Antibody response specific to the capsular polysaccharide is impaired in Streptococcus suis serotype 2-infected animals.

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.

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

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-ß-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.

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-1ß (IL-1ß), 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.

The NOD2 receptor does not play a major role in the pathogenesis of Group B Streptococcus in mice.

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 this internalization has an impact on cytokine production. The intracellular receptors or pathways underlying this response are not well understood. In this work, we investigated the role of NOD2 in the pathogenesis of GBS using a mouse model of infection. NOD2(-/-) mice showed similar levels of survival and bacteremia than control mice. Interestingly, ex vivo analysis of total spleen cells from infected animals showed that the absence of NOD2 results in reduced production of inflammatory cytokines. However this abridged inflammatory response does not seem to improve mouse survival. In conclusion, we demonstrated that NOD2 is not a crucial receptor to fight GBS infection and only partially contributes to the inflammatory 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.

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.

Nod1 promotes colorectal carcinogenesis by regulating the immunosuppressive functions of tumor-infiltrating myeloid cells.

Pioneering studies from the early 1980s suggested that bacterial peptidoglycan-derived muramyl peptides (MPs) could exert either stimulatory or immunosuppressive functions depending, in part, on chronicity of exposure. However, this Janus-faced property of MPs remains largely unexplored. Here, we demonstrate the immunosuppressive potential of Nod1, the bacterial sensor of diaminopimelic acid (DAP)-containing MPs. Using a model of self-limiting peritonitis, we show that systemic Nod1 activation promotes an autophagy-dependent reprogramming of macrophages toward an alternative phenotype. Moreover, Nod1 stimulation induces the expansion of myeloid-derived suppressor cells (MDSCs) and maintains their immunosuppressive potential via arginase-1 activity. Supporting the role of MDSCs and tumor-associated macrophages in cancer, we demonstrate that myeloid-intrinsic Nod1 expression sustains intra-tumoral arginase-1 levels to foster an immunosuppressive and tumor-permissive microenvironment during colorectal cancer (CRC) development. Our findings support the notion that bacterial products, via Nod1 detection, modulate the immunosuppressive activity of myeloid cells and fuel tumor progression in CRC.

Comparison of Co-housing and Littermate Methods for Microbiota Standardization in Mouse Models.

The intestinal microbiota is a fundamental factor that broadly influences physiology. Thus, studies using transgenic animals should be designed to limit the confounding effects of microbiota variation between strains. Here, we report the impact on intestinal microbiota of co-housed versus F2-generation littermates, two commonly used techniques to standardize microbiota in animal models. Our results establish that while fecal microbiota is partially normalized by extended co-housing, mucosal communities associated with the proximal colon and terminal ileum remain stable and distinct. In contrast, strain inter-crossing to generate F2 littermates allows robust microbiota standardization in fecal, colon, and ileum sampling locations. Using reciprocal inter-crosses of P1 parents, we identify dissymmetry in F2 community structures caused by maternal transmission, in particular of the Verrucomicrobiaceae. Thus, F2 littermate animals from a unidirectional P1 cross should be used as a standard method to minimize the influence of the microbiota in genotype-phenotype studies.

Streptococcus suis Serotype 2 Infection Impairs Interleukin-12 Production and the MHC-II-Restricted Antigen Presentation Capacity of Dendritic Cells.

Streptococcus suis is an important swine pathogen and emerging zoonotic agent. Encapsulated strains of S. suis modulate dendritic cell (DC) functions, leading to poorly activated CD4+ T cells. However, the antigen presentation ability of S. suis-stimulated DCs has not been investigated yet. In this work, we aimed to characterize the antigen presentation profiles of S. suis-stimulated DCs, both in vitro and in vivo. Upon direct activation in vitro, S. suis-stimulated murine bone marrow-derived DCs (bmDCs) preserved their antigen capture/processing capacities. However, they showed delayed kinetics of MHC-II expression compared to lipopolysaccharide-stimulated bmDCs. Meanwhile, splenic DCs from infected mice exhibited a compromised MHC-II expression, despite an appropriate expression of maturation markers. To identify potential interfering mechanisms, Class II Major Histocompatibility Complex Transactivator (CIITA) and membrane-associated RING-CH (MARCH)1/8 transcription were studied. S. suis-stimulated DCs maintained low levels of CIITA at early time points, both in vitro and in vivo, which could limit their ability to increase MHC-II synthesis. S. suis-stimulated DCs also displayed sustained/upregulated levels of MARCH1/8, thus possibly leading to MHC-II lysosomal degradation. The bacterial capsular polysaccharide played a partial role in this modulation. Finally, interleukin (IL)-12p70 production was inhibited in splenic DCs from infected mice, a profile compatible with DC indirect activation by pro-inflammatory compounds. Consequently, these cells induced lower levels of IL-2 and TNF-α in an antigen-specific CD4+ T cell presentation assay and blunted T cell CD25 expression. It remains unclear at this stage whether these phenotypical and transcriptional modulations observed in response to S. suis in in vivo infections are part of a bacterial immune evasion strategy or rather a feature common to systemic inflammatory response-inducing agents. However, it appears that the MHC-II-restricted antigen presentation and Th1-polarizing cytokine production capacities of DCs are impaired during S. suis infection. This study highlights the potential consequences of inflammation on the type and magnitude of the immune response elicited by a pathogen.

Natural Killer Cell Functions during the Innate Immune Response to Pathogenic Streptococci.

Dendritic cells (DCs) and NK cells play a crucial role in the first phase of host defense against infections. Group B Streptococcus (GBS) and Streptococcus suis are encapsulated streptococci causing severe systemic inflammation, leading to septicemia and meningitis. Yet, the involvement of NK cells in the innate immune response to encapsulated bacterial infection is poorly characterized. Here, it was observed that these two streptococcal species rapidly induce the release of IFN-γ and that NK cells are the major cell type responsible for this production during the acute phase of the infection. Albeit S. suis capacity to activate NK cells was lower than that of GBS, these cells partially contribute to S. suis systemic infection; mainly through amplification of the inflammatory loop. In contrast, such a role was not observed during GBS systemic infection. IFN-γ release by NK cells required the presence of DCs, which in turn had a synergistic effect on DC cytokine production. These responses were mainly mediated by direct DC-NK cell contact and partially dependent on soluble factors. Though IL-12 and LFA-1 were shown to be critical in S. suis-mediated activation of the DC-NK cell crosstalk, different or redundant molecular pathways modulate DC-NK interactions during GBS infection. The bacterial capsular polysaccharides also differently modulated NK cell activation. Together, these results demonstrated a role of NK cells in the innate immune response against encapsulated streptococcal infections; yet the molecular pathways governing NK activation seem to differ upon the pathogen and should not be generalized when studying bacterial infections.

The NLR Protein NLRP6 Does Not Impact Gut Microbiota Composition.

NLRP6 is a Nod-like receptor expressed in the intestinal epithelium. Previous studies reported a protective role for NLRP6 against intestinal injury and colitis-associated carcinogenesis via the regulation and establishment of a healthy microbiota. However, these results were not obtained using littermate animals, leaving the possibility that the pro-colitogenic microbiota phenotype associated with knockout (KO) mice was stochastically acquired and genotype independent. Here, we analyzed the microbiota at three intestinal locations from Nlrp6-/- and wild-type (WT) littermates, either co-caged or individually caged after weaning. Our results demonstrate that NLRP6 does not significantly influence the intestinal microbiota at homeostasis, and they support a previously reported sex-biased microbial community structure. Moreover, WT and Nlrp6-/- littermate mice displayed comparable sensitivity to dextran sulfate sodium (DSS)-induced colitis, although increased sensitivity was noted in KO females. Our results clarify the role of NLRP6 in microbiota and colitis control, and they highlight the importance of analyzing littermate animals in such studies.

Group B Streptococcus Induces a Robust IFN-γ Response by CD4(+) T Cells in an In Vitro and In Vivo Model.

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.

The common mouse protozoa Tritrichomonas muris alters mucosal T cell homeostasis and colitis susceptibility.

The mammalian gastrointestinal tract hosts a diverse community of microbes including bacteria, fungi, protozoa, helminths, and viruses. Through coevolution, mammals and these microbes have developed a symbiosis that is sustained through the host's continuous sensing of microbial factors and the generation of a tolerant or pro-inflammatory response. While analyzing T cell-driven colitis in nonlittermate mouse strains, we serendipitously identified that a nongenetic transmissible factor dramatically increased disease susceptibility. We identified the protozoan Tritrichomonas muris as the disease-exacerbating element. Furthermore, experimental colonization with T. muris induced an elevated Th1 response in the cecum of naive wild-type mice and accelerated colitis in Rag1-/- mice after T cell transfer. Overall, we describe a novel cross-kingdom interaction within the murine gut that alters immune cell homeostasis and disease susceptibility. This example of unpredicted microbial priming of the immune response highlights the importance of studying trans-kingdom interactions and serves as a stark reminder of the importance of using littermate controls in all mouse research.

Genomic Recombination Leading to Decreased Virulence of Group B Streptococcus in a Mouse Model of Adult Invasive Disease.

Adult invasive disease caused by Group B Streptococcus (GBS) is increasing worldwide. Whole-genome sequencing (WGS) now permits rapid identification of recombination events, a phenomenon that occurs frequently in GBS. Using WGS, we described that strain NGBS375, a capsular serotype V GBS isolate of sequence type (ST)297, has an ST1 genomic background but has acquired approximately 300 kbp of genetic material likely from an ST17 strain. Here, we examined the virulence of this strain in an in vivo model of GBS adult invasive infection. The mosaic ST297 strain showed intermediate virulence, causing significantly less systemic infection and reduced mortality than a more virulent, serotype V ST1 isolate. Bacteremia induced by the ST297 strain was similar to that induced by a serotype III ST17 strain, which was the least virulent under the conditions tested. Yet, under normalized bacteremia levels, the in vivo intrinsic capacity to induce the production of pro-inflammatory cytokines was similar between the ST297 strain and the virulent ST1 strain. Thus, the diminished virulence of the mosaic strain may be due to reduced capacity to disseminate or multiply in blood during a systemic infection which could be mediated by regulatory factors contained in the recombined region.

Immune-responsiveness of CD4+ T cells during Streptococcus suis serotype 2 infection.

The pathogenesis of Streptococcus suis infection, a major swine and human pathogen, is only partially understood and knowledge on the host adaptive immune response is critically scarce. Yet, S. suis virulence factors, particularly its capsular polysaccharide (CPS), enable this bacterium to modulate dendritic cell (DC) functions and potentially impair the immune response. This study aimed to evaluate modulation of T cell activation during S. suis infection and the role of DCs in this response. S. suis-stimulated total mouse splenocytes readily produced TNF-α, IL-6, IFN-γ, CCL3, CXCL9, and IL-10. Ex vivo and in vivo analyses revealed the involvement of CD4+ T cells and a Th1 response. Nevertheless, during S. suis infection, levels of the Th1-derived cytokines TNF-α and IFN-γ were very low. A transient splenic depletion of CD4+ T cells and a poor memory response were also observed. Moreover, CD4+ T cells secreted IL-10 and failed to up-regulate optimal levels of CD40L and CD69 in coculture with DCs. The CPS hampered release of several T cell-derived cytokines in vitro. Finally, a correlation was established between severe clinical signs of S. suis disease and impaired antibody responses. Altogether, these results suggest S. suis interferes with the adaptive immune response.

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.

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.