Paul Lemire

Critical Role for Streptococcus suis Cell Wall Modifications and Suilysin in Resistance to Complement-Dependent Killing by Dendritic Cells

Streptococcus suis is an emerging zoonotic agent of septicemia and meningitis. Knowledge on host immune responses toward S. suis and strategies used by this pathogen for subversion of these responses is scarce. Here, S. suis modulation of dendritic cell (DC) functions were assessed for the first time. Using S. suis knockout mutants in capsular polysaccharide (CPS) expression, it was shown that CPS blocks DC phagocytosis and impairs cytokine release by hindering cell wall components. Mutants impaired in D-alanylation of lipoteichoic acid (LTA) or N-deacetylation of peptidoglycan (PG) further demonstrated the importance of cell wall in modulation of DC activation. Notably, LTA/PG modifications were identified as major players in resistance to complement-dependent killing by DCs. Finally, S. suis hemolysin was partially involved in cytokine release and also contributed to bacterial escape of opsonophagocytosis. Overall, S. suis uses its arsenal of virulence factors to modulate DC functions and escape immune surveillance.

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.

Role of capsular polysaccharide in Group B Streptococccus interactions with dendritic cells

Group B Streptococcus (GBS) type III is an important agent of life-threatening invasive infections. Albeit the immune system plays a dual role in development and protection against disease, mechanisms leading to an efficient immune response against GBS remain obscure. Mouse bone marrow-derived dendritic cells (DCs) and primary spleen DCs were used to evaluate GBS capacity to modulate the functions of these important antigen-presenting cells. The role of capsular polysaccharide (CPS), one of the most important GBS virulence factors, in bacterial-DC interactions was evaluated by using a non-encapsulated mutant. Phagocytosis assays, confocal and electron microscopy showed that DCs efficiently internalize encapsulated GBS, but the latter possesses strong intracellular survival capacity. GBS devoid of CPS was internalized and killed at higher and faster rates than encapsulated GBS early after infection. Among several cytokines tested, GBS internalization was required for modulation of IL-12, IL-10 and CXCL10 pathways. In contrast, GBS induced DC expression of co-stimulatory molecules in a phagocytosis-independent manner. Finally, the production of pro-inflammatory and Th1 cytokines by GBS-stimulated DCs was differentially modulated by CPS expression, depending on DC origin. Our data suggest multiple mechanisms involved in GBS modulation of DC functions, which were selectively regulated by the presence of CPS.

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.

Antibody Response Specific to the Capsular Polysaccharide Is Impaired in Streptococcus suis Serotype 2-Infected Animals

ABSTRACT Streptococcus suis serotype 2 is an extracellular encapsulated bacterium that causes severe septicemia and meningitis in swine and humans. Albeit crucial in the fight against encapsulated bacteria, the nature of the capsular polysaccharide (CPS)-specific antibody (Ab) response during S. suis type 2 infection is unknown. We compared for the first time the features of CPS-specific versus protein-specific Ab responses during experimental infections with live virulent S. suis type 2 in mice. The primary protein-specific Ab response was dominated by both type 1 and 2 IgG subclasses, whereas IgM titers were more modest. The secondary protein-specific Ab response showed all of the features of a memory response with faster kinetics and boosted the titers of all Ig isotypes. In contrast, the primary CPS-specific Ab response was either inexistent or had titers only slightly higher than those in noninfected animals and was essentially composed of IgM. A poor CPS-specific memory response was observed, with only a moderate boost in IgM titers and no IgG. Both protein- and CPS-specific Ab responses were Toll-like receptor 2 independent. By using S. suis type 2 strains of European or North American origin, the poor CPS-specific Ab response was demonstrated to be independent of the genotypic/phenotypic diversity of the strain within serotype 2. Finally, the CPS-specific Ab response was also impaired and lacked isotype switching in S. suis-infected pigs, the natural host of the bacterium. The better resistance of preinfected animals to reinfection with the same strain of S. suis type 2 might thus more likely be related to the development of a protein rather than CPS Ab response.

Encapsulated group B Streptococcus modulates dendritic cell functions via lipid rafts and clathrin-mediated endocytosis

Group B Streptococcus (GBS) capsular type III is an important agent of life‐threatening invasive infections. It has been previously shown that encapsulated GBS is easily internalized by dendritic cells (DCs) and can persist inside these immune cells. The mechanisms underlying these processes are unknown. Here, colocalization studies and the use of endocytosis inhibitors and caveolin−/− mice, demonstrated that GBS uses multiple endocytosis mechanisms to enter mouse DCs. The capsular polysaccharide (CPS) selectively drives GBS internalization via caveolae‐independent but lipid raft‐dependent pathways. Non‐encapsulated bacteria failed to engage lipid rafts. GBS internalization by DCs also occurs via clathrin‐mediated endocytosis in a process independent of bacterial CPS. Albeit caveolae are not required for GBS internalization, signalling events through caveolin‐1 are involved in production of the inflammatory chemokine CCL2 by DCs infected with encapsulated GBS only. This study addresses for the first time endocytosis pathways implicated in DC internalization of encapsulated GBS and suggests a complex interplay between GBS and DCs, which was selectively modulated by the presence of CPS.

Implication of TLR- but Not of NOD2-Signaling Pathways in Dendritic Cell Activation by Group B Streptococcus Serotypes III and V

Group B Streptococcus (GBS) is an important agent of life-threatening invasive infection. It has been previously shown that encapsulated type III GBS is easily internalized by dendritic cells (DCs), and that this internalization had an impact on cytokine production. The receptors underlying these processes are poorly characterized. Knowledge on the mechanisms used by type V GBS to activate DCs is minimal. In this work, we investigated the role of Toll-like receptor (TLR)/MyD88 signaling pathway, the particular involvement of TLR2, and that of the intracellular sensing receptor NOD2 in the activation of DCs by types III and V GBS. The role of capsular polysaccharide (CPS, one of the most important GBS virulence factors) in bacterial-DC interactions was evaluated using non-encapsulated mutants. Despite differences in the role of CPS between types III and V GBS in bacterial internalization and intracellular survival, no major differences were observed in their capacity to modulate release of cytokines by DC. For both serotypes, CPS had a minor role in this response. Production of cytokines by DCs was shown to strongly rely on MyD88-dependent signaling pathways, suggesting that DCs recognize GBS and become activated mostly through TLR signaling. Yet, GBS-infected TLR2-/- DCs only showed a partial reduction in the production of IL-6 and CXCL1 compared to control DCs. Surprisingly, CXCL10 release by type III or type V GBS-infected DCs was MyD88-independent. No differences in DC activation were observed between NOD2-/- and control DCs. These results demonstrate the involvement of various receptors and the complexity of the cytokine production pathways activated by GBS upon DC infection.

Differentiation of equine induced pluripotent stem cells into a keratinocyte lineage

REASONS FOR PERFORMING STUDY Skin trauma in horses often leads to the development of chronic nonhealing wounds that lack a keratinocyte cover, vital for healing. Reports in mouse and man confirm the possibility of generating functional keratinocytes from induced pluripotent stem cells (iPSC), thus presenting myriad potential applications for wound management or treatment of skin disease. Similarly, differentiation of equine iPSC (eiPSC) into a keratinocyte lineage should provide opportunities for the advancement of veterinary regenerative medicine. OBJECTIVES The purpose of this study was to develop an efficient method for the differentiation of eiPSC into a keratinocyte lineage. It was hypothesised that eiPSC can form differentiated keratinocytes (eiPSC-KC) comparable with primary equine keratinocytes (PEK) in their morphological and functional characteristics. STUDY DESIGN Experimental in vitro study. METHODS Equine iPSC established using a nonviral system were treated for 30 days with retinoic acid and bone morphogenetic protein-4 to induce directed differentiation into iPSC-KC. Temporospatial gene and protein expression by eiPSC-KC was measured at weekly intervals of differentiation and in response to calcium switch. Proliferative and migratory capacities of eiPSC-KC were compared with those of PEK. RESULTS Equine iPSC, upon directed differentiation, showed loss of pluripotency genes and progressive increase in pancytokeratin expression indicating ectodermal specification into keratinocytes. High differentiation efficiency was achieved, with 82.5% of eiPSC expressing keratin 14, a marker of epidermal-specific basal stem cells, after 30 days of directed differentiation. Moreover, the proliferative capacity of eiPSC-KC was superior, while the migratory capacity (measured as the ability to epithelise in vitro wounds) was comparable with that of PEK. CONCLUSIONS This proof of concept study suggests that eiPSC can successfully be differentiated into equine keratinocytes (eiPSC-KC) with features that are promising to the development of a stem cell-based skin construct, with the potential to regenerate lost or damaged skin.

Group BStreptococcusInduces a Robust IFN-γResponse by CD4+T Cells in anIn VitroandIn VivoModel

Group B Streptococcus (GBS) serotype III causes life-threatening infections. Cytokines have emerged as important players for the control of disease, particularly IFN-γ. Although potential sources of this cytokine have been proposed, no specific cell line has ever been described as a leading contributor. In this study, CD4+ T cell activation profiles in response to GBS were evaluated through in vivo, ex vivo, and in vitro approaches. Total splenocytes readily produce a type 1 proinflammatory response by releasing IFN-γ, TNF-α, and IL-6 and actively recruit T cells via chemokines like CXCL9, CXCL10, and CCL3. Responding CD4+ T cells differentiate into Th1 cells producing large amounts of IFN-γ, TNF-α, and IL-2. In vitro studies using dendritic cell and CD4+ T cell cocultures infected with wild-type GBS or a nonencapsulated mutant suggested that GBS capsular polysaccharide, one of the major bacterial virulence factors, differentially modulates surface expression of CD69 and IFN-γ production. Overall, CD4+ T cells are important producers of IFN-γ and might thus influence the course of GBS infection through the expression balance of this cytokine.

Immune potential of allogeneic equine induced pluripotent stem cells.

REASONS FOR PERFORMING STUDY Induced pluripotent stem cells (iPSC) have brought immense hope to cellular therapy and regenerative medicine. However, the antigenicity of iPSC has not been well documented and remains a hurdle for clinical applications. Expression of major histocompatibility complex (MHC) molecules by human and murine iPSC is downregulated, making these cells potentially safe for transplantation. No such data are available for any large animal model. OBJECTIVES To measure expression of MHC molecules on equine iPSC (eiPSC) and describe their antigenicity using intradermal testing. The hypothesis was that allogeneic eiPSC weakly express MHC molecules and would not elicit a rejection response when injected intradermally. STUDY DESIGN Experimental study involving both in vitro and in vivo components. METHODS Two green fluorescent protein-expressing eiPSC lines were analysed by flow cytometry for MHC expression. One line was then transplanted intradermally, along with appropriate controls, into 2 unrelated experimental horses. Blood was collected pre- and 7 days post transplantation. The wheals formed at the sites of injection were measured at regular intervals beginning at 0.25 h until 4 weeks. Tissue samples of the injected sites were obtained at 2, 3, 7 and 30 days post transplantation and analysed by histopathology and immunofluorescence. RESULTS Both eiPSC lines weakly expressed MHC molecules. eiPSC were detectable up to 7 days following allogeneic transplantation and elicited no apparent systemic response. Injection of eiPSC caused small wheal formation at the skin surface. Skin sections revealed CD4(+) and CD8(+) mononuclear cells up to 30 days post transplantation. CONCLUSIONS These data suggest that while transplantation of allogeneic eiPSC elicits a moderate cellular response, it does not cause acute rejection. The feasibility of banking allogeneic iPSC for regenerative medicine applications should be explored.