IL-17 and IFN-γ-producing respiratory tissue resident memory CD4†T cells persist for decades in adults immunized as children with whole cell pertussis vaccines.

The objective was to determine if antigen-specific tissue resident memory T (TRM) cells persist in respiratory tissues of adults immunized as children with whole cell pertussis (wP) or acellular pertussis (aP) vaccines. Mononuclear cells from tonsil or nasal tissue cells were cultured with Bordetella pertussis antigens and TRM cells quantified by flow cytometry. Adults immunized with wP vaccines as children had significantly more IL-17A and IFN-y-producing TRM cells that respond to B. pertussis antigens in respiratory tissues when compared with aP-primed donors. Our findings demonstrate that wP vaccines induce CD4 TRM cells that can persist in respiratory tissues for decades.

Bystander activation of Bordetella pertussis-induced nasal tissue-resident memory CD4 T cells confers heterologous immunity to Klebsiella pneumoniae.

Tissue-resident memory CD4 T (TRM ) cells induced by infection with Bordetella pertussis persist in respiratory tissues and confer long-term protective immunity against reinfection. However, it is not clear how they are maintained in respiratory tissues. Here, we demonstrate that B. pertussis-specific CD4 TRM cells produce IL-17A in response to in vitro stimulation with LPS or heat-killed Klebsiella pneumoniae (HKKP) in the presence of dendritic cells. Furthermore, IL-17A-secreting CD4 TRM cells expand in the lung and nasal tissue of B. pertussis convalescent mice following in vivo administration of LPS or HKKP. Bystander activation of CD4 TRM cells was suppressed by anti-IL-12p40 but not by anti-MHCII antibodies. Furthermore, purified respiratory tissue-resident, but not circulating, CD4 T cells from convalescent mice produced IL-17A following direct stimulation with IL-23 and IL-1ß or IL-18. Intranasal immunization of mice with a whole-cell pertussis vaccine induced respiratory CD4 TRM cells that were reactivated following stimulation with K. pneumoniae. Furthermore, the nasal pertussis vaccine conferred protective immunity against B. pertussis but also attenuated infection with K. pneumoniae. Our findings demonstrate that CD4 TRM cells induced by respiratory infection or vaccination can undergo bystander activation and confer heterologous immunity to an unrelated respiratory pathogen.

Staphylococcus aureus-induced immunosuppression mediated by IL-10 and IL-27 facilitates nasal colonisation.

Staphylococcus aureus persistently colonises the anterior nares of a significant proportion of the healthy population, however the local immune response elicited during S. aureus nasal colonisation remains ill-defined. Local activation of IL-17/IL-22 producing T cells are critical for controlling bacterial clearance from the nasal cavity. However, recurrent and long-term colonisation is commonplace indicating efficient clearance does not invariably occur. Here we identify a central role for the regulatory cytokine IL-10 in facilitating bacterial persistence during S. aureus nasal colonisation in a murine model. IL-10 is produced rapidly within the nasal cavity following S. aureus colonisation, primarily by myeloid cells. Colonised IL-10-/- mice demonstrate enhanced IL-17+ and IL-22+ T cell responses and more rapidly clear bacteria from the nasal tissues as compared with wild-type mice. S. aureus also induces the regulatory cytokine IL-27 within the nasal tissue, which acts upstream of IL-10 promoting its production. IL-27 blockade reduces IL-10 production within the nasal cavity and improves bacterial clearance. TLR2 signalling was confirmed to be central to controlling the IL-10 response. Our findings conclude that during nasal colonisation S. aureus creates an immunosuppressive microenvironment through the local induction of IL-27 and IL-10, to dampen protective T cell responses and facilitate its persistence.

Targeting Skin-Resident Memory T Cells via Vaccination to Combat Staphylococcus aureus Infections.

Tissue-resident memory T cells are important in adaptive immunity against many infections, rendering these cells attractive potential targets in vaccine development. Genetic and experimental evidence highlights the importance of cellular immunity in protection from Staphylococcus aureus skin infections, yet skin-resident memory T cells are, thus far, an untested component of immunity during such infections. Novel methods of generating and sampling vaccine-induced skin memory T cells are paralleled by discoveries of global, skin-wide immunosurveillance. We propose skin-resident memory CD4+ T cells as a potential missing link in the search for correlates of protection during S. aureus infections. A better appreciation of their phenotypes and functions could accelerate the development of preventive vaccines against this highly virulent and antibiotic-resistant pathogen.

Interleukin-6 signaling drives fibrosis in unresolved inflammation.

Fibrosis in response to tissue damage or persistent inflammation is a pathological hallmark of many chronic degenerative diseases. By using a model of acute peritoneal inflammation, we have examined how repeated inflammatory activation promotes fibrotic tissue injury. In this context, fibrosis was strictly dependent on interleukin-6 (IL-6). Repeat inflammation induced IL-6-mediated T helper 1 (Th1) cell effector commitment and the emergence of STAT1 (signal transducer and activator of transcription-1) activity within the peritoneal membrane. Fibrosis was not observed in mice lacking interferon-γ (IFN-γ), STAT1, or RAG-1. Here, IFN-γ and STAT1 signaling disrupted the turnover of extracellular matrix by metalloproteases. Whereas IL-6-deficient mice resisted fibrosis, transfer of polarized Th1 cells or inhibition of MMP activity reversed this outcome. Thus, IL-6 causes compromised tissue repair by shifting acute inflammation into a more chronic profibrotic state through induction of Th1 cell responses as a consequence of recurrent inflammation.

Activation of Human Vδ2+ γδ T Cells by Staphylococcus aureus Promotes Enhanced Anti-Staphylococcal Adaptive Immunity.

Murine studies have shown the potential for γδ T cells to mediate immunity to Staphylococcus aureus in multiple tissue settings by the secretion of diverse cytokines. However, the role played by γδ T cells in human immune responses to S. aureus is almost entirely unknown. In this study, we establish the capacity of human Vδ2+ γδ T cells for rapid activation in response to S. aureus In coculture with S. aureus-infected monocyte-derived dendritic cells (DCs), Vδ2+ cells derived from peripheral blood rapidly upregulate CD69 and secrete high levels of IFN-γ. DCs mediate this response through direct contact and IL-12 secretion. In turn, IFN-γ released by Vδ2+ cells upregulates IL-12 secretion by DCs in a positive feedback loop. Furthermore, coculture with γδ T cells results in heightened expression of the costimulatory molecule CD86 and the lymph node homing molecule CCR7 on S. aureus-infected DCs. In cocultures of CD4+ T cells with S. aureus-infected DCs, the addition of γδ T cells results in heightened CD4+ T cell activation. Our findings identify γδ T cells as potential key players in the early host response to S. aureus during bloodstream infection, promoting enhanced responses by both innate and adaptive immune cell populations, and support their consideration in the development of host-directed anti-S. aureus treatments.

Clumping factor B is an important virulence factor during Staphylococcus aureus skin infection and a promising vaccine target.

Staphylococcus aureus expresses a number of cell wall-anchored proteins that mediate adhesion and invasion of host cells and tissues and promote immune evasion, consequently contributing to the virulence of this organism. The cell wall-anchored protein clumping factor B (ClfB) has previously been shown to facilitate S. aureus nasal colonization through high affinity interactions with the cornified envelope in the anterior nares. However, the role of ClfB during skin and soft tissue infection (SSTI) has never been investigated. This study reveals a novel role for ClfB during SSTIs. ClfB is crucial in determining the abscess structure and bacterial burden early in infection and this is dependent upon a specific interaction with the ligand loricrin which is expressed within the abscess tissue. Targeting ClfB using a model vaccine that induced both protective humoral and cellular responses, leads to protection during S. aureus skin infection. This study therefore identifies ClfB as an important antigen for future SSTI vaccines.

Toll-like receptor 2-dependent endosomal signaling by Staphylococcus aureus in monocytes induces type I interferon and promotes intracellular survival.

Pathogen activation of innate immune pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) stimulates cellular signaling pathways. This often leads to outcomes that contribute to pathogen clearance. Alternatively, activation of specific PRR pathways can aid pathogen survival. The human pathogen Staphylococcus aureus is a case in point, employing strategies to escape innate immune recognition and killing by the host. As for other bacteria, PRR-stimulated type I interferon (IFN-I) induction has been proposed as one such immune escape pathway that may favor S. aureus Cell wall components of S. aureus elicit TLR2-dependent cellular responses, but the exact signaling pathways activated by S. aureus-TLR2 engagement and the consequences of their activation for the host and bacterium are not fully known. We previously showed that TLR2 activates both a cytoplasmic and an endosome-dependent signaling pathway, the latter leading to IFN-I production. Here, we demonstrate that S. aureus infection of human monocytes activates a TLR2-dependent endosomal signaling pathway, leading to IFN-I induction. We mapped the signaling components of this pathway and identified roles in IFN-I stimulation for the Toll-interleukin-1 receptor (TIR) adaptor Myd88 adaptor-like (Mal), TNF receptor-associated factor 6 (TRAF6), and IκB kinase (IKK)-related kinases, but not for TRIF-related adaptor molecule (TRAM) and TRAF3. Importantly, monocyte TLR2-dependent endosomal signaling enabled immune escape for S. aureus, because this pathway, but not IFN-I per se, contributed to intracellular bacterial survival. These results reveal a TLR2-dependent mechanism in human monocytes whereby S. aureus manipulates innate immune signaling for its survival in cells.

A Small Membrane Stabilizing Protein Critical to the Pathogenicity of Staphylococcus aureus.

Staphylococcus aureus is a major human pathogen, and the emergence of antibiotic-resistant strains is making all types of S. aureus infections more challenging to treat. With a pressing need to develop alternative control strategies to use alongside or in place of conventional antibiotics, one approach is the targeting of established virulence factors. However, attempts at this have had little success to date, suggesting that we need to better understand how this pathogen causes disease if effective targets are to be identified. To address this, using a functional genomics approach, we have identified a small membrane-bound protein that we have called MspA. Inactivation of this protein results in the loss of the ability of S. aureus to secrete cytolytic toxins, protect itself from several aspects of the human innate immune system, and control its iron homeostasis. These changes appear to be mediated through a change in the stability of the bacterial membrane as a consequence of iron toxicity. These pleiotropic effects on the ability of the pathogen to interact with its host result in significant impairment in the ability of S. aureus to cause infection in both a subcutaneous and sepsis model of infection. Given the scale of the effect the inactivation of MspA causes, it represents a unique and promising target for the development of a novel therapeutic approach.

Would hemodialysis patients benefit from a Staphylococcus aureus vaccine?

Staphylococcus aureus bloodstream infection can have potentially catastrophic consequences for patients on hemodialysis. Consequently, an effective vaccine to prevent S aureus infection would have a significant influence on morbidity and mortality in this group. To date, however, efforts to develop a vaccine have been unsuccessful. Previous antibody-inducing vaccine candidates did not prevent or attenuate S aureus infection in clinical trials. Recent advances have helped to elucidate the role of specific T-cell subsets, notably T-helper cell 1 and T-helper cell 17, in the immune response to S aureus. These cells are essential for coordinating an effective phagocytic response via cytokine production, indirectly leading to destruction of the organism. It is now widely accepted that next-generation S aureus vaccines must also induce effective T-cell-mediated immunity. However, there remains a gap in our knowledge: how will an S aureus vaccine drive these responses in those patients most at risk? Given that patients on hemodialysis are an immunocompromised population, in particular with specific T-cell defects, including defects in T-helper cell subsets, this is likely to affect their ability to respond to an S aureus vaccine. We urgently need a better understanding of T-cell-mediated immunity in this cohort if an efficacious vaccine is ever to be realized for these patients.

Bacterial toxins: Offensive, defensive, or something else altogether?

The secretion of proteins that damage host tissue is well established as integral to the infectious processes of many bacterial pathogens. However, recent advances in our understanding of the activity of toxins suggest that the attributes we have assigned to them from early in vitro experimentation have misled us into thinking of them as merely destructive tools. Here, we will discuss the multifarious ways in which toxins contribute to the lifestyle of bacteria and, by considering their activity from an evolutionary perspective, demonstrate how this extends far beyond their ability to destroy host tissue.

Memory γδ T Cells-Newly Appreciated Protagonists in Infection and Immunity.

Despite the potential for diversity in their T cell receptor, γδ T cells are primarily considered to be innate immune cells. Recently, memory-like γδ T cell responses have been identified in murine models of infection and autoimmunity. Similar memory responses have also been described in human and non-human primate γδ T cells. It has thus become clear that subpopulations of γδ T cells can develop long-lasting memory akin to conventional αß T cells, with protective and pathogenic consequences. Hence, a re-evaluation of their true capabilities and role in infection and immunity is required. This review discusses recent reports of memory-type responses attributed to γδ T cells and assesses this underappreciated facet of these enigmatic cells.

IL-10 Plays Opposing Roles during Staphylococcus aureus Systemic and Localized Infections.

IL-10 is a potent anti-inflammatory mediator that plays a crucial role in limiting host immunopathology during bacterial infections by controlling effector T cell activation. Staphylococcus aureus has previously been shown to manipulate the IL-10 response as a mechanism of immune evasion during chronic systemic and biofilm models of infection. In the present study, we demonstrate divergent roles for IL-10 depending on the site of infection. During acute systemic S. aureus infection, IL-10 plays an important protective role and is required to prevent bacterial dissemination and host morbidity by controlling effector T cells and the associated downstream hyperactivation of inflammatory phagocytes, which are capable of host tissue damage. CD19+CD11b+CD5+ B1a regulatory cells were shown to rapidly express IL-10 in a TLR2-dependent manner in response to S. aureus, and adoptive transfer of B1a cells was protective during acute systemic infection in IL-10-deficient hosts. In contrast, during localized s.c. infection, IL-10 production plays a detrimental role by facilitating bacterial persistence via the same mechanism of controlling proinflammatory T cell responses. Our findings demonstrate that induction of IL-10 has a major influence on disease outcome during acute S. aureus infection. Too much IL-10 at one end of the scale may suppress otherwise protective T cell responses, thus facilitating persistence of the bacteria, and at the other end, too little IL-10 may tend toward fatal host-mediated pathology through excessive activation of T cells and associated phagocyte-mediated damage.

The Staphylococcus aureus Cell Wall-Anchored Protein Clumping Factor A Is an Important T Cell Antigen.

Staphylococcus aureus has become increasingly resistant to antibiotics, and vaccines offer a potential solution to this epidemic of antimicrobial resistance. Targeting of specific T cell subsets is now considered crucial for next-generation anti-S. aureus vaccines; however, there is a paucity of information regarding T cell antigens of S. aureus This study highlights the importance of cell wall-anchored proteins as human CD4+ T cell activators capable of driving antigen-specific Th1 and Th17 cell activation. Clumping factor A (ClfA), which contains N1, N2, and N3 binding domains, was found to be a potent human T cell activator. We further investigated which subdomains of ClfA were involved in T cell activation and found that the full-length ClfA N123 and N23 were potent Th1 and Th17 activators. Interestingly, the N1 subdomain was capable of exclusively activating Th1 cells. Furthermore, when these subdomains were used in a model vaccine, N23 and N1 offered Th1- and Th17-mediated systemic protection in mice upon intraperitoneal challenge. Overall, however, full-length ClfA N123 is required for maximal protection both locally and systemically.

The circadian protein BMAL1 in myeloid cells is a negative regulator of allergic asthma.

Our body clock drives rhythms in the expression of genes that have a 24-h periodicity. The transcription factor BMAL1 is a crucial component of the molecular clock. A number of physiological processes, including immune function, are modulated by the circadian clock. Asthma, a disease with very strong clinical evidence demonstrating regulation by circadian variation, is of particular relevance to circadian control of immunity. Airway hypersensitivity and asthma attacks are more common at night in humans. The molecular basis for this is unknown, and there is no model of asthma in animals with genetic distortion of the molecular clock. We used mice lacking BMAL1 in myeloid cells (BMAL1-LysM-/-) to determine the role of BMAL1 in allergic asthma. Using the ovalbumin model of allergic asthma, we demonstrated markedly increased asthma features, such as increased lung inflammation, demonstrated by drastically higher numbers of eosinophils and increased IL-5 levels in the lung and serum, in BMAL1-LysM-/- mice. In vitro studies demonstrated increased proinflammatory chemokine and mannose receptor expression in IL-4- as well as LPS-treated macrophages from BMAL1-LysM-/- mice compared with wild-type controls. This suggests that Bmal1 is a potent negative regulator in myeloid cells in the context of allergic asthma. Our findings might explain the increase in asthma incidents during the night, when BMAL1 expression is low.

Memory Th1 Cells Are Protective in Invasive Staphylococcus aureus Infection.

Mechanisms of protective immunity to Staphylococcus aureus infection in humans remain elusive. While the importance of cellular immunity has been shown in mice, T cell responses in humans have not been characterised. Using a murine model of recurrent S. aureus peritonitis, we demonstrated that prior exposure to S. aureus enhanced IFNγ responses upon subsequent infection, while adoptive transfer of S. aureus antigen-specific Th1 cells was protective in naïve mice. Translating these findings, we found that S. aureus antigen-specific Th1 cells were also significantly expanded during human S. aureus bloodstream infection (BSI). These Th1 cells were CD45RO+, indicative of a memory phenotype. Thus, exposure to S. aureus induces memory Th1 cells in mice and humans, identifying Th1 cells as potential S. aureus vaccine targets. Consequently, we developed a model vaccine comprising staphylococcal clumping factor A, which we demonstrate to be an effective human T cell antigen, combined with the Th1-driving adjuvant CpG. This novel Th1-inducing vaccine conferred significant protection during S. aureus infection in mice. This study notably advances our understanding of S. aureus cellular immunity, and demonstrates for the first time that a correlate of S. aureus protective immunity identified in mice may be relevant in humans.

TRAM is required for TLR2 endosomal signaling to type I IFN induction.

Detection of microbes by TLRs on the plasma membrane leads to the induction of proinflammatory cytokines such as TNF-α, via activation of NF-κB. Alternatively, activation of endosomal TLRs leads to the induction of type I IFNs via IFN regulatory factors (IRFs). TLR4 signaling from the plasma membrane to NF-κB via the Toll/IL-1R (TIR) adaptor protein MyD88 requires the TIR sorting adaptor Mal, whereas endosomal TLR4 signaling to IRF3 via the TIR domain-containing adaptor-inducing IFN-ß (TRIF) requires the TRIF-related adaptor molecule (TRAM). Similar to TLR4 homodimers, TLR2 heterodimers can also induce both proinflammatory cytokines and type I IFNs. TLR2 plasma membrane signaling to NF-κB is known to require MyD88 and Mal, whereas endosomal IRF activation by TLR2 requires MyD88. However, it was unclear whether TLR2 requires a sorting adaptor for endosomal signaling, like TLR4 does. In this study, we show that TLR2-dependent IRF7 activation at the endosome is both Mal- and TRAM-dependent, and that TRAM is required for the TLR2-dependent movement of MyD88 to endosomes following ligand engagement. TRAM interacted with both TLR2 and MyD88, suggesting that TRAM can act as a bridging adapter between these two molecules. Furthermore, infection of macrophages lacking TRAM with herpes viruses or the bacterium Staphylococcus aureus led to impaired induction of type I IFN, indicating a role for TRAM in TLR2-dependent responses to human pathogens. Our work reveals that TRAM acts as a sorting adaptor not only for TLR4, but also for TLR2, to facilitate signaling to IRF7 at the endosome, which explains how TLR2 is capable of causing type I IFN induction.

Staphylococcus aureus infection of mice expands a population of memory γδ T cells that are protective against subsequent infection.

The development of vaccines against Staphylococcus aureus has consistently failed in clinical trials, likely due to inefficient induction of cellular immunity. T cell-derived IL-17 is one of the few known correlates of antistaphylococcoal immunity, conferring protection against S. aureus infections through its ability to promote phagocytic cell effector functions. A comprehensive understanding of the discrete T cell subsets critical for site-specific IL-17-mediated bacterial clearance will therefore be necessary to inform the development of vaccines that efficiently target cellular immunity. In this study, we have identified a population of CD44+ CD27- memory γδ T cells, expanded upon infection of C57BL/6 mice with S. aureus, which produce high levels of IL-17 and mediate enhanced bacterial clearance upon reinfection with the bacterium. These cells are comprised largely of the Vγ4+ subset and accumulate at the site of infection subsequent to an initial Vγ1.1+ and Vγ2+ T cell response. Moreover, these Vγ4+ T cells are retained in the peritoneum and draining mediastinal lymph nodes for a prolonged period following bacterial clearance. In contrast to its critical requirement for γδ T cell activation during the primary infection, IL-1 signaling was dispensable for activation and expansion of memory γδ T cells upon re-exposure to S. aureus. Our findings demonstrate that a γδ T cell memory response can be induced upon exposure to S. aureus, in a fashion analogous to that associated with classical αß T cells, and suggest that induction of IL-17-expressing γδ T cells may be an important property of a protective vaccine against S. aureus.

Manipulation of Autophagy in Phagocytes Facilitates Staphylococcus aureus Bloodstream Infection.

The capacity for intracellular survival within phagocytes is likely a critical factor facilitating the dissemination of Staphylococcus aureus in the host. To date, the majority of work on S. aureus-phagocyte interactions has focused on neutrophils and, to a lesser extent, macrophages, yet we understand little about the role played by dendritic cells (DCs) in the direct killing of this bacterium. Using bone marrow-derived DCs (BMDCs), we demonstrate for the first time that DCs can effectively kill S. aureus but that certain strains of S. aureus have the capacity to evade DC (and macrophage) killing by manipulation of autophagic pathways. Strains with high levels of Agr activity were capable of causing autophagosome accumulation, were not killed by BMDCs, and subsequently escaped from the phagocyte, exerting significant cytotoxic effects. Conversely, strains that exhibited low levels of Agr activity failed to accumulate autophagosomes and were killed by BMDCs. Inhibition of the autophagic pathway by treatment with 3-methyladenine restored the bactericidal effects of BMDCs. Using an in vivo model of systemic infection, we demonstrated that the ability of S. aureus strains to evade phagocytic cell killing and to survive temporarily within phagocytes correlated with persistence in the periphery and that this effect is critically Agr dependent. Taken together, our data suggest that strains of S. aureus exhibiting high levels of Agr activity are capable of blocking autophagic flux, leading to the accumulation of autophagosomes. Within these autophagosomes, the bacteria are protected from phagocytic killing, thus providing an intracellular survival niche within professional phagocytes, which ultimately facilitates dissemination.

Relative contribution of Th1 and Th17 cells in adaptive immunity to Bordetella pertussis: towards the rational design of an improved acellular pertussis vaccine.

Whooping cough caused by Bordetella pertussis is a re-emerging infectious disease despite the introduction of safer acellular pertussis vaccines (Pa). One explanation for this is that Pa are less protective than the more reactogenic whole cell pertussis vaccines (Pw) that they replaced. Although Pa induce potent antibody responses, and protection has been found to be associated with high concentrations of circulating IgG against vaccine antigens, it has not been firmly established that host protection induced with this vaccine is mediated solely by humoral immunity. The aim of this study was to examine the relative contribution of Th1 and Th17 cells in host immunity to infection with B. pertussis and in immunity induced by immunization with Pw and Pa and to use this information to help rationally design a more effective Pa. Our findings demonstrate that Th1 and Th17 both function in protective immunity induced by infection with B. pertussis or immunization with Pw. In contrast, a current licensed Pa, administered with alum as the adjuvant, induced Th2 and Th17 cells, but weak Th1 responses. We found that IL-1 signalling played a central role in protective immunity induced with alum-adsorbed Pa and this was associated with the induction of Th17 cells. Pa generated strong antibody and Th2 responses, but was fully protective in IL-4-defective mice, suggesting that Th2 cells were dispensable. In contrast, Pa failed to confer protective immunity in IL-17A-defective mice. Bacterial clearance mediated by Pa-induced Th17 cells was associated with cell recruitment to the lungs after challenge. Finally, protective immunity induced by an experimental Pa could be enhanced by substituting alum with a TLR agonist that induces Th1 cells. Our findings demonstrate that alum promotes protective immunity through IL-1ß-induced IL-17A production, but also reveal that optimum protection against B. pertussis requires induction of Th1, but not Th2 cells.