL‐10 knockout male mice was significantly higher than that observed in their wildtype male counterparts and we show that neutralization of IL‐10 in infected female mice for a prolonged period of time leads to increased susceptibility to infection. Results reported in this study clearly demonstrate that females, both wildtype and IL‐10 knockout mice are more susceptible to Pseudomonas aeruginosa infection than males, and that they mount a stronger inflammatory response in the lungs.

In Vitro Characterization of the Microglial Inflammatory Response to Streptococcus suis, an Important Emerging Zoonotic Agent of Meningitis

ABSTRACT Streptococcus suis is an important swine and human pathogen responsible for septicemia and meningitis. In vivo research in mice suggested that in the brain, microglia might be involved in activating the inflammatory response against S. suis. The aim of this study was to better understand the interactions between S. suis and microglia. Murine microglial cells were infected with a virulent wild-type strain of S. suis. Two isogenic mutants deficient at either capsular polysaccharide (CPS) or hemolysin production were also included. CPS contributed to S. suis resistance to phagocytosis and regulated the inflammatory response by hiding proinflammatory components from the bacterial cell wall, while the absence of hemolysin, a potential cytotoxic factor, did not have a major impact on S. suis interactions with microglia. Wild-type S. suis induced enhanced expression of Toll-like receptor 2 by microglial cells, as well as phophotyrosine, protein kinase C, and different mitogen-activated protein kinase signaling events. However, cells infected with the CPS-deficient mutant showed overall stronger and more sustained phosphorylation profiles. CPS also modulated inducible nitric oxide synthase expression and further nitric oxide production from S. suis-infected microglia. Finally, S. suis-induced NF-κB translocation was faster for cells stimulated with the CPS-deficient mutant, suggesting that bacterial cell wall components are potent inducers of NF-κB. These results contribute to increase the knowledge of mechanisms underlying S. suis inflammation in the brain and will be useful in designing more efficient anti-inflammatory strategies for meningitis.

Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 (MK2) Contributes to Secondary Damage after Spinal Cord Injury

The inflammatory response contributes importantly to secondary tissue damage and functional deficits after spinal cord injury (SCI). In this work, we identified mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MAPKAPK2 or MK2), a downstream substrate of p38 MAPK, as a potential target using microarray analysis of contused spinal cord tissue taken at the peak of the inflammatory response. There was increased expression and phosphorylation of MK2 after SCI, with phospho-MK2 expressed in microglia/macrophages, neurons and astrocytes. We examined the role of MK2 in spinal cord contusion injury using MK2−/− mice. These results show that locomotor recovery was significantly improved in MK2−/− mice, compared with wild-type controls. MK2−/− mice showed reduced neuron and myelin loss, and increased sparing of serotonergic fibers in the ventral horn caudal to the injury site. We also found differential expression of matrix metalloproteinase-2 and 9 in MK2−/− and wild-type mice after SCI. Significant reduction was also seen in the expression of proinflammatory cytokines and protein nitrosylation in the injured spinal cord of MK2−/− mice. Our previous work has shown that macrophages lacking MK2 have an anti-inflammatory phenotype. We now show that there is no difference in the number of macrophages in the injured spinal cord between the two mouse strains and little if any difference in their phagocytic capacity, suggesting that macrophages lacking MK2 have a beneficial phenotype. These findings suggest that a lack of MK2 can reduce tissue damage after SCI and improve locomotor recovery. MK2 may therefore be a useful target to treat acute SCI.

Characterization of porcine dendritic cell response to Streptococcus suis

Streptococcus suis is a major swine pathogen and important zoonotic agent causing mainly septicemia and meningitis. However, the mechanisms involved in host innate and adaptive immune responses toward S. suis as well as the mechanisms used by S. suis to subvert these responses are unknown. Here, and for the first time, the ability of S. suis to interact with bone marrow-derived swine dendritic cells (DCs) was evaluated. In addition, the role of S. suis capsular polysaccharide in modulation of DC functions was also assessed. Well encapsulated S. suis was relatively resistant to phagocytosis, but it increased the relative expression of Toll-like receptors 2 and 6 and triggered the release of several cytokines by DCs, including IL-1β, IL-6, IL-8, IL-12p40 and TNF-α. The capsular polysaccharide was shown to interfere with DC phagocytosis; however, once internalized, S. suis was readily destroyed by DCs independently of the presence of the capsular polysaccharide. Cell wall components were mainly responsible for DC activation, since the capsular polysaccharide-negative mutant induced higher cytokine levels than the wild-type strain. The capsular polysaccharide also interfered with the expression of the co-stimulatory molecules CD80/86 and MHC-II on DCs. To conclude, our results show for the first time that S. suis interacts with swine origin DCs and suggest that these cells might play a role in the development of host innate and adaptive immunity during an infection with S. suis serotype 2.

Capsular Sialic Acid of Streptococcus suis Serotype 2 Binds to Swine Influenza Virus and Enhances Bacterial Interactions with Virus-Infected Tracheal Epithelial Cells

ABSTRACT Streptococcus suis serotype 2 is an important swine bacterial pathogen, and it is also an emerging zoonotic agent. It is unknown how S. suis virulent strains, which are usually found in low quantities in pig tonsils, manage to cross the first host defense lines to initiate systemic disease. Influenza virus produces a contagious infection in pigs which is frequently complicated by bacterial coinfections, leading to significant economic impacts. In this study, the effect of a preceding swine influenza H1N1 virus (swH1N1) infection of swine tracheal epithelial cells (NTPr) on the ability of S. suis serotype 2 to adhere to, invade, and activate these cells was evaluated. Cells preinfected with swH1N1 showed bacterial adhesion and invasion levels that were increased more than 100-fold compared to those of normal cells. Inhibition studies confirmed that the capsular sialic acid moiety is responsible for the binding to virus-infected cell surfaces. Also, preincubation of S. suis with swH1N1 significantly increased bacterial adhesion to/invasion of epithelial cells, suggesting that S. suis also uses swH1N1 as a vehicle to invade epithelial cells when the two infections occur simultaneously. Influenza virus infection may facilitate the transient passage of S. suis at the respiratory tract to reach the bloodstream and cause bacteremia and septicemia. S. suis may also increase the local inflammation at the respiratory tract during influenza infection, as suggested by an exacerbated expression of proinflammatory mediators in coinfected cells. These results give new insight into the complex interactions between influenza virus and S. suis in a coinfection model.

Exacerbated type II interferon response drives hypervirulence and toxic shock by an emergent epidemic strain of Streptococcus suis.

ABSTRACT Streptococcus suis, a major porcine pathogen, can be transmitted to humans and cause severe symptoms. A large human outbreak associated with an unusual streptococcal toxic shock-like syndrome (STSLS) was described in China. Albeit an early burst of proinflammatory cytokines following Chinese S. suis infection was suggested to be responsible for STSLS case severity, the mechanisms involved are still poorly understood. Using a mouse model, the host response to S. suis infection with a North American intermediately pathogenic strain, a European highly pathogenic strain, and the Chinese epidemic strain was investigated by a whole-genome microarray approach. Proinflammatory genes were expressed at higher levels in mice infected with the Chinese strain than those infected with the European strain. The Chinese strain induced a fast and strong gamma interferon (IFN-γ) response by natural killer (NK) cells. In fact, IFN-γ-knockout mice infected with the Chinese strain showed significantly better survival than wild-type mice. Conversely, infection with the less virulent North American strain resulted in an IFN-β-subjugated, low inflammatory response that might be beneficial for the host to clear the infection. Overall, our data suggest that a highly virulent epidemic strain has evolved to massively activate IFN-γ production, mainly by NK cells, leading to a rapid and lethal STSLS.

Group B Streptococcus and Streptococcus suis Capsular Polysaccharides Induce Chemokine Production by Dendritic Cells via Toll-Like Receptor 2- and MyD88-Dependent and -Independent Pathways

ABSTRACT Streptococcus agalactiae (also known as group B Streptococcus [GBS]) and Streptococcus suis are encapsulated streptococci causing severe septicemia and meningitis. Bacterial capsular polysaccharides (CPSs) are poorly immunogenic, but anti-CPS antibodies are essential to the host defense against encapsulated bacteria. The mechanisms underlying anti-CPS antibody responses are not fully elucidated, but the biochemistry of CPSs, particularly the presence of sialic acid, may have an immunosuppressive effect. We investigated the ability of highly purified S. suis and GBS native (sialylated) CPSs to activate dendritic cells (DCs), which are crucial actors in the initiation of humoral immunity. The influence of CPS biochemistry was studied using CPSs extracted from different serotypes within these two streptococcal species, as well as desialylated CPSs. No interleukin-1β (IL-1β), IL-6, IL-12p70, tumor necrosis factor alpha (TNF-α), or IL-10 production was observed in S. suis or GBS CPS-stimulated DCs. Moreover, these CPSs exerted immunosuppressive effects on DC activation, as a diminution of gamma interferon (IFN-γ)-induced B cell-activating factor of the tumor necrosis factor family (BAFF) expression was observed in CPS-pretreated cells. However, S. suis and GBS CPSs induced significant production of CCL3, via partially Toll-like receptor 2 (TLR2)- and myeloid differentiation factor 88 (MyD88)-dependent pathways, and CCL2, via TLR-independent mechanisms. No major influence of CPS biochemistry was observed on the capacity to induce chemokine production by DCs, indicating that DCs respond to these CPSs in a patterned way rather than a structure-dedicated manner.

Clearance of Infection with Mycobacterium bovis BCG in Mice Is Enhanced by Treatment with S28463 (R-848), and Its Efficiency Depends on Expression of Wild-Type Nramp1 (Resistance Allele)

ABSTRACT The mouse bcg host resistance gene is known to control the activation of host macrophages for killing of intracellular parasites like Leishmania donovani as well as intracellular bacteria, including Mycobacterium bovisBCG and Salmonella enterica serovar Typhimurium. TheNramp1 gene has been mapped to this locus and affects the efficiency of macrophage activation. It has been shown that imidazoquinoline compounds, including S28463, are able to improve the clearance of a number of intracellular pathogens such as herpes simplex virus 2, human papillomavirus, and Leishmania. The goal of this study was to determine whether S28463 is efficient against infection with another intracellular pathogen, M. bovisBCG, and to determine the molecular basis underlying this effect. To achieve this, B10A.Nramp1r and B10A.Nramp1−/− mice were infected with M. bovis BCG and treated with S28463. The bacterial content in the spleen from these mice was assayed by a colony-forming assay. In addition, in vitro experiments were performed using bone marrow-derived macrophage cell lines from these mice. These cells were treated with S28463 and/or gamma interferon (IFN-γ), and nitric oxide (NO) production was measured. Our study was able to show that S28463 acts in synergy with IFN-γ to increase the production of NO in vitro. We were also able to demonstrate that mice that carried the resistant allele of the Nramp1 gene and were infected withM. bovis BCG responded to treatment with S28463, resulting in a decreased bacterial load after 2 weeks of treatment. Mice that do not express the Nramp1 gene responded only to a very large dose of S28463, and the response was not as efficient as that observed in mice carrying a wild-type Nramp1allele. Our data provide evidence for the potential of S28463 as an immunomodulator that may be helpful in designing efficient strategies to improve host defense against mycobacterial infection.

Development of a two-step multiplex PCR assay for typing of capsular polysaccharide synthesis gene clusters of Streptococcus suis

ABSTRACT We developed a practical and easy two-step multiplex PCR assay to aid in serotyping of Streptococcus suis. The assay accurately typed almost all of the serotype reference strains and field isolates of various serotypes and also identified the genotypes of capsular polysaccharide synthesis gene clusters of some serologically nontypeable strains.

Toll-like receptor 2 is partially involved in the activation of murine astrocytes by Streptococcus suis, an important zoonotic agent of meningitis

The murine astrocyte response to virulent Streptococcus suis, a swine and an emerging human meningitis-causing pathogen, is reported. Albeit astrocytes do not internalize S. suis, all S. suis strains studied enhanced Toll-like receptor (TLR)2 expression and the production of pro-inflammatory cytokines and inducible nitric oxide synthase. Cell wall components and hemolysin (suilysin) are shown to be mainly responsible for cell activation. Astrocytes from TLR2 knockout mice presented a partial but significant reduction of S. suis-induced production of pro-inflammatory cytokines. These results contribute to increase the knowledge on mechanisms underlying S. suis inflammation in the brain.