Thrombosis is a common, life-threatening consequence of systemic infection; however, the underlying mechanisms that drive the formation of infection-associated thrombi are poorly understood. Here, ...using a mouse model of systemic Salmonella Typhimurium infection, we determined that inflammation in tissues triggers thrombosis within vessels via ligation of C-type lectin-like receptor-2 (CLEC-2) on platelets by podoplanin exposed to the vasculature following breaching of the vessel wall. During infection, mice developed thrombi that persisted for weeks within the liver. Bacteria triggered but did not maintain this process, as thrombosis peaked at times when bacteremia was absent and bacteria in tissues were reduced by more than 90% from their peak levels. Thrombus development was triggered by an innate, TLR4-dependent inflammatory cascade that was independent of classical glycoprotein VI-mediated (GPVI-mediated) platelet activation. After infection, IFN-γ release enhanced the number of podoplanin-expressing monocytes and Kupffer cells in the hepatic parenchyma and perivascular sites and absence of TLR4, IFN-γ, or depletion of monocytic-lineage cells or CLEC-2 on platelets markedly inhibited the process. Together, our data indicate that infection-driven thrombosis follows local inflammation and upregulation of podoplanin and platelet activation. The identification of this pathway offers potential therapeutic opportunities to control the devastating consequences of infection-driven thrombosis without increasing the risk of bleeding.
Lipopolysaccharide (LPS) O-antigen (O-Ag) is known to limit antibody binding to surface antigens, although the relationship between antibody, O-Ag and other outer-membrane antigens is poorly ...understood. Here we report, immunization with the trimeric porin OmpD from Salmonella Typhimurium (STmOmpD) protects against infection. Atomistic molecular dynamics simulations indicate this is because OmpD trimers generate footprints within the O-Ag layer sufficiently sized for a single IgG Fab to access. While STmOmpD differs from its orthologue in S. Enteritidis (SEn) by a single amino-acid residue, immunization with STmOmpD confers minimal protection to SEn. This is due to the OmpD-O-Ag interplay restricting IgG binding, with the pairing of OmpD with its native O-Ag being essential for optimal protection after immunization. Thus, both the chemical and physical structure of O-Ag are key for the presentation of specific epitopes within proteinaceous surface-antigens. This enhances combinatorial antigenic diversity in Gram-negative bacteria, while reducing associated fitness costs.
Salmonella enterica serovar Typhi expresses a capsule of Vi polysaccharide, while most Salmonella serovars, including S. Enteritidis and S. Typhimurium, do not. Both S. Typhi and S. Enteritidis ...express the lipopolysaccharide O:9 antigen, yet there is little evidence of cross-protection from anti-O:9 antibodies. Vaccines based on Vi polysaccharide have efficacy against typhoid fever, indicating that antibodies against Vi confer protection. Here we investigate the role of Vi capsule and antibodies against Vi and O:9 in antibody-dependent complement- and phagocyte-mediated killing of Salmonella. Using isogenic Vi-expressing and non-Vi-expressing derivatives of S. Typhi and S. Typhimurium, we show that S. Typhi is inherently more sensitive to serum and blood than S. Typhimurium. Vi expression confers increased resistance to both complement- and phagocyte-mediated modalities of antibody-dependent killing in human blood. The Vi capsule is associated with reduced C3 and C5b-9 deposition, and decreased overall antibody binding to S. Typhi. However, purified human anti-Vi antibodies in the presence of complement are able to kill Vi-expressing Salmonella, while killing by anti-O:9 antibodies is inversely related to Vi expression. Human serum depleted of antibodies to antigens other than Vi retains the ability to kill Vi-expressing bacteria. Our findings support a protective role for Vi capsule in preventing complement and phagocyte killing of Salmonella that can be overcome by specific anti-Vi antibodies, but only to a limited extent by anti-O:9 antibodies.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Membranes of Gram-negative bacteria, mitochondria and chloroplasts receive and fold β-barrel transmembrane proteins through the action of polypeptide transport- associated (POTRA) domains. In ...Escherichia coli, folding substrates are inserted into the outer membrane by the essential protein YaeT, a prototypic Omp85 protein. Here, the articulation between tandem POTRA domains in solution is defined by nuclear magnetic resonance (NMR) spectroscopy, indicating an unprecedented juxtaposition. The novel solution orientations of all five POTRA domains are revealed by small-angle X-ray scattering of the entire 46 kDa periplasmic region. NMR titration studies show that strands from YaeT's canonical folding substrate, PhoE, bind non-specifically along alternating sides of its mixed β sheets, thus providing an ideal platform for helping to fold nascent outer-membrane proteins. Together, this provides the first structural model of how multiple POTRA domains recruit substrates from the periplasmic solution into the outer membrane.
Invasive nontyphoidal Salmonella (NTS), including Salmonella typhimurium (STm), are major yet poorly-recognized killers of infants in sub-Saharan Africa. Death in these children is usually associated ...with bacteremia, commonly in the absence of gastrointestinal symptoms. Evidence from humans and animal studies suggest that severe infection and bacteremia occur when specific Ab is lacking. Understanding how Ab responses to Salmonella are regulated will help develop vaccines against these devastating infections. STm induces atypical Ab responses characterized by prominent, accelerated, extrafollicular T-independent (TI) Ab against a range of surface antigens. These responses develop without concomitant germinal centers, which only appear as infection resolves. Here, we show STm rapidly induces a population of TI B220⁺CD5⁻ B1b cells during infection and TI Ab from B1b cells targets the outer membrane protein (Omp) porins OmpC, OmpD and OmpF but not flagellin. When porins are used as immunogens they can ablate bacteremia and provide equivalent protection against STm as killed bacterial vaccine and this is wholly B cell-dependent. Furthermore Ab from porin-immunized chimeras, that have B1b cells, is sufficient to impair infection. Infecting with porin-deficient bacteria identifies OmpD, a protein absent from Salmonella Typhi, as a key target of Ab in these infections. This work broadens the recognized repertoire of TI protein antigens and highlights the importance of Ab from different B cell subsets in controlling STm infection. OmpD is a strong candidate vaccine target and may, in part, explain the lack of cross-protection between Salmonella Typhi and STm infections.
In mice, the IgG subclass induced after Ag encounter can reflect the nature of the Ag. Th2 Ags such as alum-precipitated proteins and helminths induce IgG1, whereas Th1 Ags, such as
Typhimurium, ...predominantly induce IgG2a. The contribution of different IgG isotypes to protection against bacteria such as
Typhimurium is unclear, although as IgG2a is induced by natural infection, it is assumed this isotype is important. Previously, we have shown that purified
Typhimurium porins including outer membrane protein OmpD, which induce both IgG1 and IgG2a in mice, provide protection to
Typhimurium infection via Ab. In this study we report the unexpected finding that mice lacking IgG1, but not IgG2a, are substantially less protected after porin immunization than wild-type controls. IgG1-deficient mice produce more porin-specific IgG2a, resulting in total IgG levels that are similar to wild-type mice. The decreased protection in IgG1-deficient mice correlates with less efficient bacterial opsonization and uptake by macrophages, and this reflects the low binding of outer membrane protein OmpD-specific IgG2a to the bacterial surface. Thus, the Th2-associated isotype IgG1 can play a role in protection against Th1-associated organisms such as
Typhimurium. Therefore, individual IgG subclasses to a single Ag can provide different levels of protection and the IgG isotype induced may need to be a consideration when designing vaccines and immunization strategies.
There are multiple, distinct B-cell populations in human beings and other animals such as mice. In the latter species, there is a well-characterized subset of B-cells known as B1 cells, which are ...enriched in peripheral sites such as the peritoneal cavity but are rare in the blood. B1 cells can be further subdivided into B1a and B1b subsets. There may be additional B1 subsets, though it is unclear if these are distinct populations or stages in the developmental process to become mature B1a and B1b cells. A limitation in understanding B1 subsets is the relative paucity of specific surface markers. In contrast to mice, the existence of B1 cells in human beings is controversial and more studies are needed to investigate the nature of these enigmatic cells. Examples of B1b antigens include pneumococcal polysaccharide and the Vi antigen from Salmonella Typhi, both used routinely as vaccines in human beings and experimental antigens such as haptenated-Ficoll. In addition to inducing classical T-dependent responses some proteins are B1b antigens and can induce T-independent (TI) immunity, examples include factor H binding protein from Borrelia hermsii and porins from Salmonella. Therefore, B1b antigens can be proteinaceous or non-proteinaceous, induce TI responses, memory, and immunity, they exist in a diverse range of pathogenic bacteria, and a single species can contain multiple B1b antigens. An unexpected benefit to studying B1b cells is that they appear to have a propensity to recognize protective antigens in bacteria. This suggests that studying B1b cells may be rewarding for vaccine design as immunoprophylactic and immunotherapeutic interventions become more important due to the decreasing efficacy of small molecule antimicrobials.
Innate immunity is regulated by cholinergic signalling through nicotinic acetylcholine receptors. We show here that signalling through the M3 muscarinic acetylcholine receptor (M3R) plays an ...important role in adaptive immunity to both Nippostrongylus brasiliensis and Salmonella enterica serovar Typhimurium, as M3R-/- mice were impaired in their ability to resolve infection with either pathogen. CD4 T cell activation and cytokine production were reduced in M3R-/- mice. Immunity to secondary infection with N. brasiliensis was severely impaired, with reduced cytokine responses in M3R-/- mice accompanied by lower numbers of mucus-producing goblet cells and alternatively activated macrophages in the lungs. Ex vivo lymphocyte stimulation of cells from intact BALB/c mice infected with N. brasiliensis and S. typhimurium with muscarinic agonists resulted in enhanced production of IL-13 and IFN-γ respectively, which was blocked by an M3R-selective antagonist. Our data therefore indicate that cholinergic signalling via the M3R is essential for optimal Th1 and Th2 adaptive immunity to infection.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Although in vitro IL-4 directs CD4 T cells to produce T helper 2 (Th2)-cytokines, these cytokines can be induced in vivo in the absence of IL-4-signalling. Thus, mechanism(s), different from the in ...vitro pathway for Th2-induction, contribute to in vivo Th2-differentiation. The pathway for in vivo IL-4-independent Th2-differentiation has yet to be characterized.
Helios (ikzf2), a member of the Ikaros transcription regulator family, is expressed in thymocytes and some antigen-matured T cells as well as in regulatory T cells. It has been proposed that Helios is a specific marker for thymus-derived regulatory T cells. Here, we show that mouse ovalbumin-specific CD4 (OTII) cells responding to alum-precipitated ovalbumin (alumOVA) upregulate Th2 features - GATA-3 and IL-4 - as well as Helios mRNA and protein. Helios is also upregulated in follicular helper T (TFh) cells in this response. By contrast, OTII cells responding to the Th1 antigen - live attenuated ovalbumin-expressing Salmonella - upregulate Th1 features - T-bet and IFN-γ - but not Helios. In addition, CD4 T cells induced to produce Th2 cytokines in vitro do not express Helios. The kinetics of Helios mRNA and protein induction mirrors that of GATA-3. The induction of IL-4, IL-13 and CXCR5 by alumOVA requires NF-κB1 and this is also needed for Helios upregulation. Importantly, Helios is induced in Th2 and TFh cells without parallel upregulation of Foxp3. These findings suggested a key role for Helios in Th2 and TFh development in response to alum-protein vaccines. We tested this possibility using Helios-deficient OTII cells and found this deficiency had no discernable impact on Th2 and TFh differentiation in response to alumOVA.
Helios is selectively upregulated in CD4 T cells during Th2 and TFh responses to alum-protein vaccines in vivo, but the functional significance of this upregulation remains uncertain.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Alum-precipitated protein (alum protein) vaccines elicit long-lasting neutralizing antibody responses that prevent bacterial exotoxins and viruses from entering cells. Typically, these vaccines ...induce CD4 T cells to become T helper 2 (Th2) cells that induce Ig class switching to IgG1. We now report that CD8 T cells also respond to alum proteins, proliferating extensively and producing IFN-γ, a key Th1 cytokine. These findings led us to question whether adoptive transfer of antigen-specific CD8 T cells alters the characteristic CD4 Th2 response to alum proteins and the switching pattern in responding B cells. To this end, WT mice given transgenic ovalbumin (OVA)-specific CD4 (OTII) or CD8 (OTI) T cells, or both, were immunized with alum-precipitated OVA. Cotransfer of antigen-specific CD8 T cells skewed switching patterns in responding B cells from IgG1 to IgG2a and IgG2b. Blocking with anti-IFN-γ antibody largely inhibited this altered B-cell switching pattern. The transcription factor T-bet is required in B cells for IFN-γ-dependent switching to IgG2a. By contrast, we show that this transcription factor is dispensable in B cells both for IFN-γ-induced switching to IgG2b and for inhibition of switching to IgG1. Thus, T-bet dependence identifies distinct transcriptional pathways in B cells that regulate IFN-γ-induced switching to different IgG isotypes.
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