Search

Infections with Staphylococcus aureus (S. aureus) have been reported from various organs ranging from asymptomatic colonization to severe infections and sepsis. Although considered an extracellular pathogen, S. aureus can invade and persist in professional phagocytes such as monocytes and macrophages. Its capability to persist and manipulate macrophages is considered a critical step to evade host antimicrobial reactions. We leveraged a recently established human liver-on-chip model to demonstrate that S. aureus specifically targets macrophages as essential niche facilitating bacterial persistence and phenotype switching to small colony variants (SCVs). In vitro, M2 polarization was found to favor SCV-formation and was associated with increased intracellular bacterial loads in macrophages, increased cell death, and impaired recruitment of circulating monocytes to sites of infection. These findings expand the knowledge about macrophage activation in the liver and its impact on bacterial persistence and dissemination in the course of infection.

Infections withStaphylococcus aureus(S. aureus) have been reported from various organs ranging from asymptomatic colonization to severe infections and sepsis associated with multiple organ dysfunction. Although considered an extracellular pathogen,S. aureuscan invade and persist in professional phagocytes such as monocytes and macrophages. Its capability to persist and manipulate phagocytes is considered a critical step to evade host antimicrobial reactions. For the first time we leveraged a human liver-on-chip model and tailored image analysis algorithms to demonstrate thatS. aureus(USA300) specifically targets macrophages in the liver models as essential niche facilitating bacterial persistence and phenotype switching to small colony variants (SCVs).In vitroM2 polarization was found to favor SCV-formation and was associated with increased intracellular bacterial loads in macrophages, increased cell death, and impaired recruitment of circulating monocytes to sites of infection. These findings expand the knowledge about the role of liver macrophages in the course of systemic infection. Further, the results might be relevant for understanding infection mechanisms in patients with chronic liver disease such as fibrosis that display increased frequencies of M2 polarized liver macrophages and have a higher risk for developing chronic infections and relapsing bacteremia.

Staphylococcus aureus is a human commensal organism and opportunist pathogen, causing potentially fatal disease. The presence of non-pathogenic microflora or their components, at the point of infection, dramatically increases S. aureus pathogenicity, a process termed augmentation. Augmentation is associated with macrophage interaction but by a hitherto unknown mechanism. Here, we demonstrate a breadth of cross-kingdom microorganisms can augment S. aureus disease and that pathogenesis of Enterococcus faecalis can also be augmented. Co-administration of augmenting material also forms an efficacious vaccine model for S. aureus. In vitro, augmenting material protects S. aureus directly from reactive oxygen species (ROS), which correlates with in vivo studies where augmentation restores full virulence to the ROS-susceptible, attenuated mutant katA ahpC. At the cellular level, augmentation increases bacterial survival within macrophages via amelioration of ROS, leading to proliferation and escape. We have defined the molecular basis for augmentation that represents an important aspect of the initiation of infection., Author summary S. aureus is a commensal inhabitant of the human skin and nares. However, it can cause serious diseases if it is able to breach our protective barriers such as the skin, often via wounds or surgery. If infection occurs via a wound, this initial inoculum contains both the pathogen, other members of the microflora and also wider environmental microbes. We have previously described “augmentation”, whereby this other non-pathogenic material can enhance the ability of S. aureus to lead to a serious disease outcome. Here we have determined the breadth of augmenting material and elucidated the cellular and molecular basis for its activity. Augmentation occurs via shielding of S. aureus from the direct bactericidal effects of reactive oxygen species produced by macrophages. This initial protection enables the effective establishment of S. aureus infection. Understanding augmentation not only explains an important facet of the interaction of S. aureus with our innate immune system, but also provides a platform for the development of novel prophylaxis approaches.

Non-alcoholic fatty liver disease ranges from steatosis to non-alcoholic steatohepatitis (NASH), potentially progressing to cirrhosis and hepatocellular carcinoma (HCC). Here, we show that platelet number, platelet activation and platelet aggregation are increased in NASH but not in steatosis or insulin resistance. Antiplatelet therapy (APT; aspirin/clopidogrel, ticagrelor) but not nonsteroidal anti-inflammatory drug (NSAID) treatment with sulindac prevented NASH and subsequent HCC development. Intravital microscopy showed that liver colonization by platelets depended primarily on Kupffer cells at early and late stages of NASH, involving hyaluronan-CD44 binding. APT reduced intrahepatic platelet accumulation and the frequency of platelet-immune cell interaction, thereby limiting hepatic immune cell trafficking. Consequently, intrahepatic cytokine and chemokine release, macrovesicular steatosis and liver damage were attenuated. Platelet cargo, platelet adhesion and platelet activation but not platelet aggregation were identified as pivotal for NASH and subsequent hepatocarcinogenesis. In particular, platelet-derived GPIbα proved critical for development of NASH and subsequent HCC, independent of its reported cognate ligands vWF, P-selectin or Mac-1, offering a potential target against NASH.

Staphylococcus aureus virulence has been associated with the production of phenol-soluble modulins (PSMs). These PSMs have distinct virulence functions and are known to activate, attract and lyse neutrophils. These PSM-associated biological functions are inhibited by lipoproteins in vitro. We set out to address whether lipoproteins neutralize staphylococcal PSM-associated virulence in experimental animal models. Serum from both LCAT an ABCA1 knockout mice strains which are characterised by near absence of high-density lipoprotein (HDL) levels, was shown to fail to protect against PSM-induced neutrophil activation and lysis in vitro. Importantly, PSM-induced peritonitis in LCAT−/− mice resulted in increased lysis of resident peritoneal macrophages and enhanced neutrophil recruitment into the peritoneal cavity. Notably, LCAT−/− mice were more likely to succumb to staphylococcal bloodstream infections in a PSM-dependent manner. Plasma from homozygous carriers of ABCA1 variants characterized by very low HDL-cholesterol levels, was found to be less protective against PSM-mediated biological functions compared to healthy humans. Therefore, we conclude that lipoproteins present in blood can protect against staphylococcal PSMs, the key virulence factor of community-associated methicillin resistant S. aureus.

SummaryThe liver is exemplar to study tissue regeneration due to its inherent ability of repair and regrowth. It replaces its lost or injured tissue by the proliferation, interaction and temporal coordination of multiple residential cell types. Until now we lacked a detailed description of the specific contributions of each cell type to the regenerative process, and therefore analyzed mouse livers 0, 3, 6, and 24 hours following two-thirds partial hepatectomy (PHx) by single cell RNA-sequencing (scRNA-seq) and mass cytometry. Our resulting genome wide temporal atlas contains the time dependent transcriptional changes in hepatocytes, endothelial cells, bone marrow-derived macrophages (BMDM) and Kupffer cells. In addition, it describes the cell specific contribution of mitogenic growth factors from biliary epithelial, endothelial and stellate cells as well as chemokines and cytokines from BMDM and granulocytes. And interestingly, Kupffer cells as opposed to hepatocytes emerged as the first cell to proliferate presenting a new dynamic in the liver following PHx. Here, we provide a robust data set at cellular resolution to uncover new elements and revisit current dogmas on the mechanisms underlying liver regeneration. To facilitate access to the data, we have launched the portal www.phxatlas.ch in which the scRNA-seq data can be visualized.

Methicillin-resistant Staphylococcus aureus (MRSA) first emerged after methicillin was introduced to combat penicillin resistance, and its prevalence in Canada has increased since the first MRSA outbreak in the early 1980s. We reviewed the existing literature on MRSA prevalence in Canada over time and in diverse populations across the country. MRSA prevalence increased steadily in the 1990s and 2000s and remains a public health concern in Canada, especially among vulnerable populations, such as rural, remote, and Indigenous communities. Antibiotic resistance patterns and risk factors for MRSA infection were also reported. All studies reported high susceptibility (>85%) to trimethoprim-sulfamethoxazole, with no significant resistance reported for vancomycin, linezolid, or rifampin. While MRSA continues to have susceptibility to several antibiotics, the high and sometimes variable resistance rates to other drugs underscores the importance of antimicrobial stewardship. Risk factors for high MRSA infection rates related to infection control measures, low socioeconomic status, and personal demographic characteristics were also reported. Additional surveillance, infection control measures, enhanced anti-microbial stewardship, and community education programs are necessary to decrease MRSA prevalence and minimize the public health risk posed by this pathogen.

Background and aims Bacterial infections are common and severe in cirrhosis, but their pathogenesis is poorly understood. Dysfunction of liver macrophages may play a role, but information about their function in cirrhosis is limited. Our aims were to investigate the specific profile and function of liver macrophages in cirrhosis and their contribution to infections. Macrophages from human cirrhotic livers were characterized phenotypically by transcriptome analysis and flow cytometry; function was assessed in vivo by single photon emission computerized tomography in patients with cirrhosis. Serum levels of specific proteins and expression in peripheral monocytes were determined by ELISA and flow cytometry. In vivo phagocytic activity of liver macrophages was measured by spinning disk intravital microscopy in a mouse model of chronic liver injury. Approach and results Liver macrophages from patients with cirrhosis overexpressed proteins related to immune exhaustion, such as programmed death ligand 1 (PD-L1), macrophage receptor with collagenous structure (MARCO), and CD163. In vivo phagocytic activity of liver macrophages in patients with cirrhosis was markedly impaired. Monocytes from patients with cirrhosis showed overexpression of PD-L1 that paralleled disease severity, correlated with its serum levels, and was associated with increased risk of infections. Blockade of PD-L1 with anti-PD-L1 antibody caused a shift in macrophage phenotype toward a less immunosuppressive profile, restored liver macrophage in vivo phagocytic activity, and reduced bacterial dissemination. Conclusion Liver cirrhosis is characterized by a remarkable impairment of phagocytic function of macrophages associated with an immunosuppressive transcriptome profile. The programmed cell death receptor 1/PD-L1 axis plays a major role in the impaired activity of liver macrophages. PD-L1 blockade reverses the immune suppressive profile and increases antimicrobial activity of liver macrophages in cirrhosis.

Macrophages seal 'em in the coelom GATA6 + macrophages resident in body cavities exhibit both phagocytic and repair functions. However, the mechanisms by which these cells can identify and migrate to sites of injury have remained unclear. Using intravital imaging of mouse peritoneal cavities, Zindel et al. report that GATA6 + macrophages rapidly assemble clot-like structures in a process strongly analogous to thrombosis (see the Perspective by Herrick and Allen). The formation of these aggregates requires the expression of macrophage scavenger receptor domains and acts to plug wounds and promote healing. This pathway can be inadvertently activated during medical procedures, when macrophage aggregates can promote the generation of abdominal scar tissue known as adhesions. Inhibition of macrophage scavenger receptors may therefore be a useful therapeutic approach after surgeries that cause injury to body cavities. Science , this issue p. eabe0595 ; see also p. 993

BACKGROUND AND AIMS Kupffer cells (KCs) are the resident intravascular phagocyte population of the liver and critical to the capture and killing of bacteria. Calcineurin/nuclear factor of activated T cells (NFAT) inhibitors (CNIs) such as tacrolimus are used to prevent rejection in solid organ transplant recipients. Although their effect on lymphocytes has been studied extensively, there are limited experimental data about if and how CNIs shape innate immunity, and whether this contributes to the higher rates of infection observed in patients taking CNIs. APPROACH AND RESULTS Here, we investigated the impact of tacrolimus treatment on innate immunity and, more specifically, on the capability of Kupffer cells (KCs) to fight infections. Retrospective analysis of data of >2,700 liver transplant recipients showed that taking calcineurin inhibitors such as tacrolimus significantly increased the likelihood of Staphylococcus aureus infection. Using a mouse model of acute methicillin-resistant S. aureus (MRSA) bacteremia, most bacteria were sequestered in the liver and we found that bacteria were more likely to disseminate and kill the host in tacrolimus-treated mice. Using imaging, we unveiled the mechanism underlying this observation: the reduced capability of KCs to capture, phagocytose, and destroy bacteria in tacrolimus-treated animals. Furthermore, in a gene expression analysis of infected KCs, the triggering receptor expressed on myeloid cells 1 (TREM1) pathway was the one with the most significant down-regulation after tacrolimus treatment. TREM1 inhibition likewise inhibited KC bacteria capture. TREM1 levels on neutrophils as well as the overall neutrophil response after infection were unaffected by tacrolimus treatment. CONCLUSIONS Our results indicate that tacrolimus treatment has a significant impact directly on KCs and on TREM1, thereby compromising their capacity to fend off infections.

While the ontogeny and recruitment of the intestinal monocyte/macrophage lineage has been studied extensively, their precise localization and function has been overlooked. Here we show by imaging the murine small and large intestines in steady-state that intestinal CX3CR1+ macrophages form an interdigitated network intimately adherent to the entire mucosal lamina propria vasculature. The macrophages form contacts with each other, which are disrupted in the absence of microbiome, monocyte recruitment (Ccr2−/−), or monocyte conversion (Nr4a1−/−). In dysbiosis, gaps exist between the perivascular macrophages correlating with increased bacterial translocation from the lamina propria into the bloodstream. The recruitment of monocytes and conversion to macrophages during intestinal injury is also dependent upon CCR2, Nr4a1 and the microbiome. These findings demonstrate a relationship between microbiome and the maturation of lamina propria perivascular macrophages into a tight anatomical barrier that might function to prevent bacterial translocation. These cells are also critical for emergency vascular repair., Lamina propria macrophages are at the frontline of defense against intestinal pathogens. Here the authors reveal that CCR2 and NR4A1-dependent CX3CR1+ macrophages form a dense network around the vessels in the lamina propria, and implicate this anatomical structure into prevention of systemic bacterial dissemination.

Humans are well equipped to defend themselves against bacteria. The innate immune system employs diverse mechanisms to recognize, control and initiate a response that can destroy millions of different microbes. Microbes that evade the sophisticated innate immune system are able to escape detection and could become pathogens. The pathogens Streptococcus pneumoniae and Staphylococcus aureus are particularly successful due to the development of a wide variety of virulence strategies for bacterial pathogenesis and they invest significant efforts towards mechanisms that allow for neutrophil evasion. Neutrophils are a primary cellular defense and can rapidly kill invading microbes, which is an indispensable function for maintaining host health. This review compares the key features of Streptococcus pneumoniae and Staphylococcus aureus in epidemiology, with a specific focus on virulence mechanisms utilized to evade neutrophils in bacterial pathogenesis. It is important to understand the complex interactions between pathogenic bacteria and neutrophils so that we can disrupt the ability of pathogens to cause disease.

Pseudomonas aeruginosa is a major cause of severe infections that lead to bacteremia and high patient mortality. P. aeruginosa has evolved numerous evasion and subversion mechanisms that work in concert to overcome immune recognition and effector functions in hospitalized and immunosuppressed individuals. Here, we have used multilaser spinning-disk intravital microscopy to monitor the blood-borne stage in a murine bacteremic model of P. aeruginosa infection. P. aeruginosa adhered avidly to lung vasculature, where patrolling neutrophils and other immune cells were virtually blind to the pathogen’s presence. This cloaking phenomenon was attributed to expression of Psl exopolysaccharide. Although an anti-Psl mAb activated complement and enhanced neutrophil recognition of P. aeruginosa, neutrophil-mediated clearance of the pathogen was suboptimal owing to a second subversion mechanism, namely the type 3 secretion (T3S) injectisome. Indeed, T3S prevented phagosome acidification and resisted killing inside these compartments. Antibody-mediated inhibition of the T3S protein PcrV did not enhance bacterial phagocytosis but did enhance killing of the few bacteria ingested by neutrophils. A bispecific mAb targeting both Psl and PcrV enhanced neutrophil uptake of P. aeruginosa and also greatly increased inhibition of T3S function, allowing for phagosome acidification and bacterial killing. These data highlight the need to block multiple evasion and subversion mechanisms in tandem to kill P. aeruginosa.

The spleen is integral for protection against encapsulated bacteria. Using intravital imaging, Deniset et al. demonstrate that resident neutrophil and macrophage populations in the spleen coordinate the rapid clearance of Streptococcus pneumoniae, ensuring sufficient time for subseqent protective antibody production., The spleen plays an integral protective role against encapsulated bacterial infections. Our understanding of the associated mechanisms is limited to thymus-independent (TI) antibody production by the marginal zone (MZ) B cells, leaving the contribution of other splenic compartments such as the red pulp (RP) largely unexplored despite asplenic patients succumbing to the infection in the first 24 h, suggesting important antibody-independent mechanisms. In this study, using time-lapse intravital imaging of the spleen, we identify a tropism for Streptococcus pneumoniae in this organ mediated by tissue-resident MZ and RP macrophages and a protective role for two distinct splenic neutrophil populations (Ly6Ghi and Ly6Gintermediate) residing in the splenic RP. Splenic mature neutrophils mediated pneumococcal clearance in the spleen by plucking bacteria off the surface of RP macrophages that caught the majority of bacteria in a complement-dependent manner. This neutrophil phagocytic capacity was further enhanced after TI antibody production. Resident immature neutrophils (Ly6Gintermediate) in the spleen undergo emergency proliferation and mobilization from their splenic niche after pneumococcal stimulation to increase the effector mature neutrophil pool. We demonstrate that splenic neutrophils together with two macrophage populations and MZ B cells regulate systemic S. pneumoniae clearance through complementary mechanisms.

Staphylococcus aureus and its toxins have been linked to disease progression and mortality in advanced stages of cutaneous T-cell lymphoma (CTCL). CD8+ T cells play a crucial role in anti-cancer responses and high CD8+ T cell numbers in tumor lesions are associated with a favorable prognosis in CTCL. Here, we show that CD8+ T cells from both healthy donors and Sézary syndrome patients are highly susceptible to cell death induced by Staphylococcal alpha-toxin, whereas malignant T cells are not. Importantly, alpha-toxin almost completely blocks cytotoxic killing of CTCL tumor cells by peptide-specific CD8+ T cells, leading to their escape from induced cell death and continued proliferation. These findings suggest that alpha-toxin may favor the persistence of malignant CTCL cells in vivo by inhibiting CD8+ T cell cytotoxicity. Thus, we propose a novel mechanism by which colonization with Staphylococcus aureus may contribute to cancer immune evasion and disease progression in CTCL.

Summary Neutrophils with immunoregulatory properties, also referred to as type-2 neutrophils (N2), myeloid-derived suppressor cells (MDSCs), or tumor-associated neutrophils (TANs), comprise a heterogeneous subset of cells that arise from unknown precursors in response to poorly understood cues. Here, we find that, in several models of liver autoimmunity, pharmacologically induced, autoantigen-specific T regulatory type-1 (TR1) cells and TR1-cell-induced B regulatory (Breg) cells use five immunoregulatory cytokines to coordinately recruit neutrophils into the liver and program their transcriptome to generate regulatory neutrophils. The liver-associated neutrophils from the treated mice, unlike their circulating counterparts or the liver neutrophils of sick mice lacking antigen-specific TR1 cells, are proliferative, can transfer disease protection to immunocompromised hosts engrafted with pathogenic effectors, and blunt antigen-presentation and local autoimmune responses via cathelin-related anti-microbial peptide (CRAMP), a cathelicidin, in a CRAMP-receptor-dependent manner. These results, thus, identify antigen-specific regulatory T cells as drivers of tissue-restricted regulatory neutrophil formation and CRAMP as an effector of regulatory neutrophil-mediated immunoregulation.

Kupffer cells (KCs), the vast pool of intravascular macrophages in the liver, help to clear blood-borne pathogens. The mechanisms by which KCs capture circulating pathogens remain unknown. Here we use intra-vital imaging of mice infected with Staphylococcus aureus to directly visualize the dynamic process of bacterial capture in the liver. Circulating S. aureus were captured by KCs in a manner dependent on the macrophage complement receptor CRIg, but the process was independent of complement. CRIg bound Staphylococcus aureus specifically through recognition of lipoteichoic acid (LTA), but not cell-wall-anchored surface proteins or peptidoglycan. Blocking the recognition between CRIg and LTA in vivo diminished the bacterial capture in liver and led to systemic bacterial dissemination. All tested Gram-positive, but not Gram-negative, bacteria bound CRIg in a complement-independent manner. These findings reveal a pattern recognition role for CRIg in the direct capture of circulating Gram-positive bacteria from the bloodstream.

Methicillin-resistant Staphylococcus aureus (MRSA) bacteremia is reaching epidemic proportions causing morbidity, mortality, and chronic disease due to relapses, suggesting an intracellular reservoir. Using spinning-disk confocal intravital microscopy to track MRSA-GFP in vivo, we identified that within minutes after intravenous infection MRSA is primarily sequestered and killed by intravascular Kupffer cells (KCs) in the liver. However, a minority of the Staphylococci overcome the KC’s antimicrobial defenses. These bacteria survive and proliferate for many days within this intracellular niche, where they remain undetected by recruited neutrophils. Over time, the KCs lyse, releasing bacteria into the circulation, enabling dissemination to other organs such as the kidneys. Vancomycin, the antibiotic of choice to treat MRSA bacteremia, could not penetrate the KCs to eradicate intracellular MRSA. However, based on the intravascular location of these specific macrophages, we designed a liposomal formulation of vancomycin that is efficiently taken up by KCs and diminished the intracellular MRSA. Targeting the source of the reservoir dramatically protected the liver but also dissemination to other organs, and prevented mortality. This vancomycin formulation strategy could help treat patients with Staphylococcal bacteremia without a need for novel antibiotics by targeting the previously inaccessible intracellular reservoir in KCs.

SUMMARY Skin is one of the most common sites of host immune response against Staphylococcus aureus infection. Here, through a combination of in vitro assays, mouse models, and intravital imaging, we find that S. aureus immune evasion in skin is controlled by a cascade composed of the ArlRS two-component regulatory system and its downstream effector, MgrA. S. aureus lacking either ArlRS or MgrA is less virulent and unable to form correct abscess structure due to de-repression of a giant surface protein, Ebh. These S. aureus mutants also have decreased expression of immune evasion factors (leukocidins, chemotaxis-inhibitory protein of S. aureus [CHIPS], staphylococcal complement inhibitor [SCIN], and nuclease) and are unable to kill neutrophils, block their chemotaxis, degrade neutrophil extracellular traps, and survive direct neutrophil attack. The combination of disrupted abscess structure and reduced immune evasion factors makes S. aureus susceptible to host defenses. ArlRS and MgrA are therefore the main regulators of S. aureus immune evasion and promising treatment targets., In brief Kwiecinski et al. show that Staphylococcus aureus uses the ArlRS-MgrA regulatory system to coordinate gene expression during skin infection. This cascade is required for proper abscess structuring and evasion of the host innate immune system, which together are essential for full S. aureus virulence., Graphical Abstract

Essentially all Staphylococcus aureus (S. aureus) bacteria that gain access to the circulation are plucked out of the bloodstream by the intravascular macrophages of the liver - the Kupffer cells. It is also thought that these bacteria are disseminated via the bloodstream to other organs. Our data show that S. aureus inside Kupffer cells grew and escaped across the mesothelium into the peritoneal cavity and immediately infected GATA-binding factor 6-positive (GATA6+) peritoneal cavity macrophages. These macrophages provided a haven for S. aureus, thereby delaying the neutrophilic response in the peritoneum by 48 hours and allowing dissemination to various peritoneal and retroperitoneal organs including the kidneys. In mice deficient in GATA6+ peritoneal macrophages, neutrophils infiltrated more robustly and reduced S. aureus dissemination. Antibiotics administered i.v. did not prevent dissemination into the peritoneum or to the kidneys, whereas peritoneal administration of vancomycin (particularly liposomal vancomycin with optimized intracellular penetrance capacity) reduced kidney infection and mortality, even when administered 24 hours after infection. These data indicate that GATA6+ macrophages within the peritoneal cavity are a conduit of dissemination for i.v. S. aureus, and changing the route of antibiotic delivery could provide a more effective treatment for patients with peritonitis-associated bacterial sepsis.

Staphylococcus aureus is implicated in disease progression in cutaneous T-cell lymphoma (CTCL). Here, we demonstrate that malignant T cell lines derived from CTCL patients as well as primary malignant CD4+ T cells from Sézary syndrome patients are considerably more resistant to alpha-toxin-induced cell death than their non-malignant counterparts. Thus, in a subset of Sézary syndrome patients the ratio between malignant and non-malignant CD4+ T cells increases significantly following exposure to alpha-toxin. Whereas toxin-induced cell death is ADAM10 dependent in healthy CD4+ T cells, resistance to alpha-toxin in malignant T cells involves both downregulation of ADAM10 as well as other resistance mechanisms. In conclusion, we provide first evidence that Staphylococcus aureus derived alpha-toxin can tilt the balance between malignant and non-malignant CD4+ T cells in CTCL patients. Consequently, alpha-toxin may promote disease progression through positive selection of malignant CD4+ T cells, identifying alpha-toxin as a putative drug target in CTCL.

Females have an overall advantage over males in resisting Gram-negative bacteremias, thus hinting at sexual dimorphism of immunity during infections. Here, through intravital microscopy, we observed a sex-biased difference in the capture of blood-borne bacteria by liver macrophages, a process that is critical for the clearance of systemic infections. Complement opsonization was indispensable for the capture of enteropathogenic Escherichia coli (EPEC) in male mice; however, a faster complement component 3-independent process involving abundant preexisting antibodies to EPEC was detected in female mice. These antibodies were elicited predominantly in female mice at puberty in response to estrogen regardless of microbiota-colonization conditions. Estrogen-driven antibodies were maternally transferrable to offspring and conferred protection during infancy. These antibodies were conserved in humans and recognized specialized oligosaccharides integrated into the bacterial lipopolysaccharide and capsule. Thus, an estrogen-driven, innate antibody-mediated immunological strategy conferred protection to females and their offspring.

Staphylococcus lugdunensis is a commensal bacterium that can cause serious infection suggesting an ability to circumvent aspects of host immunity. We demonstrate here that macrophages fail to kill ingested S. lugdunensis and the bacteria persist for extended periods, without replicating, within mature LAMP-1-positive phagolysosomes. Phagocytosed S. lugdunensis also do not intoxicate host cells in contrast to Staphylococcus aureus. Optimal survival of S. lugdunensis requires O-acetylated peptidoglycan because an oatA mutant, which is more sensitive to killing by lysozyme than wild type, survived to a lesser extent in macrophages. In vitro models of macrophage infection reveal that viable intracellular S. lugdunensis bacteria can be made to grow by pharmacologic perturbation of phagosome function or by phagocyte intoxication by S. aureus toxins. Remarkably, replicating S. lugdunensis is not constrained by LAMP-1 and phosphatidylserine-positive endomembranes, which is distinct from S. aureus that replicates within phagolysosomes. In vivo, S. lugdunensis can also reside in the murine Kupffer cell where the bacteria persist without replicating and require oatA to resist killing in vivo. The intracellular environment of the macrophage represents a niche where S. lugdunensis can exist while protected from extracellular immune factors and may serve as a reservoir from which these bacteria could disseminate.

Summary During sepsis, small blood vessels can become occluded by large platelet aggregates of poorly understood etiology. During Staphylococcal aureus infection, sepsis severity is linked to the bacterial α-toxin (α-hemolysin, AT) through unclear mechanisms. In this study, we visualized intravascular events in the microcirculation and found that intravenous AT injection induces rapid platelet aggregation, forming dynamic micro-thrombi in the microcirculation. These aggregates are retained in the liver sinusoids and kidney glomeruli, causing multi-organ dysfunction. Acute staphylococcal infection results in sequestration of most bacteria by liver macrophages. Platelets are initially recruited to these macrophages and help eradicate S. aureus. However, at later time points, AT causes aberrant and damaging thrombosis throughout the liver. Treatment with an AT neutralizing antibody (MEDI4893∗) prevents platelet aggregation and subsequent liver damage, without affecting the initial and beneficial platelet recruitment. Thus, AT neutralization may represent a promising approach to combat staphylococcal-induced intravascular coagulation and organ dysfunction.

It is central to the field of bacterial pathogenesis to define how bacteria are killed by phagocytic cells. During phagocytosis, the microbe is localized to the phagolysosome where crucial defense mechanisms such as acidification and production of reactive oxygen species (ROS) are initiated. This process has extensively been studied in vitro, however many resident tissue phagocytes will phenotypically change upon isolation from their natural environment. Therefore, interrogation of phagocytosis and phagosomal function of cells in the context of their natural tissue environment enhances our understanding of the biological process in vivo. This article outlines a real-time intravital microscopy protocol that utilizes fluorescent dyes to study the process of phagocytosis, which reveals acidification and oxidation of individual bacteria inside host cells of living animals. The novelty of this technique exists in use of bacteria that are covalently labelled with the fluorescent dyes Oxyburst and pHrodo, which respectively report on oxidation or acidification. Intravital microscopy is applied to visualize the uptake and subsequent oxidation or acidification of reporter bacteria in the organ of interest. Fluorescently labelled antibodies can be used to counter stain for host immune cells such as neutrophils and macrophages, along with reference stains to identify all bacteria. Although these assays were originally developed to assess the uptake and survival of Staphylococcus aureus in liver resident macrophages (Kupffer cells), this protocol may be adapted to investigate any bacterium-host cell interaction.

Radiographic contrast agents cause acute kidney injury (AKI), yet the underlying pathogenesis is poorly understood. Nod-like receptor pyrin containing 3-deficient (Nlrp3-deficient) mice displayed reduced epithelial cell injury and inflammation in the kidney in a model of contrast-induced AKI (CI-AKI). Unexpectedly, contrast agents directly induced tubular epithelial cell death in vitro that was not dependent on Nlrp3. Rather, contrast agents activated the canonical Nlrp3 inflammasome in macrophages. Intravital microscopy revealed diatrizoate (DTA) uptake within minutes in perivascular CX3CR1+ resident phagocytes in the kidney. Following rapid filtration into the tubular luminal space, DTA was reabsorbed and concentrated in tubular epithelial cells via the brush border enzyme dipeptidase-1 in volume-depleted but not euvolemic mice. LysM-GFP+ macrophages recruited to the kidney interstitial space ingested contrast material transported from the urine via direct interactions with tubules. CI-AKI was dependent on resident renal phagocytes, IL-1, leukocyte recruitment, and dipeptidase-1. Levels of the inflammasome-related urinary biomarkers IL-18 and caspase-1 were increased immediately following contrast administration in patients undergoing coronary angiography, consistent with the acute renal effects observed in mice. Taken together, these data show that CI-AKI is a multistep process that involves immune surveillance by resident and infiltrating renal phagocytes, Nlrp3-dependent inflammation, and the tubular reabsorption of contrast via dipeptidase-1.

All bacterial infections occur within a polymicrobial environment, from which a pathogen population emerges to establish disease within a host. Emphasis has been placed on prevention of pathogen dominance by competing microflora acting as probiotics1. Here we show that the virulence of the human pathogen Staphylococcus aureus is augmented by native, polymicrobial, commensal skin flora and individual species acting as ‘proinfectious agents’. The outcome is pathogen proliferation, but not commensal. Pathogenesis augmentation can be mediated by particulate cell wall peptidoglycan, reducing the S. aureus infectious dose by over 1,000-fold. This phenomenon occurs using a range of S. aureus strains and infection models and is not mediated by established receptor-mediated pathways including Nod1, Nod2, Myd88 and the NLPR3 inflammasome. During mouse sepsis, augmentation depends on liver-resident macrophages (Kupffer cells) that capture and internalize both the pathogen and the proinfectious agent, leading to reduced production of reactive oxygen species, pathogen survival and subsequent multiple liver abscess formation. The augmented infection model more closely resembles the natural situation and establishes the role of resident environmental microflora in the initiation of disease by an invading pathogen. As the human microflora is ubiquitous2, its role in increasing susceptibility to infection by S. aureus highlights potential strategies for disease prevention.

Many key components of innate immunity to infection are shared between Drosophila and humans. However, the fly Toll ligand Spaetzle is not thought to have a vertebrate equivalent. We have found that the structurally related cystine-knot protein, nerve growth factor β (NGFβ), plays an unexpected Spaetzle-like role in immunity to Staphylococcus aureus infection in chordates. Deleterious mutations of either human NGFβ or its high-affinity receptor tropomyosin-related kinase receptor A (TRKA) were associated with severe S. aureus infections. NGFβ was released by macrophages in response to S. aureus exoproteins through activation of the NOD-like receptors NLRP3 and NLRC4 and enhanced phagocytosis and superoxide-dependent killing, stimulated proinflammatory cytokine production, and promoted calcium-dependent neutrophil recruitment. TrkA knockdown in zebrafish increased susceptibility to S. aureus infection, confirming an evolutionarily conserved role for NGFβ-TRKA signaling in pathogen-specific host immunity.

Summary Bacterial biofilm infections are difficult to eradicate because of antibiotic insusceptibility and high recurrence rates. Biofilm formation by Pseudomonas aeruginosa, a leading cause of bacterial keratitis, is facilitated by the bacterial Psl exopolysaccharide and associated with heightened virulence. Using intravital microscopy, we observed that neutrophilic recruitment to corneal infections limits P. aeruginosa biofilms to the outer eye surface, preventing bacterial dissemination. Neutrophils moved to the base of forming biofilms, where they underwent neutrophil extracellular trap formation (NETosis) in response to high expression of the bacterial type-3 secretion system (T3SS). NETs formed a barrier “dead zone,” confining bacteria to the external corneal environment and inhibiting bacterial dissemination into the brain. Once formed, ocular biofilms were resistant to antibiotics and neutrophil killing, advancing eye pathology. However, blocking both Psl and T3SS together with antibiotic treatment broke down the biofilm and reversed keratitis, suggesting future therapeutic strategies for this intractable infection.

Staphylococcus aureus community-acquired (CA) MRSA strains are highly virulent and can cause infections in otherwise healthy individuals. The most important mechanism of the host for clearing S. aureus is phagocytosis by neutrophils and subsequent killing of the pathogen. Especially CA-MRSA strains are very efficient in circumventing this neutrophil killing. Interestingly, only a relative small number of virulence factors have been associated with CA-MRSA, one of which are the phenol soluble modulins (PSMs). We have recently shown that the PSMs are functionally inhibited by serum lipoproteins, indicating that PSMs may exert their cytolytic function primarily in the intracellular environment. To further investigate the intracellular role of the PSMs we measured the effect of the α-type and β-type PSMs on neutrophil killing after phagocytosis. Using fluorescently labelled S. aureus, we measured bacterial survival after phagocytosis in a plate reader, which was employed next to flow cytometry and time-lapse microscopy. Phagocytosis of the CA-MRSA strain MW2 by human neutrophils resulted in rapid host cell death. Using mutant strains of MW2, we demonstrated that in the presence of serum, the intracellular expression of only the psmα operon is both necessary and sufficient for both increased neutrophil cell death and increased survival of S. aureus. Our results identify PSMα peptides as prominent contributors to killing of neutrophils after phagocytosis, a finding with major implications for our understanding of S. aureus pathogenesis and strategies for S. aureus vaccine development.

Kubes et al. show that methicillin-resistant Staphylococcus aureus (MRSA) survive and proliferate inside Kupffer cells. Intracellular MRSA is resistant to neutrophil-killing and antibiotics treatment and, when released into the circulation, can infect other organs., Methicillin-resistant Staphylococcus aureus (MRSA) bacteremia is reaching epidemic proportions causing morbidity, mortality, and chronic disease due to relapses, suggesting an intracellular reservoir. Using spinning-disk confocal intravital microscopy to track MRSA-GFP in vivo, we identified that within minutes after intravenous infection MRSA is primarily sequestered and killed by intravascular Kupffer cells (KCs) in the liver. However, a minority of the Staphylococci overcome the KC’s antimicrobial defenses. These bacteria survive and proliferate for many days within this intracellular niche, where they remain undetected by recruited neutrophils. Over time, the KCs lyse, releasing bacteria into the circulation, enabling dissemination to other organs such as the kidneys. Vancomycin, the antibiotic of choice to treat MRSA bacteremia, could not penetrate the KCs to eradicate intracellular MRSA. However, based on the intravascular location of these specific macrophages, we designed a liposomal formulation of vancomycin that is efficiently taken up by KCs and diminished the intracellular MRSA. Targeting the source of the reservoir dramatically protected the liver but also dissemination to other organs, and prevented mortality. This vancomycin formulation strategy could help treat patients with Staphylococcal bacteremia without a need for novel antibiotics by targeting the previously inaccessible intracellular reservoir in KCs.

Neutrophil extracellular traps (NETs) composed of DNA decorated with histones and proteases trap and kill bacteria but also injure host tissue. Here we show that during a bloodstream infection with methicillin-resistant Staphylococcus aureus, the majority of bacteria are sequestered immediately by hepatic Kupffer cells, resulting in transient increases in liver enzymes, focal ischaemic areas and a robust neutrophil infiltration into the liver. The neutrophils release NETs into the liver vasculature, which remain anchored to the vascular wall via von Willebrand factor and reveal significant neutrophil elastase (NE) proteolytic activity. Importantly, DNase although very effective at DNA removal, and somewhat effective at inhibiting NE proteolytic activity, fails to remove the majority of histones from the vessel wall and only partly reduces injury. By contrast, inhibition of NET production as modelled by PAD4-deficiency, or prevention of NET formation and proteolytic activity as modelled in NE−/− mice prevent collateral host tissue damage.

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation. To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSMα operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

The pathogen Staphylococcus aureus is well adapted to its human host. Neutrophil-mediated killing is a crucial defense system against S. aureus; however, the pathogen has evolved many strategies to resist killing. We first describe the discrete steps of neutrophil activation and migration to the site of infection and the killing of microbes by neutrophils in general. We then highlight the different approaches utilized by S. aureus to resist the different steps of neutrophil attack. Various molecules are discussed in their evolutionary context. Most of the molecules secreted by S. aureus to combat neutrophil attacks at the site of infection show clear human specificity. Many elements of human neutrophil defenses appear redundant, and so the evasion strategies of staphylococci display redundant functions as well. All efforts by S. aureus to resist neutrophil-mediated killing stress the importance of these mechanisms in the pathophysiology of staphylococcal diseases. However, the highly human-specific nature of most host-pathogen interactions hinders the in vivo establishment of their contribution to staphylococcal pathophysiology.

Methicillin-resistant Staphylococcus aureus (MRSA) strains of the pulsed-field type USA300 are primarily responsible for the current community-associated epidemic of MRSA infections in the United States. The success of USA300 is partly attributed to the ability of the pathogen to avoid destruction by human neutrophils (polymorphonuclear leukocytes [PMNs]), which are crucial to the host immune response to S. aureus infection. In this work, we investigated the contribution of bicomponent pore-forming toxins to the ability of USA300 to withstand attack from primary human PMNs. We demonstrate that in vitro growth conditions influence the expression, production, and availability of leukotoxins by USA300, which in turn impact the cytotoxic potential of this clone toward PMNs. Interestingly, we also found that upon exposure to PMNs, USA300 preferentially activates the promoter of the lukAB operon, which encodes the recently identified leukocidin AB (LukAB). LukAB elaborated by extracellular S. aureus forms pores in the plasma membrane of PMNs, leading to PMN lysis, highlighting a contribution of LukAB to USA300 virulence. We now show that LukAB also facilitates the escape of bacteria engulfed within PMNs, in turn enabling the replication and outgrowth of S. aureus . Together, these results suggest that upon encountering PMNs S. aureus induces the production of LukAB, which serves as an extra- and intracellular weapon to protect the bacterium from destruction by human PMNs.

Staphylococcus aureus virulence has been associated with the production of phenol soluble modulins (PSM). PSM are known to activate, attract and lyse neutrophils. However, the functional characterizations were generally performed in the absence of human serum. Here, we demonstrate that human serum can inhibit all the previously-described activities of PSM. We observed that serum can fully block both the cell lysis and FPR2 activation of neutrophils. We show a direct interaction between PSM and serum lipoproteins in human serum and whole blood. Subsequent analysis using purified high, low, and very low density lipoproteins (HDL, LDL, and VLDL) revealed that they indeed neutralize PSM. The lipoprotein HDL showed highest binding and antagonizing capacity for PSM. Furthermore, we show potential intracellular production of PSM by S. aureus upon phagocytosis by neutrophils, which opens a new area for exploration of the intracellular lytic capacity of PSM. Collectively, our data show that in a serum environment the function of PSM as important extracellular toxins should be reconsidered., Author Summary Infections with methicillin-resistant Staphylococcus aureus (MRSA) are difficult to treat because of resistance against standard antibiotics. In contrast to the traditional healthcare-associated (HA-) MRSA strains, community-associated (CA-) MRSA strains cause severe infections in otherwise healthy individuals. CA-MRSA strains display enhanced virulence, spreading more rapidly and causing more severe illness than HA-MRSA strains. Enhanced virulence of CA-MRSA is thought to be associated with the production of several toxins, such as Phenol Soluble Modulins (PSM). PSM have been described to activate, attract and lyse neutrophils. Thus far, previous studies characterizing the functions of PSM were performed in the absence of body fluids. In the current study, we show that human serum strongly inhibits many functions attributed to PSM. We demonstrate that serum lipoprotein particles are responsible for the binding and inhibition of PSM, even when PSM are produced by growing S. aureus in whole blood. Finally, we show production of PSM by S. aureus within neutrophils, suggesting that PSM may play a role intracellularly in a serum-free environment. These findings significantly contribute to our understanding of the function of PSM and strongly suggest that PSM, instead of performing as extracellular toxins, most likely act as intracellular toxins.