Armelle Phalipon

Caspase-1 Activation of IL-1β and IL-18 Are Essential for Shigella flexneri–Induced Inflammation

Caspases are intracellular proteases that mediate mammalian cell apoptosis. Caspase-1 (Casp-1) is a unique caspase because it activates the proinflammatory cytokines interleukin (IL)-1beta and IL-18. Shigella flexneri, the etiological agent of bacillary dysentery, induces macrophage apoptosis, which requires Casp-1 and results in the release of mature IL-1beta and IL-18. Here we show that casp-1(-/-) mice infected with S. flexneri do not develop the acute inflammation characteristic of shigellosis and are unable to resolve the bacterial infection. Using casp-1(-/-) mice supplemented with recombinant cytokines and experiments with IL-1beta(-/-) and IL-18(-/-) mice, we show that IL-1beta and IL-18 are both required to mediate inflammation in S. flexneri infections. Together, these data demonstrate the importance of Casp-1 in acute inflammation and show the different roles of its substrates, IL-1beta and IL-18, in this response.

Secretory component: a new role in secretory IgA-mediated immune exclusion in vivo.

Secretory immunoglobulin (Ig) A (SIgA) is essential in protecting mucosal surfaces. It is composed of at least two monomeric IgA molecules, covalently linked through the J chain, and secretory component (SC). We show here that a dimeric/polymeric IgA (IgA(d/p)) is more efficient when bound to SC in protecting mice against bacterial infection of the respiratory tract. We demonstrate that SC ensures, through its carbohydrate residues, the appropriate tissue localization of SIgA by anchoring the antibody to mucus lining the epithelial surface. This in turn impacts the localization and the subsequent clearance of bacteria. Thus, SC is directly involved in the SIgA function in vivo. Therefore, binding of IgA(d/p) to SC during the course of SIgA-mediated mucosal response constitutes a crucial step in achieving efficient protection of the epithelial barrier by immune exclusion.

Shigella’ s ways of manipulating the host intestinal innate and adaptive immune system: a tool box for survival?

Shigella, a Gram‐negative invasive enteropathogenic bacterium, causes the rupture, invasion and inflammatory destruction of the human colonic epithelium. This complex and aggressive process accounts for the symptoms of bacillary dysentery. The so‐called invasive phenotype of Shigella is linked to expression of a type III secretory system (TTSS) injecting effector proteins into the epithelial cell membrane and cytoplasm, thereby inducing local but massive changes in the cell cytoskeleton that lead to bacterial internalization into non‐phagocytic intestinal epithelial cells. The invasive phenotype also accounts for the potent pro‐inflammatory capacity of the microorganism. Recent evidence indicates that a large part of the mucosal inflammation is initiated by intracellular sensing of bacterial peptidoglycan by cytosolic leucine‐rich receptors of the NOD family, particularly NOD1, in epithelial cells. This causes activation of the nuclear factor kappa B and c‐JunNH2‐terminal‐kinase pathways, with interleukin‐8 appearing as a major chemokine mediating the inflammatory burst that is dominated by massive infiltration of the mucosa by polymorphonuclear leukocytes. Not unexpectedly, this inflammatory response, which is likely to be very harmful for the invading microbe, is regulated by the bacterium itself. A group of proteins encoded by Shigella, which are injected into target cells by the TTSS, has been recently recognized as a family of potent regulators of the innate immune response. These enzymes target key cellular functions that are essential in triggering the inflammatory response, and more generally defense responses of the intestinal mucosa. This review focuses on the mechanisms employed by Shigella to manipulate the host innate response in order to escape early bacterial killing, thus ensuring establishment of its infectious process. The escape strategies, the possible direct effect of Shigella on B and T lymphocytes, their impact on the development of adaptive immunity, and how they may help explain the limited protection induced by natural infection are discussed.

Anti-inflammatory role for intracellular dimeric immunoglobulin a by neutralization of lipopolysaccharide in epithelial cells.

Intestinal epithelial cells (IEC) play a central role in innate and acquired mucosal immunity. They ensure early signaling to trigger an inflammatory response against pathogens. Moreover, IEC mediate transcytosis of dimeric IgA (dIgA), through the polymeric-immunoglobulin receptor (pIgR), to provide secretory IgA, the major protective Ig in mucosal secretions. Using an in vitro model of polarized IEC, we describe an additional anti-inflammatory mechanism of dIgA-mediated protection against intracellular bacterial components involved in the proinflammatory activation of IEC. Specific dIgA colocalizes to lipopolysaccharide (LPS) in the apical recycling endosome compartment, preventing LPS-induced NF-kappaB translocation and subsequent proinflammatory response. Thus, intracellular neutralization by dIgA limits the acute local inflammation induced by proinflammatory pathogen-associated molecular patterns such as LPS.

Secretory IgA-mediated neutralization of Shigella flexneri prevents intestinal tissue destruction by down-regulating inflammatory circuits.

Shigella, a Gram-negative invasive enteropathogenic bacterium responsible for bacillary dysentery, causes the rupture, invasion, and inflammatory destruction of the human colonic mucosa. We explored the mechanisms of protection mediated by Shigella LPS-specific secretory IgA (SIgA), the major mucosal Ab induced upon natural infection. Bacteria, SIgA, or SIgA-S. flexneri immune complexes were administered into rabbit ligated intestinal loops containing a Peyer’s patch. After 8 h, localizations of bacteria, SIgA, and SIgA-S. flexneri immune complexes were examined by immunohistochemistry and confocal microscopy imaging. We found that anti-Shigella LPS SIgA, mainly via immune exclusion, prevented Shigella-induced inflammation responsible for the destruction of the intestinal barrier. Besides this luminal trapping, a small proportion of SIgA-S. flexneri immune complexes were shown to enter the rabbit Peyer’s patch and were internalized by dendritic cells of the subepithelial dome region. Local inflammatory status was analyzed by quantitative RT-PCR using newly designed primers for rabbit pro- and anti-inflammatory mediator genes. In Peyer’s patches exposed to immune complexes, limited up-regulation of the expression of proinflammatory genes, including TNF-α, IL-6, Cox-2, and IFN-γ, was observed, consistent with preserved morphology. In contrast, in Peyer’s patches exposed to Shigella alone, high expression of the same mediators was measured, indicating that neutralizing SIgA dampens the proinflammatory properties of Shigella. These results show that in the form of immune complexes, SIgA guarantees both immune exclusion and neutralization of translocated bacteria, thus preserving the intestinal barrier integrity by preventing bacterial-induced inflammation. These findings add to the multiple facets of the noninflammatory properties of SIgA.

Characterization of Functional Oligosaccharide Mimics of the Shigella flexneri Serotype 2a O-Antigen: Implications for the Development of a Chemically Defined Glycoconjugate Vaccine

Protection against reinfection with noncapsulated Gram-negative bacteria, such as Shigella, an enteroinvasive bacterium responsible for bacillary dysentery, is mainly achieved by Abs specific for the O-Ag, the polysaccharide part of the LPS, the major bacterial surface Ag. The use of chemically defined glycoconjugates encompassing oligosaccharides mimicking the protective determinants carried by the O-Ag, thus expected to induce an efficient anti-LPS Ab response, has been considered an alternative to detoxified LPS-protein conjugate vaccines. The aim of this study was to identify such functional oligosaccharide mimics of the S. flexneri serotype 2a O-Ag. Using protective murine mAbs specific for S. flexneri serotype 2a and synthetic oligosaccharides designed to analyze the contribution of each sugar residue of the branched pentasaccharide repeating unit of the O-Ag, we demonstrated that the O-Ag exhibited an immunodominant serotype-specific determinant. We also showed that elongating the oligosaccharide sequence improved Ab recognition. From these antigenicity data, selected synthetic oligosaccharides were assessed for their potential to mimic the O-Ag by analyzing their immunogenicity in mice when coupled to tetanus toxoid via single point attachment. Our results demonstrated that induction of an efficient serotype 2a-specific anti-O-Ag Ab response was dependent on the length of the oligosaccharide sequence. A pentadecasaccharide representing three biological repeating units was identified as a potential candidate for further development of a chemically defined glycoconjugate vaccine against S. flexneri 2a infection.

A synthetic carbohydrate-protein conjugate vaccine candidate against Shigella flexneri 2a infection.

The protective Ag of Shigella, the Gram-negative enteroinvasive bacterium causing bacillary dysentery, or shigellosis, is its O-specific polysaccharide (O-SP) domain of the LPS, the major bacterial surface component. As an alternative to the development of detoxified LPS-based conjugate vaccines, recent effort was put into the investigation of neoglycoproteins encompassing synthetic oligosaccharides mimicking the protective Ags of the O-SP. We previously reported that when coupled to tetanus toxoid via single point attachment, a synthetic pentadecasaccharide representing three biological repeating units of the O-SP of Shigella flexneri 2a (SF2a), one of the most common Shigella serotypes, elicits a better serum anti-LPS 2a Ab response in mice than shorter synthetic O-SP sequences. In this study, we show that the pentadecasaccharide-induced anti-LPS 2a Abs protect passively administered naive mice from Shigella infection. Therefore, this three repeating units sequence, which is recognized by anti-SF2a sera from infected patients, acts as a functional mimic of the native polysaccharide Ag. Analyses of parameters influencing immunogenicity revealed that an investigational SF2a vaccine displaying a pentadecasaccharide:tetanus toxoid molar loading of 14:1 triggers a high and sustained anti-LPS Ab response, without inducing anti-linker Ab, when administered four times at a dose corresponding to 1 μg of carbohydrate. In addition, the profile of the anti-LPS Ab response, dominated by IgG1 production (Th2-type response), mimics that observed in human upon natural SF2a infection. This synthetic carbohydrate-based conjugate may be a candidate for a SF2a vaccine.

Roles for T and NK Cells in the Innate Immune Response to Shigella flexneri

Shigella flexneri, an enteroinvasive Gram-negative bacterium, is responsible for the worldwide endemic form of bacillary dysentery. The host response to primary infection is characterized by the induction of an acute inflammation, which is accompanied by polymorphonuclear cell (PMN) infiltration, resulting in massive destruction of the colonic mucosa. However, PMN play a major role in the recovery from primary infection, by restricting the bacterial infection at the intestinal mucosa. In this study, we assessed the roles for T and NK cells in the control of primary S. flexneri infection, using an alymphoid mouse strain (Rag°γc°) devoid of B, T, and NK cells. Using the mouse pulmonary model of Shigella infection, we showed that alymphoid Rag°γc° mice were highly susceptible to S. flexneri infection in comparison with wild-type (wt) mice. Whereas PMN recruitment upon infection was similar, macrophage recruitment and production of proinflammatory cytokines were significantly decreased in Rag°γc° mice compared with wt mice. Upon selective engraftment of Rag°γc° mice with polyclonal αβ T cells, but not with αβ T cells from IFN-γ°, S. flexneri infection could be subsequently controlled. Rag° mice devoid of B and T cells but harboring NK cells could control infection. Local IFN-γ production by T and NK cells recruited to the lung was demonstrated in S. flexneri-infected wt mice. These data demonstrate that both αβ T cells and NK cells contribute to the early control of S. flexneri infection through amplification of an inflammatory response. This cellular lymphocyte redundancy assures IFN-γ production, which is central to innate immunity against Shigella infection.

Th17 Cells Are the Dominant T Cell Subtype Primed by Shigella flexneri Mediating Protective Immunity

The T cell response to Shigella, the causative agent of bacillary dysentery, remains poorly understood. Using a murine model of infection, we report that Shigella flexneri primes predominately IL-17A– and IL-22–producing Th17 cells. Shigella-specific Th1 cells are only significantly induced on secondary infection, whereas specific Th2 and CD8+ T cells are undetectable. Apart from Th17 cells that are primed in a MHC class II- and IL-6–dependent, but IL12/23p40-independent manner, we identified γδ T cells as an additional but minor source of IL-17A. Priming of IL-17A+ γδ T cells is dependent on IL12/23p40, but independent of MHC-class II and IL-6. Th17 cells have emerged as important players in inflammatory, autoimmune, and infectious diseases. Among the yet unresolved questions is their role in long-term immunity to pathogens. In this study, we show that the elicited S. flexneri-specific Th17 pool gives rise to an enhanced recall response up to 12 mo after priming, suggesting the presence of a long-term memory state. The clearance of primary infection is impaired in the absence of T cells, but independently of IL-17A. However, after reinfection, IL-17A produced by S. flexneri-specific Th17 cells becomes important to ultimately restrict bacterial growth. These findings bring new insights into the adaptive immune response to Shigella infection and highlight the importance of pathogen-specific Th17 cell immunity for secondary immune protection.

A Pathogen-specific Epitope Inserted into Recombinant Secretory Immunoglobulin A Is Immunogenic by the Oral Route

Oral administration of rabbit secretory IgA (sIgA) to adult BALB/c mice induced IgA+, IgM+, and IgG+ lymphoblasts in the Peyers patches, whose fusion with myeloma cells resulted in hybridomas producing IgA, IgM, and IgG1 antibodies to the secretory component (SC). This suggests that SC could serve as a vector to target protective epitopes into mucosal lymphoid tissue and elicit an immune response. We tested this concept by inserting a Shigella flexneri invasin B epitope into SC, which, following reassociation with IgA, was delivered orally to mice. To identify potential insertion sites at the surface of SC, we constructed a molecular model of the first and second Ig-like domains of rabbit SC. A surface epitope recognized by an SC-specific antibody was mapped to the loop connecting the E and F β strands of domain I. This 8-amino acid sequence was replaced by a 9-amino acid linear epitope from S. flexneri invasin B. We found that cellular trafficking of recombinant SC produced in mammalian CV-1 cells was drastically altered and resulted in a 50-fold lower rate of secretion. However, purification of chimeric SC could be achieved by Ni2+-chelate affinity chromatoraphy. Both wild-type and chimeric SC bound to dimeric IgA, but not to monomeric IgA. Reconstituted sIgA carrying the invasin B epitope within the SC moiety triggers the appearance of seric and salivary invasin B-specific antibodies. Thus, neo-antigenized sIgA can serve as a mucosal vaccine delivery system inducing systemic and mucosal immune responses.