Long-term persistence of immunity induced by OVA-coupled gas-filled microbubble vaccination partially protects mice against infection by OVA-expressing Listeria

Vaccination aims at generating memory immune responses able to protect individuals against pathogenic challenges over long periods of time. Subunit vaccine formulations based on safe, but poorly immunogenic, antigenic entities must be combined with adjuvant molecules to make them efficient against infections. We have previously shown that gas-filled microbubbles (MB) are potent antigen-delivery systems. This study compares the ability of various ovalbumin-associated MB (OVA-MB) formulations to induce antigen-specific memory immune responses and evaluates long-term protection toward bacterial infections. When initially testing dendritic cells reactivity to MB constituents, palmitic acid exhibited the highest degree of activation. Subcutaneous immunization of naïve wild-type mice with the OVA-MB formulation comprising the highest palmitic acid content and devoid of PEG2000 was found to trigger the more pronounced Th1-type response, as reflected by robust IFN-γ and IL-2 production. Both T cell and antibody responses persisted for at least 6 months after immunization. At that time, systemic infection with OVA-expressing Listeria monocytgenes was performed. Partial protection of vaccinated mice was demonstrated by reduction of the bacterial load in both the spleen and liver. We conclude that antigen-bound MB exhibit promising properties as a vaccine candidate ensuring prolonged maintenance of protective immunity.     

Histamine and mast cell activator compound 48/80 are safe but inefficient systemic adjuvants for gilthead seabream vaccination

Histamine has a key role in the regulation of inflammatory and innate immune responses in vertebrates. Gilthead seabream (Sparus aurata L.), a marine hermaphrodite teleost of great commercial value, was the first fish species shown to possess histamine-containing mast cells (MCs) at mucosal tissues. MCs are highly abundant in the peritoneal exudate of gilthead seabream and compound 48/80 (Co 48/80), often used to promote MC activation and histamine release, is able to promote histamine release from gilthead seabream MCs in vitro and in vivo. The aim of the present study was to analyze the effect of histamine and Co 48/80 on the immune responses of gilthead seabream. For this purpose, histamine and Co 48/80 were intraperitoneally injected alone or combined with 10⁹ heat-killed Vibrio anguillarum cells and their effects on head kidney and peritoneal exudate were analyzed. The results indicated that although histamine and Co 48/80 were both able to alter the percentage of peritoneal exudate and head kidney immune cell types, only Co 48/80 increased reactive oxygen species production by peritoneal leukocytes. In addition, histamine, but not Co 48/80, was able to slightly impair the humoral adaptive immune response, i.e. production of specific IgM to V. anguillarum. Notably, both histamine and Co 48/80 reduced the expression of the gene encoding histamine receptor H2 in peritoneal exudate leukocytes. These results show for the first time in fish that although systemic administration of histamine and Co 48/80 is safe, neither compound can be regarded as an efficient adjuvant for gilthead seabream vaccination.     

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.     

Progress in the use of swine in developmental immunology of B and T lymphocytes

The adaptive immune system of higher vertebrates is believed to have evolved to counter the ability of pathogens to avoid expulsion because their high rate of germline mutations. Vertebrates developed this adaptive immune response through the evolution of lymphocytes capable of somatic generation of a diverse repertoire of their antigenic receptors without the need to increase the frequency of germline mutation. The focus of our research and this article is on the ontogenetic development of the lymphocytes, and the repertoires they generate in swine. Several features are discussed including (a) the “closed” porcine placenta means that de novo fetal development can be studied for 114 days without passive influence from the mother, (b) newborn piglets are precocial permitting them to be reared without their mothers in germ-free isolators, (c) swine are members of the γδ−high group of mammals and thus provides a greater opportunity to characterize the role of γδ T cells and (d) because swine have a simplified variable heavy and light chain genome they offer a convenient system to study antibody repertoire development.     

Breakdown of immune privilege and spontaneous autoimmunity in mice expressing a transgenic T cell receptor specific for a retinal autoantigen

Despite presence of circulating retina-specific T cells in healthy individuals, ocular immune privilege usually averts development of autoimmune uveitis. To study the breakdown of immune privilege and development of disease, we generated transgenic (Tg) mice that express a T cell receptor (TCR) specific for interphotoreceptor retinoid-binding protein (IRBP), which serves as an autoimmune target in uveitis induced by immunization. Three lines of TCR Tg mice, with different levels of expression of the transgenic R161 TCR and different proportions of IRBP-specific CD4⁺ T cells in their peripheral repertoire, were successfully established. Importantly, two of the lines rapidly developed spontaneous uveitis, reaching 100% incidence by 2 and 3 months of age, respectively, whereas the third appeared “poised” and only developed appreciable disease upon immune perturbation. Susceptibility roughly paralleled expression of the R161 TCR. In all three lines, peripheral CD4⁺ T cells displayed a naïve phenotype, but proliferated in vitro in response to IRBP and elicited uveitis upon adoptive transfer. In contrast, CD4⁺ T cells infiltrating uveitic eyes mostly showed an effector/memory phenotype, and included Th1, Th17 as well as T regulatory cells that appeared to have been peripherally converted from conventional CD4⁺ T cells rather than thymically derived. Thus, R161 mice provide a new and valuable model of spontaneous autoimmune disease that circumvents the limitations of active immunization and adjuvants, and allows to study basic mechanisms involved in maintenance and breakdown of immune homeostasis affecting immunologically privileged sites such as the eye.     

Thymic B cells promote thymus-derived regulatory T cell development and proliferation

Thymic CD4⁺ FoxP3⁺ regulatory T (Treg) cells are critical for the development of immunological tolerance and immune homeostasis and requires contributions of both thymic dendritic and epithelial cells. Although B cells have been reported to be present within the thymus, there has not hitherto been a definition of their role in immune cell development and, in particular, whether or how they contribute to the Treg cellular thymic compartment. Herein, using both phenotypic and functional approaches, we demonstrate that thymic B cells contribute to the maintenance of thymic Treg cells and, using an in vitro culture system, demonstrate that thymic B cells contribute to the size of the thymic Treg compartment via cell–cell MHC II contact and the involvement of two independent co-stimulatory pathways that include interactions between the CD40/CD80/CD86 co-stimulatory molecules. Our data also suggest that thymic B cells promote the generation of thymic Treg cell precursors (pre-Treg cells), but not the conversion of FoxP3⁺ Treg cells from pre-Treg cells. In addition, thymic B cells directly promote the proliferation of thymic Treg cells that is MHC II contact dependent with a minimal if any role for co-stimulatory molecules including CD40/CD80/CD86. Both pathways are independent of TGFβ. In conclusion, we rigorously define the critical role of thymic B cells in the development of thymic Treg cells from non-Treg to precursor stage and in the proliferation of mature thymic Treg cells.     

Evolutionary history of the T cell receptor complex as revealed by small-spotted catshark (Scyliorhinus canicula)

In every jawed vertebrate species studied so far, the T cell receptor (TCR) complex is composed of two different TCR chains (α/β or γ/δ) and a number of CD3 subunits responsible for transmitting signals into the T cell. In this study, we characterised all of the TCR and CD3 genes of small-spotted catshark (Scyliorhinus canicula) and analysed their expression in a broad range of tissues. While the TCR complex is highly conserved across jawed vertebrates, we identified a number of differences in catshark, most notably the presence of two copies of both TCRβ and CD3γδ, and the absence of a functionally-important proline rich region from CD3ε. We also demonstrate that TCRβ has duplicated independently multiple times in jawed vertebrate evolution, bringing additional diversity to the TCR complex. This study reveals new insights about the evolutionary history of the TCR complex and raises new avenues for future exploration.     

Silkworm ferritin 1 heavy chain homolog is involved in defense against bacterial infection through regulation of haemolymph iron homeostasis

Iron functions as a nutrient and a potential toxin in all organisms. It plays a key role in the interaction between microbes and their hosts as well. Microbial infection disrupts iron homeostasis in the host; meanwhile the host endeavors to keep the homeostasis through iron transport and storage. Transferrins and ferritins are the major iron-binding proteins that affect iron distribution in insects. In this study, we investigated a possible involvement of Bombyx mori ferritin 1 (BmFer1) heavy chain homolog in the defense against bacterial infection in the silkworm larvae. The BmFer1 mRNA abundance was up-regulated in hemocytes, but not in fat body, after Pseudomonas aeruginosa or Staphylococcus aureus infection. The infection resulted in elevated iron levels in the hemolymph. Injection of recombinant BmFer1 protein into hemocoel reduced the plasma iron level after infection, limited the bacterial growth in the hemolymph, and resulted in a lower mortality caused by infection. Our study indicated that B. mori ferritin-1 may restrict iron access of the invading bacteria to block their growth as a defense strategy.     

Expansion and differentiation of IgM+ B cells in the rainbow trout peritoneal cavity in response to different antigens

To date, intraperitoneal (i.p.) injection seems to be the most effective vaccination route in aquaculture, as many i.p. administered fish vaccines are capable of conferring strong and long-lasting immune responses. Despite this, how peritoneal leukocytes are regulated upon antigen encounter has only been scarcely studied in fish. Although, in the past, myeloid cells were thought to be the main responders to peritoneal inflammation, a recent study revealed that IgM⁺ B cells are one of the main cell types in the teleost peritoneal cavity in response to pathogenic bacteria. Thus, in the current work, we have focused on establishing how IgM⁺ B cells are recruited into the peritoneum in rainbow trout (Oncorhynchus mykiss) comparing different antigens: Escherichia coli as a bacterial model, E. coli-derived lipopolysaccharide (LPS) or viral hemorrhagic septicemia virus (VHSV). In addition to studying their capacity to dominate the peritoneal cavity, we have established how these IgM⁺ B cells are regulated in response to the different antigens, determining their levels of IgM secretion, surface MHC II expression, cell size and phagocytic abilities. Our results reveal that IgM⁺ B cells are one of the main cell types amplified in the peritoneum in response to either bacterial or viral antigens and that these immunogenic stimulations provoke a differentiation of some of these cells towards plasmablasts/plasma cells whereas others seem to be implicated in antigen presentation. These findings contribute to a better understanding of the immune processes that regulate peritoneal inflammation in teleost fish.     

Thymic CCL2 influences induction of T-cell tolerance

Thymic epithelial cells (TEC) and dendritic cells (DC) play a role in T cell development by controlling the selection of the T cell receptor repertoire. DC have been described to take up antigens in the periphery and migrate into the thymus where they mediate tolerance via deletion of autoreactive T cells, or by induction of natural regulatory T cells. Migration of DC to thymus is driven by chemokine receptors. CCL2, a major ligand for the chemokine receptor CCR2, is an inflammation-associated chemokine that induces the recruitment of immune cells in tissues. CCL2 and CCR2 are implicated in promoting experimental autoimmune encephalomyelitis (EAE), a mouse model for multiple sclerosis. We here show that CCL2 is constitutively expressed by endothelial cells and TEC in the thymus. Transgenic mice overexpressing CCL2 in the thymus showed an increased number of thymic plasmacytoid DC and pronounced impairment of T cell development. Consequently, CCL2 transgenic mice were resistant to EAE. These findings demonstrate that expression of CCL2 in thymus regulates DC homeostasis and controls development of autoreactive T cells, thus preventing development of autoimmune diseases.     

The RND protein is involved in the vulnibactin export system in Vibrio vulnificus M2799

Vibrio vulnificus, an opportunistic marine bacterium that causes a serious, often fatal, infection in humans, requires iron for its pathogenesis. This bacterium exports vulnibactin for iron acquisition from the environment. The mechanisms of vulnibactin biosynthesis and ferric-vulnibactin uptake systems have recently been reported, while the vulnibactin export system has not been reported. Mutant growth under low-iron concentration conditions and a bioassay of the culture supernatant indicate that the VV1_0612 protein plays a crucial role in the vulnibactin secretion as a component of the resistance-nodulation-division (RND)-type efflux system in V. vulnificus M2799. To identify which RND protein(s) together with VV1_0612 TolC constituted the RND efflux system for vulnibactin secretion, deletion mutants of 11 RND protein-encoding genes were constructed. The growth inhibition of a multiple mutant (Δ11) of the RND protein-encoding genes was observed 6 h after the beginning of the culture. Furthermore, ΔVV1_1681 exhibited a growth curve that was similar to that of Δ11, while the multiple mutant except ΔVV1_1681 showed the same growth as the wild-type strain. These results indicate that the VV1_1681 protein is involved in the vulnibactin export system of V. vulnificus M2799. This is the first genetic evidence that vulnibactin is secreted through the RND-type efflux systems in V. vulnificus.