Charlotte N. Cook

Systemic Flagellin Immunization Stimulates Mucosal CD103+ Dendritic Cells and Drives Foxp3+ Regulatory T Cell and IgA Responses in the Mesenteric Lymph Node

Mucosal immunity is poorly activated after systemic immunization with protein Ags. Nevertheless, induction of mucosal immunity in such a manner would be an attractive and simple way to overcome the intrinsic difficulties in delivering Ag to such sites. Flagellin from Salmonella enterica serovar Typhimurium (FliC) can impact markedly on host immunity, in part via its recognition by TLR5. In this study, we show that systemic immunization with soluble FliC (sFliC) drives distinct immune responses concurrently in the mesenteric lymph nodes (MLN) and the spleen after i.p. and s.c. immunization. In the MLN, but not the spleen, sFliC drives a TLR5-dependent recruitment of CD103+ dendritic cells (DCs), which correlates with a diminution in CD103+ DC numbers in the lamina propria. In the MLN, CD103+ DCs carry Ag and are the major primers of endogenous and transgenic T cell priming. A key consequence of these interactions with CD103+ DCs in the MLN is an increase in local regulatory T cell differentiation. In parallel, systemic sFliC immunization results in a pronounced switching of FliC-specific B cells to IgA in the MLN but not elsewhere. Loss of TLR5 has more impact on MLN than splenic Ab responses, reflected in an ablation of IgA, but not IgG, serum Ab titers. Therefore, systemic sFliC immunization targets CD103+ DCs and drives distinct mucosal T and B cell responses. This offers a potential “Trojan horse” approach to modulate mucosal immunity by systemically immunizing with sFliC.

Inflammation drives thrombosis after Salmonella infection via CLEC-2 on platelets

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.

Natural and vaccine-mediated immunity to Salmonella typhimurium is impaired by the helminth Nippostrongylus brasiliensis

Background The impact of exposure to multiple pathogens concurrently or consecutively on immune function is unclear. Here, immune responses induced by combinations of the bacterium Salmonella Typhimurium (STm) and the helminth Nippostrongylus brasiliensis (Nb), which causes a murine hookworm infection and an experimental porin protein vaccine against STm, were examined. Methodology/Principal Findings Mice infected with both STm and Nb induced similar numbers of Th1 and Th2 lymphocytes compared with singly infected mice, as determined by flow cytometry, although lower levels of secreted Th2, but not Th1 cytokines were detected by ELISA after re-stimulation of splenocytes. Furthermore, the density of FoxP3+ T cells in the T zone of co-infected mice was lower compared to mice that only received Nb, but was greater than those that received STm. This reflected the intermediate levels of IL-10 detected from splenocytes. Co-infection compromised clearance of both pathogens, with worms still detectable in mice weeks after they were cleared in the control group. Despite altered control of bacterial and helminth colonization in co-infected mice, robust extrafollicular Th1 and Th2-reflecting immunoglobulin-switching profiles were detected, with IgG2a, IgG1 and IgE plasma cells all detected in parallel. Whilst extrafollicular antibody responses were maintained in the first weeks after co-infection, the GC response was less than that in mice infected with Nb only. Nb infection resulted in some abrogation of the longer-term development of anti-STm IgG responses. This suggested that prior Nb infection may modulate the induction of protective antibody responses to vaccination. To assess this we immunized mice with porins, which confer protection in an antibody-dependent manner, before challenging with STm. Mice that had resolved a Nb infection prior to immunization induced less anti-porin IgG and had compromised protection against infection. Conclusion These findings demonstrate that co-infection can radically alter the development of protective immunity during natural infection and in response to immunization.

B1b Cells Recognize Protective Antigens after Natural Infection and Vaccination

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.

Soluble flagellin coimmunization attenuates Th1 priming to Salmonella and clearance by modulating dendritic cell activation and cytokine production

Soluble flagellin (sFliC) from Salmonella Typhimurium (STm) can induce a Th2 response to itself and coadministered antigens through ligation of TLR5. These properties suggest that sFliC could potentially modulate responses to Th1 antigens like live STm if both antigens are given concurrently. After coimmunization of mice with sFliC and STm there was a reduction in Th1 T cells (T‐bet+IFN‐γ+ CD4 T cells) compared to STm alone and there was impaired clearance of STm. In contrast, there was no significant defect in the early extrafollicular B‐cell response to STm. These effects are dependent upon TLR5 and flagellin expression by STm. The mechanism for these effects is not related to IL‐4 induced to sFliC but rather to the effects of sFliC coimmunization on DCs. After coimmunization with STm and sFliC, splenic DCs had a lower expression of costimulatory molecules and profoundly altered kinetics of IL‐12 and TNFα expression. Ex vivo experiments using in vivo conditioned DCs confirmed the effects of sFliC were due to altered DC function during a critical window in the coordinated interplay between DCs and naïve T cells. This has marked implications for understanding how limits in Th1 priming can be achieved during infection‐induced, Th1‐mediated inflammation.

Maintenance of the marginal-zone B cell compartment specifically requires the RNA-binding protein ZFP36L1

RNA-binding proteins of the ZFP36 family are best known for inhibiting the expression of cytokines through binding to AU-rich elements in the 3′ untranslated region and promoting mRNA decay. Here we identified an indispensable role for ZFP36L1 as the regulator of a post-transcriptional hub that determined the identity of marginal-zone B cells by promoting their proper localization and survival. ZFP36L1 controlled a gene-expression program related to signaling, cell adhesion and locomotion; it achieved this in part by limiting expression of the transcription factors KLF2 and IRF8, which are known to enforce the follicular B cell phenotype. These mechanisms emphasize the importance of integrating transcriptional and post-transcriptional processes by RNA-binding proteins for maintaining cellular identity among closely related cell types.

Resolving Salmonella infection reveals dynamic and persisting changes in murine bone marrow progenitor cell phenotype and function.

The generation of immune cells from BM precursors is a carefully regulated process. This is essential to limit the potential for oncogenesis and autoimmunity yet protect against infection. How infection modulates this is unclear. Salmonella can colonize systemic sites including the BM and spleen. This resolving infection has multiple IFN‐γ‐mediated acute and chronic effects on BM progenitors, and during the first week of infection IFN‐γ is produced by myeloid, NK, NKT, CD4+ T cells, and some lineage‐negative cells. After infection, the phenotype of BM progenitors rapidly but reversibly alters, with a peak ∼30‐fold increase in Sca‐1hi progenitors and a corresponding loss of Sca‐1lo/int subsets. Most strikingly, the capacity of donor Sca‐1hi cells to reconstitute an irradiated host is reduced; the longer donor mice are exposed to infection, and Sca‐1hic‐kitint cells have an increased potential to generate B1a‐like cells. Thus, Salmonella can have a prolonged influence on BM progenitor functionality not directly related to bacterial persistence. These results reflect changes observed in leucopoiesis during aging and suggest that BM functionality can be modulated by life‐long, periodic exposure to infection. Better understanding of this process could offer novel therapeutic opportunities to modulate BM functionality and promote healthy aging.

CD103 + CD11b + mucosal classical dendritic cells initiate long-term switched antibody responses to flagellin

Antibody responses induced at mucosal and nonmucosal sites demonstrate a significant level of autonomy. Here, we demonstrate a key role for mucosal interferon regulatory factor-4 (IRF4)-dependent CD103+CD11b+ (DP), classical dendritic cells (cDCs) in the induction of T-dependent immunoglobulin G (IgG) and immunoglobulin A (IgA) responses in the mesenteric lymph node (MLN) following systemic immunization with soluble flagellin (sFliC). In contrast, IRF8-dependent CD103+CD11b− (SP) are not required for these responses. The lack of this response correlated with a complete absence of sFliC-specific plasma cells in the MLN, small intestinal lamina propria, and surprisingly also the bone marrow (BM). Many sFliC-specific plasma cells accumulating in the BM of immunized wild-type mice expressed α4β7+, suggesting a mucosal origin. Collectively, these results suggest that mucosal DP cDC contribute to the generation of the sFliC-specific plasma cell pool in the BM and thus serve as a bridge linking the mucosal and systemic immune system.

IgG Responses to Porins and Lipopolysaccharide within an Outer Membrane-Based Vaccine against Nontyphoidal Salmonella Develop at Discordant Rates

ABSTRACT Antibodies acquired after vaccination or natural infection with Gram-negative bacteria, such as invasive Salmonella enterica serovar Typhimurium, can protect against disease. Immunization with naturally shed outer membrane vesicles from Gram-negative bacteria is being studied for its potential to protect against many infections, since antigens within vesicles maintain their natural conformation and orientation. Shedding can be enhanced through genetic modification, and the resulting particles, generalized modules for membrane antigens (GMMA), not only offer potential as vaccines but also can facilitate the study of B-cell responses to bacterial antigens. Here we show that the response to immunization with GMMA from S. Typhimurium (STmGMMA) provides B-cell-dependent protection and induces antibodies to two immunodominant antigens, lipopolysaccharide (LPS) and porins. Antibodies to LPS O antigen (O-Ag) markedly enhance protection in the spleen, but this effect is less marked in the liver. Strikingly, IgG responses to LPS and porins develop with distinct kinetics. In the first week after immunization, there is a dramatic T-cell-independent B1b-cell-associated induction of all IgG isotypes, except IgG1, to porins but not to LPS. In contrast, production of IgG1 to either antigen was delayed and T cell dependent. Nevertheless, after 1 month, cells in the bone marrow secreting IgG against porins or LPS were present at a similar frequency. Unexpectedly, immunization with O-Ag-deficient STmGMMA did not substantially enhance the anti-porin response. Therefore, IgG switching to all antigens does not develop synchronously within the same complex and so the rate of IgG switching to a single component does not necessarily reflect its frequency within the antigenic complex. IMPORTANCE Vaccines save millions of lives, yet for some infections there are none. This includes some types of Salmonella infections, killing hundreds of thousands of people annually. We show how a new type of vaccine, called GMMA, that is made from blebs shed from the Salmonella cell wall, works to protect against infection in mice by inducing host proteins (antibodies) specifically recognizing bacterial components (antigens). The rate of development of IgG antibody to antigens within GMMA occurred with different kinetics. However, the antibody response to GMMA persists and is likely to provide prolonged protection for those who need it. These results help show how antibody responses to bacterial antigens develop and how vaccines like GMMA can work and help prevent infection. IMPORTANCE Vaccines save millions of lives, yet for some infections there are none. This includes some types of Salmonella infections, killing hundreds of thousands of people annually. We show how a new type of vaccine, called GMMA, that is made from blebs shed from the Salmonella cell wall, works to protect against infection in mice by inducing host proteins (antibodies) specifically recognizing bacterial components (antigens). The rate of development of IgG antibody to antigens within GMMA occurred with different kinetics. However, the antibody response to GMMA persists and is likely to provide prolonged protection for those who need it. These results help show how antibody responses to bacterial antigens develop and how vaccines like GMMA can work and help prevent infection.

Humoral immunity to memory antigens and pathogens is maintained in patients with chronic kidney disease

Patients with chronic kidney disease (CKD) have an increased risk of infection and poorer responses to vaccination. This suggests that CKD patients have an impaired responsiveness to all antigens, even those first encountered before CKD onset. To examine this we evaluated antibody responses against two childhood vaccine antigens, tetanus (TT) and diphtheria toxoids (DT) and two common pathogens, cytomegalovirus (CMV) and Salmonella enterica serovar Enteritidis (SEn) in two independent cohorts consisting of age-matched individuals with and without CKD. Sera were evaluated for antigen-specific IgG titres and the functionality of antibody to SEn was assessed in a serum bactericidal assay. Surprisingly, patients with CKD and control subjects had comparable levels of IgG against TT and DT, suggesting preserved humoral memory responses to antigens encountered early in life. Lipopolysaccharide-specific IgG titres and serum bactericidal activity in patients with CKD were also not inferior to controls. CMV-specific IgG titres in seropositive CKD patients were similar or even increased compared to controls. Therefore, whilst responses to new vaccines in CKD are typically lower than expected, antibody responses to antigens commonly encountered prior to CKD onset are not. The immunodeficiency of CKD is likely characterised by failure to respond to new antigenic challenges and efforts to improve patient outcomes should be focussed here.