Staphylococcus aureus is a leading cause of skin and soft tissue infections (SSTIs), yet the cellular mediators of protective memory remain incompletely defined. Using a self-resolving murine SSTI model, we combined longitudinal single-cell RNA sequencing with flow cytometry to characterize immune memory within the skin following infection. We identified a persistent population of CD4+ T cells that remained in the skin after bacterial clearance and acquired a transcriptional program consistent with tissue residency, including expression of genes associated with tissue retention and long-term residence. Phenotypic analysis confirmed the emergence of a skin-resident memory T cell (Trm) population that persisted well beyond resolution of infection and displayed characteristics of a clonal T cell response. Upon reinfection, protective immunity was associated with rapid recall responses from these resident cells and was maintained despite FTY720-mediated blockade of lymphocyte egress from secondary lymphoid organs, demonstrating that circulating lymphocytes were dispensable for protection. Together, these findings identify infection-induced CD4+ Trms as a durable component of immune memory following S. aureus SSTI and support a role for tissue-resident immunity in protection against recurrent infection, highlighting CD4+ Trms as a potential target for future vaccine strategies.

Abstract Staphylococcus aureus persistently colonises the nasal tissue (NT) of a significant proportion of the population. The long-lasting impact that asymptomatic S. aureus exposure has on immune memory at colonised barrier sites is incompletely understood, potentially impacting vaccine responsiveness in a pre-exposed population. Tissue resident memory (TRM) cells are long-lived T-cells which remain poised at barrier sites for localised reactivation following antigen exposure. This study demonstrates an increase in NT CD4+ and γδ+ TRM cells in response to S. aureus colonisation, which undergo expansion and IL-17 production upon secondary S. aureus exposure. Interestingly, these cells were also capable of non-specific reactivation, with IL-17+ TRM cells in S. aureus colonised mice enhancing protection against K. pneumoniae infection. Ex-vivo data suggest that non-specific CD4+ TRM cell re-activation is pro-inflammatory cytokine dependent, but antigen independent. Overall, these findings demonstrate that S. aureus nasal colonisation shapes long-lasting TRM cell responses in the NT, which have the potential for non-specific bystander reactivation during subsequent heterologous infection.

Staphylococcus aureus bloodstream infections are a significant risk for hemodialysis patients, who would significantly benefit from a preventative vaccine. To-date S. aureus vaccine trials have failed, in part due to lack of consideration of pre-existing immune imprints in relevant patient cohorts. Using a machine learning algorithm this study revealed, prior S. aureus exposure in a cohort of 180 hemodialysis patients, was associated with an increase in circulating S. aureus antigen-specific exTh17 cells and reduced antigen-specific IL-10 producing CD4 cells and circulating NK cells and Vδ2 cells. It provides novel insights into pre-existing S. aureus immunity in hemodialysis patients that could inform next generation vaccine development.

Infectious diseases remain a global health challenge, driven by increasing antimicrobial resistance and the threat of emerging epidemics. Mycobacterium tuberculosis and Staphylococcus aureus are leading causes of mortality worldwide. Trained immunity - a form of innate immune memory - offers a promising approach to enhance pathogen clearance. Here, we demonstrate that IFN-γ induces trained immunity in human monocytes through a mechanism involving mTORC1 activation, glutaminolysis, and epigenetic remodeling. Macrophages derived from IFN-γ-trained monocytes exhibited increased glycolytic activity with enhanced cytokine and chemokine responses upon stimulation or infection. Crucially, trained macrophages had increased production of reactive oxygen species, which mediated enhanced bactericidal activity against methicillin-resistant S. aureus and M. tuberculosis. Furthermore, ATAC-sequencing analysis of IFN-γ-trained macrophages revealed increased chromatin accessibility in regions associated with host defense. Last, IFN-γ training restored impaired innate responses in macrophages from individuals homozygous for the TIRAP 180L polymorphism, a genetic variant associated with increased susceptibility to infection. These findings establish IFN-γ as a potent inducer of trained immunity in human monocytes and support its potential as a host-directed strategy to strengthen antimicrobial defenses, particularly in genetically susceptible individuals and high-risk clinical contexts.

ABSTRACTModel‐based clustering is a statistical approach to cluster analysis, which has been successfully deployed in a number of domains due to its principled framework, clear assumptions, and adaptability. For these reasons, there has been substantial interest in applying model‐based clustering methods to flow cytometry and mass cytometry data. The identification of relevant cell populations is a crucial step in the analysis of cytometry data for immunological research. Technological advances have led to a rapid increase in the dimensionality and complexity of cytometry data, prompting significant interest in the use of clustering algorithms in place of traditional manual data analysis techniques for cell population identification. This article highlights how model‐based clustering methods, such as mixture models, have been adapted to meet the many interesting and unusual challenges that present themselves to the researcher when analyzing flow and mass cytometry data. These innovations demonstrate that there is considerable potential for further methodological development and collaboration between the cytometry and model‐based clustering research communities.

Staphylococcus aureus is a formidable pathogen and major challenge to human health. However, the bacterium is dichotomous and also acts as an asymptomatic coloniser. Given its ubiquity, every individual has been exposed to the bacterium, which may 'tune' the host immune system. This prior exposure potentially hampers the efficacy of anti-S. aureus vaccines, diminishing their protective effect. Conversely, by preserving its niche as a commensal coloniser, the bacterium simultaneously contributes to host defence through interbacterial competition and stimulation of host immune responses, potentially fending off pathogenic threats. This review examines how S. aureus shapes host immunity via infection and colonisation and explores how S. aureus-associated immune tuning can be both problematic and beneficial for the host.

Staphylococcus aureus nasal colonisation is commonplace among healthy individuals, yet the immune mechanisms enabling bacterial persistence remain unclear. S. aureus drives local immunosuppression during nasal colonisation to facilitate persistence. This study reveals that S. aureus subverts microRNA-21 activity to promote IL-10 production within nasal tissue, while simultaneously impeding local pro-inflammatory responses. MiR-21 activity helps establish a S. aureus-induced immunosuppressive microenvironment, which supports S. aureus persistence. Macrophages, which are key IL-10 producers, rapidly upregulate miR-21 upon S. aureus exposure. MiR-21 expression also coincides with an increase in intracellular survival of S. aureus within macrophages. Furthermore, S. aureus represses macrophage glycolysis to promote intracellular survival, which is dependent upon miR-21. Upon S. aureus colonisation, miR-21- /- mice demonstrate an overall improved bacterial clearance compared to their wild-type counterparts. These findings highlight the targeting of miR-21, which controls glycolytic activity in macrophages, as a potential avenue to reducing bacterial persistence during S. aureus colonisation.

Staphylococcus aureus is a major human pathogen. An effective anti-S. aureus vaccine remains elusive as the correlates of protection are ill-defined. Targeting specific T cell populations is an important strategy for improving anti-S. aureus vaccine efficacy. Potential bottlenecks that remain are S. aureus-induced immunosuppression and the impact this might have on vaccine-induced immunity. S. aureus induces IL-10, which impedes effector T cell responses, facilitating persistence during both colonization and infection. Thus, it was hypothesized that transient targeting of IL-10 might represent an innovative way to improve vaccine efficacy. In this study, IL-10 expression was elevated in the nares of persistent carriers of S. aureus, and this was associated with reduced systemic S. aureus-specific Th1 responses. This suggests that systemic responses are remodeled because of commensal exposure to S. aureus, which negatively implicates vaccine function. To provide proof of concept that targeting immunosuppressive responses during immunization may be a useful approach to improve vaccine efficacy, we immunized mice with T cell-activating vaccines in combination with IL-10-neutralizing antibodies. Blocking IL-10 during vaccination enhanced effector T cell responses and improved bacterial clearance during subsequent systemic and subcutaneous infection. Taken together, these results reveal a potentially novel strategy for improving anti-S. aureus vaccine efficacy.

Staphylococcus aureus can induce trained immunity in murine macrophages offering protection against repeat exposure during S. aureus skin infection. Here we demonstrate that S. aureus exposure can result in non-specific trained immunity in humans and mice, enhancing macrophage responsiveness and bacterial clearance in a heterologous challenge. In humans, the enhanced macrophage responsiveness was accompanied by metabolic changes and histone modification. In mice, the enhanced responsiveness of macrophages occurred in conjunction with enhanced myelopoiesis. This report provides further insights on the host’s response to the bacterium S. aureus, indicating that exposure to this organism induces heterologous protection against subsequent gram-negative infection that is provided by macrophages. These findings support the hypothesis that S. aureus has evolved to develop a mutualistic relationship with the host, imbuing the host with enhanced capacity to protect itself from attack by alternative pathogens, while potentially allowing S. aureus to exert its dominance within its niche.

Staphylococcus aureus is an important human commensal which persistently colonizes up to 30% of the human population, predominantly within the nasal cavity. The commensal lifestyle of S. aureus is complex, and the mechanisms underpinning colonization are not fully understood. S. aureus can induce an immunosuppressive environment in the nasal tissue (NT) by driving IL-10 and IL-27 to facilitate nasal colonization, indicating that S. aureus has the capacity to modulate the local immune environment for its commensal habitation. Mounting evidence suggests commensal bacteria drive type 1 interferons (IFN-I) to establish an immunosuppressive environment and whilst S. aureus can induce IFN-I during infection, its role in colonization has not yet been examined. Here, we show that S. aureus preferentially induces IFN signaling in macrophages. This IFN-I in turn upregulates expression of proapoptotic genes within macrophages culminating in caspase-3 cleavage. Importantly, S. aureus was found to drive phagocytic cell apoptosis in the nasal tissue during nasal colonization in an IFN-I dependent manner with colonization significantly reduced under caspase-3 inhibition. Overall, loss of IFN-I signaling significantly diminished S. aureus nasal colonization implicating a pivotal role for IFN-I in controlling S. aureus persistence during colonization through its ability to induce phagocyte apoptosis. Together, this study reveals a novel strategy utilized by S. aureus to circumvent host immunity in the nasal mucosa to facilitate nasal colonization.

Atopic dermatitis (AD) is an inflammatory skin condition with a childhood prevalence of up to 25%. Microbial dysbiosis is characteristic of AD, with Staphylococcus aureus the most frequent pathogen associated with disease flares and increasingly implicated in disease pathogenesis. Therapeutics to mitigate the effects of S. aureus have had limited efficacy and S. aureus-associated temporal disease flares are synonymous with AD. An alternative approach is an anti-S. aureus vaccine, tailored to AD. Experimental vaccines have highlighted the importance of T cells in conferring protective anti-S. aureus responses; however, correlates of T cell immunity against S. aureus in AD have not been identified. We identify a systemic and cutaneous immunological signature associated with S. aureus skin infection (ADS.aureus) in a pediatric AD cohort, using a combined Bayesian multinomial analysis. ADS.aureus was most highly associated with elevated cutaneous chemokines IP10 and TARC, which preferentially direct Th1 and Th2 cells to skin. Systemic CD4+ and CD8+ T cells, except for Th2 cells, were suppressed in ADS.aureus, particularly circulating Th1, memory IL-10+ T cells, and skin-homing memory Th17 cells. Systemic γδ T cell expansion in ADS.aureus was also observed. This study suggests that augmentation of protective T cell subsets is a potential therapeutic strategy in the management of S. aureus in AD.

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 interleukin 17A (IL-17A) and interferon-gamma (IFN-gamma)-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. Most studies on T-cell responses to mucosal pathogens focus on the blood. Here we demonstrate tissue-resident memory T cells persist in the tonsil and nasal tissue of humans for decades after immunization with whole-cell pertussis vaccines.

AbstractThe respiratory tract is home to a diverse microbial community whose influence on local and systemic immune responses is only beginning to be appreciated. The airways have been linked with trafficking of myelin-specific T cells in the pre-clinical stages of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Th17 cells are important pathogenic effectors in MS and EAE but are innocuous immediately following differentiation. Upregulation of the cytokine GM-CSF appears to be a critical step in their acquisition of pathogenic potential, but little is known about the mechanisms that mediate this process. Here, primed myelin-specific Th17 cells were transferred to congenic recipient mice prior to exposure to various human respiratory tract-associated bacteria and T cell trafficking, phenotype and the severity of resulting EAE monitored. Disease was exacerbated in mice exposed to the ProteobacteriaMoraxella catarrhalisandKlebsiella pneumoniae,but not the FirmicuteVeillonella parvula, and this was associated with a significant increase in GM-CSF+and GM-CSF+IFNγ+ex-Th17-like donor CD4 T cells in the lungs and CNS of these mice. These findings support the concept that respiratory bacteria may contribute to the pathophysiology of CNS autoimmunity by modulating pathogenicity in crucial T cell subsets that orchestrate neuroinflammation.

Abstract Background The National Clinical Programme for Older Adults (NCPOP) advocates for timely access to integrated care that is attuned to the complex needs of older adults. Frailty Intervention teams have been introduced in Emergency Departments (ED) around the country, and have proven effectiveness in reducing wait times and hospital length of stay in older patients. The introduction of the Pathfinder service—a collaboration between acute hospitals and the National Ambulance Service—is also proving effective in reducing ED attendances for low-acuity problems in older adults. Methods We aimed to develop pathways between a Gerontological Emergency Department Intervention (GEDI) team, and a new Pathfinder team within our hospital catchment area, in order to support older adults to remain at home where safe and feasible. A mapping exercise of potential pathways between these services was conducted. Suitability criteria were developed and a communication strategy for case discussion was formulated. A collaborative approach was taken to ensure continuous two-way communication, and a patient-focussed ethos maintained throughout. Results A total of four open-ended pathways were identified between the two services: (1) Pathfinder convey patient to ED via GEDI team, and patient discharged from ED back to Pathfinder for follow up; (2) GEDI identify patient suitable for ED discharge, supported by Pathfinder team; (3) Patients discharged outside of GEDI operational hours (08:00–18:30) supported by Pathfinder team (where already assessed by GEDI); (4) Patients conveyed by Pathfinder, identified as suitable for Age-Related ANP-led Assessment Unit. Conclusion Close collaboration between two frailty-focussed older persons’ teams has helped to avoid unnecessary hospital admissions among frail older adults, and provide more gerontologically-attuned care on the acute floor. Through this close collaboration, we are reflecting the NCPOP aims of using integration and Comprehensive Geriatric Assessment to support older people to live well in their own homes.

The bloodstream represents a hostile environment that bacteria must overcome to cause bacteraemia. To understand how the major human pathogen Staphylococcus aureus manages this we have utilised a functional genomics approach to identify a number of new loci that affect the ability of the bacteria to survive exposure to serum, the critical first step in the development of bacteraemia. The expression of one of these genes, tcaA, was found to be induced upon exposure to serum, and we show that it is involved in the elaboration of a critical virulence factor, the wall teichoic acids (WTA), within the cell envelope. The activity of the TcaA protein alters the sensitivity of the bacteria to cell wall attacking agents, including antimicrobial peptides, human defence fatty acids, and several antibiotics. This protein also affects the autolytic activity and lysostaphin sensitivity of the bacteria, suggesting that in addition to changing WTA abundance in the cell envelope, it also plays a role in peptidoglycan crosslinking. With TcaA rendering the bacteria more susceptible to serum killing, while simultaneously increasing the abundance of WTA in the cell envelope, it was unclear what effect this protein may have during infection. To explore this, we examined human data and performed murine experimental infections. Collectively, our data suggests that whilst mutations in tcaA are selected for during bacteraemia, this protein positively contributes to the virulence of S. aureus through its involvement in altering the cell wall architecture of the bacteria, a process that appears to play a key role in the development of bacteraemia.

The respiratory tract is home to a diverse microbial community whose influence on local and systemic immune responses is only beginning to be appreciated. The airways have been linked with the trafficking of myelin-specific T-cells in the preclinical stages of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Th17 cells are important pathogenic effectors in MS and EAE but are innocuous immediately following differentiation. Upregulation of the cytokine GM-CSF appears to be a critical step in their acquisition of pathogenic potential, but little is known about the mechanisms that mediate this process. Here, primed myelin-specific Th17 cells were transferred to congenic recipient mice prior to exposure to various human respiratory tract-associated bacteria and T-cell trafficking, phenotype and the severity of resulting EAE were monitored. Disease was exacerbated in mice exposed to the Proteobacteria Moraxella catarrhalis and Klebsiella pneumoniae, but not the Firmicute Veillonella parvula, and this was associated with significantly increased GM-CSF+ and GM-CSF+IFN gamma+ ex-Th17-like donor CD4 T cells in the lungs and central nervous system (CNS) of these mice. These findings support the concept that respiratory bacteria may contribute to the pathophysiology of CNS autoimmunity by modulating pathogenicity in crucial T-cell subsets that orchestrate neuroinflammation.

Inflammatory bowel diseases (IBD) are chronic intestinal disorders that result from an inappropriate inflammatory response to the microbiota in genetically susceptible individuals, often triggered by environmental stressors. Part of this response is the persistent inflammation and tissue injury associated with deficiency or excess of reactive oxygen species (ROS). The NADPH oxidase NOX1 is highly expressed in the intestinal epithelium, and inactivating NOX1 missense mutations are considered a risk factor for developing very early onset IBD. Albeit NOX1 has been linked to wound healing and host defence, many questions remain about its role in intestinal homeostasis and acute inflammatory conditions. Here, we used in vivo imaging in combination with inhibitor studies and germ-free conditions to conclusively identify NOX1 as essential superoxide generator for microbiota-dependent peroxynitrite production in homeostasis and during early endotoxemia. NOX1 loss-of-function variants cannot support peroxynitrite production, suggesting that the gut barrier is persistently weakened in these patients. One of the loss-of-function NOX1 variants, NOX1 p. Asn122His, features replacement of an asparagine residue located in a highly conserved HxxxHxxN motif. Modelling the NOX1-p22phox complex revealed near the distal heme an internal pocket restricted by His119 and Asn122 that is part of the oxygen reduction site. Functional studies in several human NADPH oxidases show that substitution of asparagine with amino acids with larger side chains is not tolerated, while smaller side chains can support catalytic activity. Thus, we identified a previously unrecognized structural feature required for the electron transfer mechanism in human NADPH oxidases.

Excessive inflammation-associated coagulation is a feature of infectious diseases, occurring in such conditions as bacterial sepsis and COVID-19. It can lead to disseminated intravascular coagulation, one of the leading causes of mortality worldwide. Recently, type I interferon (IFN) signaling has been shown to be required for tissue factor (TF; gene name F3 ) release from macrophages, a critical initiator of coagulation, providing an important mechanistic link between innate immunity and coagulation. The mechanism of release involves type I IFN-induced caspase-11 which promotes macrophage pyroptosis. Here we find that F3 is a type I IFN-stimulated gene. Furthermore, F3 induction by lipopolysaccharide (LPS) is inhibited by the anti-inflammatory agents dimethyl fumarate (DMF) and 4-octyl itaconate (4-OI). Mechanistically, inhibition of F3 by DMF and 4-OI involves suppression of Ifnb1 expression. Additionally, they block type I IFN- and caspase-11-mediated macrophage pyroptosis, and subsequent TF release. Thereby, DMF and 4-OI inhibit TF-dependent thrombin generation. In vivo, DMF and 4-OI suppress TF-dependent thrombin generation, pulmonary thromboinflammation, and lethality induced by LPS, E. coli , and S. aureus , with 4-OI additionally attenuating inflammation-associated coagulation in a model of SARS-CoV-2 infection. Our results identify the clinically approved drug DMF and the pre-clinical tool compound 4-OI as anticoagulants that inhibit TF-mediated coagulopathy via inhibition of the macrophage type I IFN-TF axis.

Introductory Paragraph / AbstractThe bloodstream represents a hostile environment that bacteria must overcome to cause bacteraemia. To understand how the major human pathogenStaphylococcus aureusmanages this we have utilised a functional genomics approach to identify a number of new loci that affect the ability of the bacteria to survive exposure to serum, the critical first step in the development of bacteraemia. The expression of one of these genes,tcaA,was found to be induced upon exposure to serum, and we show that it is involved in the elaboration of a critical virulence factor, the wall teichoic acids (WTA), within the cell envelope. The activity of the TcaA protein alters the sensitivity of the bacteria to cell wall attacking agents, including antimicrobial peptides, human defence fatty acids, and several antibiotics. This protein also affects the autolytic activity and lysostaphin sensitivity of the bacteria, suggesting that in addition to changing WTA abundance in the cell envelope, it also plays a role in peptidoglycan crosslinking. With TcaA rendering the bacteria more susceptible to serum killing, while simultaneously increasing the abundance of WTA in the cell envelope, it was unclear what effect this protein may have during infection. To explore this, we examined human data and performed murine experimental infections. Collectively, our data suggests that whilst mutations intcaAare selected for during bacteraemia, this protein positively contributes to the virulence ofS. aureusthrough its involvement in altering the cell wall architecture of the bacteria, a process that appears to play a key role in the development of bacteraemia.