Hexokinase Is an Innate Immune Receptor for the Detection of Bacterial Peptidoglycan.

Degradation of Gram-positive bacterial cell wall peptidoglycan in macrophage and dendritic cell phagosomes leads to activation of the NLRP3 inflammasome, a cytosolic complex that regulates processing and secretion of interleukin (IL)-1ß and IL-18. While many inflammatory responses to peptidoglycan are mediated by detection of its muramyl dipeptide component in the cytosol by NOD2, we report here that NLRP3 inflammasome activation is caused by release of N-acetylglucosamine that is detected in the cytosol by the glycolytic enzyme hexokinase. Inhibition of hexokinase by N-acetylglucosamine causes its dissociation from mitochondria outer membranes, and we found that this is sufficient to activate the NLRP3 inflammasome. In addition, we observed that glycolytic inhibitors and metabolic conditions affecting hexokinase function and localization induce inflammasome activation. While previous studies have demonstrated that signaling by pattern recognition receptors can regulate metabolic processes, this study shows that a metabolic enzyme can act as a pattern recognition receptor. PAPERCLIP.

Candida-induced asthma steps up to the plate-lets.

Fungal proteases are well-known allergens. In this issue of Immunity, Wu et al. (2021) observe that allergic airway responses to Candida albicans are mediated by the peptide toxin candidalysin rather than proteases. Candidalysin promotes these responses by stimulating platelets to release the Wnt antagonist Dickkopf-1.

Profiling phagosome proteins identifies PD-L1 as a fungal-binding receptor.

Phagocytosis is the process by which myeloid phagocytes bind to and internalize potentially dangerous microorganisms1. During phagocytosis, innate immune receptors and associated signalling proteins are localized to the maturing phagosome compartment, forming an immune information processing hub brimming with microorganism-sensing features2-8. Here we developed proximity labelling of phagosomal contents (PhagoPL) to identify proteins localizing to phagosomes containing model yeast and bacteria. By comparing the protein composition of phagosomes containing evolutionarily and biochemically distinct microorganisms, we unexpectedly identified programmed death-ligand 1 (PD-L1) as a protein that specifically enriches in phagosomes containing yeast. We found that PD-L1 directly binds to yeast upon processing in phagosomes. By surface display library screening, we identified the ribosomal protein Rpl20b as a fungal protein ligand for PD-L1. Using an auxin-inducible depletion system, we found that detection of Rpl20b by macrophages cross-regulates production of distinct cytokines including interleukin-10 (IL-10) induced by the activation of other innate immune receptors. Thus, this study establishes PhagoPL as a useful approach to quantifying the collection of proteins enriched in phagosomes during host-microorganism interactions, exemplified by identifying PD-L1 as a receptor that binds to fungi.

Immune Interactions with Pathogenic and Commensal Fungi: A Two-Way Street.

We are exposed to a wide spectrum of fungi including innocuous environmental organisms, opportunistic pathogens, commensal organisms, and fungi that can actively and explicitly cause disease. Much less is understood about effective host immunity to fungi than is generally known about immunity to bacterial and viral pathogens. Innate and adaptive arms of the immune system are required for effective host defense against Candida, Aspergillus, Cryptococcus, and others, with specific elements of the host response regulating specific types of fungal infections (e.g., mucocutaneous versus systemic). Here we will review themes and controversies that are currently shaping investigation of antifungal immunity (primarily to Candida and Aspergillus) and will also examine the emerging field of the role of fungi in the gut microbiome.

Harnessing antifungal immunity in pursuit of a Staphylococcus aureus vaccine strategy.

Staphylococcus aureus (S. aureus) is one of the most common bacterial infections worldwide, and antibiotic resistant strains such as Methicillin-Resistant S. aureus (MRSA) are a major threat and burden to public health. MRSA not only infects immunocompromised patients but also healthy individuals and has rapidly spread from the healthcare setting to the outside community. However, all vaccines tested in clinical trials to date have failed. Immunocompromised individuals such as patients with HIV or decreased levels of CD4+ T cells are highly susceptible to S. aureus infections, and they are also at increased risk of developing fungal infections. We therefore wondered whether stimulation of antifungal immunity might promote the type of immune responses needed for effective host defense against S. aureus. Here we show that vaccination of mice with a fungal ß-glucan particle (GP) loaded with S. aureus antigens provides protective immunity to S. aureus. We generated glucan particles loaded with the four S. aureus proteins ClfA, IsdA, MntC, and SdrE, creating the 4X-SA-GP vaccine. Vaccination of mice with three doses of 4X-SA-GP promoted protection in a systemic model of S. aureus infection with a significant reduction in the bacterial burden in the spleen and kidneys. 4X-SA-GP vaccination induced antigen-specific Th1 and Th17 CD4+ T cell and antibody responses and provided long-term protection. This work suggests that the GP vaccine system has potential as a novel approach to developing vaccines for S. aureus.

C-Type Lectin Receptors in Phagocytosis.

C-type lectin receptors (CLRs) are a family of transmembrane proteins having at least one C-type lectin-like domain (CTLD) on the cell surface and either a short intracellular signaling tail or a transmembrane domain that facilitates interaction with a second protein, often the Fc receptor common gamma chain (FcRγ), that mediates signaling. Many CLRs directly recognize microbial cell walls and influence innate immunity by activating inflammatory and antimicrobial responses in phagocytes. In this review, we examine the contributions of certain CLRs to activation and regulation of phagocytosis in cells such as macrophages, dendritic cells and neutrophils.

Oxidized mitochondrial DNA activates the NLRP3 inflammasome during apoptosis.

We report that in the presence of signal 1 (NF-κB), the NLRP3 inflammasome was activated by mitochondrial apoptotic signaling that licensed production of interleukin-1ß (IL-1ß). NLRP3 secondary signal activators such as ATP induced mitochondrial dysfunction and apoptosis, resulting in release of oxidized mitochondrial DNA (mtDNA) into the cytosol, where it bound to and activated the NLRP3 inflammasome. The antiapoptotic protein Bcl-2 inversely regulated mitochondrial dysfunction and NLRP3 inflammasome activation. Mitochondrial DNA directly induced NLRP3 inflammasome activation, because macrophages lacking mtDNA had severely attenuated IL-1ß production, yet still underwent apoptosis. Both binding of oxidized mtDNA to the NLRP3 inflammasome and IL-1ß secretion could be competitively inhibited by the oxidized nucleoside 8-OH-dG. Thus, our data reveal that oxidized mtDNA released during programmed cell death causes activation of the NLRP3 inflammasome. These results provide a missing link between apoptosis and inflammasome activation, via binding of cytosolic oxidized mtDNA to the NLRP3 inflammasome.

Expansion of commensal fungus Wallemia mellicola in the gastrointestinal mycobiota enhances the severity of allergic airway disease in mice.

The gastrointestinal microbiota influences immune function throughout the body. The gut-lung axis refers to the concept that alterations of gut commensal microorganisms can have a distant effect on immune function in the lung. Overgrowth of intestinal Candida albicans has been previously observed to exacerbate allergic airways disease in mice, but whether subtler changes in intestinal fungal microbiota can affect allergic airways disease is less clear. In this study we have investigated the effects of the population expansion of commensal fungus Wallemia mellicola without overgrowth of the total fungal community. Wallemia spp. are commonly found as a minor component of the commensal gastrointestinal mycobiota in both humans and mice. Mice with an unaltered gut microbiota community resist population expansion when gavaged with W. mellicola; however, transient antibiotic depletion of gut microbiota creates a window of opportunity for expansion of W. mellicola following delivery of live spores to the gastrointestinal tract. This phenomenon is not universal as other commensal fungi (Aspergillus amstelodami, Epicoccum nigrum) do not expand when delivered to mice with antibiotic-depleted microbiota. Mice with Wallemia-expanded gut mycobiota experienced altered pulmonary immune responses to inhaled aeroallergens. Specifically, after induction of allergic airways disease with intratracheal house dust mite (HDM) antigen, mice demonstrated enhanced eosinophilic airway infiltration, airway hyperresponsiveness (AHR) to methacholine challenge, goblet cell hyperplasia, elevated bronchoalveolar lavage IL-5, and enhanced serum HDM IgG1. This phenomenon occurred with no detectable Wallemia in the lung. Targeted amplicon sequencing analysis of the gastrointestinal mycobiota revealed that expansion of W. mellicola in the gut was associated with additional alterations of bacterial and fungal commensal communities. We therefore colonized fungus-free Altered Schaedler Flora (ASF) mice with W. mellicola. ASF mice colonized with W. mellicola experienced enhanced severity of allergic airways disease compared to fungus-free control ASF mice without changes in bacterial community composition.

Autocrine Type I IFN Signaling in Dendritic Cells Stimulated with Fungal ß-Glucans or Lipopolysaccharide Promotes CD8 T Cell Activation.

Type I IFNs are key mediators of immune defense against viruses and bacteria. Type I IFNs were also previously implicated in protection against fungal infection, but their roles in antifungal immunity have not been thoroughly investigated. A recent study demonstrated that bacterial and fungal ß-glucans stimulate IFN-ß production by dendritic cells (DCs) following detection by the Dectin-1 receptor, but the effects of ß-glucan-induced type I IFNs have not been defined. We investigated whether type I IFNs regulate CD8 T cell activation by fungal ß-glucan particle-stimulated DCs. We demonstrate that ß-glucan-stimulated DCs induce CD8 T cell proliferation, activation marker (CD44 and CD69) expression, and production of IFN-γ, IL-2, and granzyme B. Moreover, we show that type I IFNs support robust CD8 T cell activation (proliferation and IFN-γ and granzyme B production) by ß-glucan-stimulated DCs in vitro and in vivo due to autocrine effects on the DCs. Specifically, type I IFNs promote Ag presentation on MHC I molecules, CD86 and CD40 expression, and the production of IL-12 p70, IL-2, IL-6, and TNF-α by ß-glucan-stimulated DCs. We also demonstrate a role for autocrine type I IFN signaling in bacterial LPS-induced DC maturation, although, in the context of LPS stimulation, this mechanism is not so critical for CD8 T cell activation (promotes IFN-γ production but not proliferation or granzyme B production). This study provides insight into the mechanisms underlying CD8 T cell activation during infection, which may be useful in the rational design of vaccines directed against pathogens and tumors.

Direct Antimicrobial Activity of IFN-ß.

Type I IFNs are a cytokine family essential for antiviral defense. More recently, type I IFNs were shown to be important during bacterial infections. In this article, we show that, in addition to known cytokine functions, IFN-ß is antimicrobial. Parts of the IFN-ß molecular surface (especially helix 4) are cationic and amphipathic, both classic characteristics of antimicrobial peptides, and we observed that IFN-ß can directly kill Staphylococcus aureus Further, a mutant S. aureus that is more sensitive to antimicrobial peptides was killed more efficiently by IFN-ß than was the wild-type S. aureus, and immunoblotting showed that IFN-ß interacts with the bacterial cell surface. To determine whether specific parts of IFN-ß are antimicrobial, we synthesized IFN-ß helix 4 and found that it is sufficient to permeate model prokaryotic membranes using synchrotron x-ray diffraction and that it is sufficient to kill S. aureus These results suggest that, in addition to its well-known signaling activity, IFN-ß may be directly antimicrobial and be part of a growing family of cytokines and chemokines, called kinocidins, that also have antimicrobial properties.

Myeloid ATG16L1 Facilitates Host-Bacteria Interactions in Maintaining Intestinal Homeostasis.

Intact ATG16L1 plays an essential role in Paneth cell function and intestinal homeostasis. However, the functional consequences of ATG16L1 deficiency in myeloid cells, particularly macrophages, are not fully characterized. We generated mice with Atg16l1 deficiency in myeloid and dendritic cells and showed that mice with myeloid Atg16l1 deficiency had exacerbated colitis in two acute and one chronic model of colitis with increased proinflammatory to anti-inflammatory macrophage ratios, production of proinflammatory cytokines, and numbers of IgA-coated intestinal microbes. Mechanistic analyses using primary murine macrophages showed that Atg16l1 deficiency led to increased reactive oxygen species production, impaired mitophagy, reduced microbial killing, impaired processing of MHC class II Ags, and altered intracellular trafficking to the lysosomal compartments. Increased production of reactive oxygen species and reduced microbial killing may be general features of the myeloid compartment, as they were also observed in Atg16l1-deficient primary murine neutrophils. A missense polymorphism (Thr300Ala) in the essential autophagy gene ATG16L1 is associated with Crohn disease (CD). Previous studies showed that this polymorphism leads to enhanced cleavage of ATG16L1 T300A protein and thus reduced autophagy. Similar findings were shown in primary human macrophages from controls and a population of CD patients carrying the Atg16l1 T300A risk variant and who were controlled for NOD2 CD-associated variants. This study revealed that ATG16L1 deficiency led to alterations in macrophage function that contribute to the severity of CD.

Mucosal immune responses to fungi and the implications for inflammatory bowel disease.

PURPOSE OF REVIEW: The intestinal microbiota plays a central role in inflammatory diseases of the gut. Although most investigations regarding how the mucosal immune system interacts with the microbiota have focused on bacteria, recent studies are elucidating the additional role of commensal fungi in health and disease in the gut. RECENT FINDINGS: New technical approaches are defining the makeup of the fungal communities in the intestines of humans and mice. The reported composition of these communities is influenced by the approaches used to define the fungi. Changes in the intestinal mycobiota are associated with gut inflammation in patients with inflammatory bowel disease and in mouse models of colitis. Recent studies are beginning to elucidate the mechanisms by which the mucosal immune system interacts with and is influenced by intestinal fungi. SUMMARY: Studies clearly demonstrate the presence of intestinal fungi and document the ability of the mucosal immune system to recognize and respond to fungi. Future studies will further investigate whether intestinal fungi directly influence intestinal disease and what cellular, molecular, and genetic mechanisms contribute.

Activation of the innate immune receptor Dectin-1 upon formation of a 'phagocytic synapse'.

Innate immune cells must be able to distinguish between direct binding to microbes and detection of components shed from the surface of microbes located at a distance. Dectin-1 (also known as CLEC7A) is a pattern-recognition receptor expressed by myeloid phagocytes (macrophages, dendritic cells and neutrophils) that detects ß-glucans in fungal cell walls and triggers direct cellular antimicrobial activity, including phagocytosis and production of reactive oxygen species (ROS). In contrast to inflammatory responses stimulated upon detection of soluble ligands by other pattern-recognition receptors, such as Toll-like receptors (TLRs), these responses are only useful when a cell comes into direct contact with a microbe and must not be spuriously activated by soluble stimuli. In this study we show that, despite its ability to bind both soluble and particulate ß-glucan polymers, Dectin-1 signalling is only activated by particulate ß-glucans, which cluster the receptor in synapse-like structures from which regulatory tyrosine phosphatases CD45 and CD148 (also known as PTPRC and PTPRJ, respectively) are excluded (Supplementary Fig. 1). The 'phagocytic synapse' now provides a model mechanism by which innate immune receptors can distinguish direct microbial contact from detection of microbes at a distance, thereby initiating direct cellular antimicrobial responses only when they are required.

Cutting edge: FYCO1 recruitment to dectin-1 phagosomes is accelerated by light chain 3 protein and regulates phagosome maturation and reactive oxygen production.

L chain 3 (LC3)-associated phagocytosis is a process in which LC3, a protein canonically involved in engulfing intracellular materials (autophagy), is recruited to traditional phagosomes during internalization of extracellular payloads. LC3's association with phagosomes has been implicated in regulating microbial killing, Ag processing, and phagosome maturation; however, the mechanism by which LC3 influences these processes has not been clear. In this study, we report that FYVE and coiled-coil domain containing 1 (FYCO1), a protein previously implicated in autophagosome trafficking, is recruited directly by LC3 to Dectin-1 phagosomes. During LC3-associated phagocytosis, FYCO1 recruitment facilitates maturation of early p40phox(+) phagosomes into late LAMP1(+) phagosomes. When FYCO1 is lacking, phagosomes stay p40phox(+) longer and produce more reactive oxygen.

Mechanisms of Fc receptor and dectin-1 activation for phagocytosis.

Phagocytosis is a key cellular process, both during homeostasis and upon infection or tissue damage. Receptors on the surface of professional phagocytic cells bind to target particles either directly or through opsonizing ligands, and trigger actin-mediated ingestion of the particles. The process must be carefully controlled to ensure that phagocytosis is triggered efficiently and specifically, and that the antimicrobial cytotoxic responses that often accompany it are initiated only when required. In this review, we will describe and compare the molecular mechanisms that regulate phagocytosis triggered by Fcγ receptors, which mediate the uptake of immunoglobulin G-opsonized targets, and Dectin-1, which is responsible for internalization of fungi with exposed cell wall ß-glucan. We will examine how these receptors detect their ligands, how signal transduction is initiated and regulated, and how internalization is instructed to achieve rapid and yet controlled uptake of their targets.

Detection of a TLR2 agonist by hematopoietic stem and progenitor cells impacts the function of the macrophages they produce.

Several groups have shown that detection of microbial components by TLRs on hematopoietic stem and progenitor cells (HSPCs) instructs myeloid cell generation, raising interest in the possibility of targeting TLRs on HSPCs to boost myelopoiesis. However, although "TLR-derived" cells exhibit myeloid cell characteristics (phagocytosis, cytokine production, antigen presentation), it is not clear whether they are functionally equivalent to macrophages derived in the absence of TLR activation. Our in vitro and in vivo studies show that macrophages derived from mouse and human HSPC subsets (including stem cells) exposed to a TLR2 agonist prior to or during macrophage differentiation produce lower levels of inflammatory cytokines (TNF-α, IL-6, and IL-1ß) and reactive oxygen species. This is in contrast to prior exposure of differentiated macrophages to the TLR2 agonist ("tolerance"), which suppresses inflammatory cytokine production, but elevates reactive oxygen species. Soluble factors produced following exposure of HSPCs to a TLR2 agonist can also act in a paracrine manner to influence the function of macrophages derived from unexposed HSPCs. Our data demonstrate that macrophage function can be influenced by TLR signaling in the HSPCs from which they are derived, and that this may impact the clinical utility of targeting TLRs on HSPCs to boost myelopoiesis.

Failure to induce IFN-ß production during Staphylococcus aureus infection contributes to pathogenicity.

The importance of type I IFNs in the host response to viral infection is well established; however, their role in bacterial infection is not fully understood. Several bacteria (both Gram-positive and -negative) have been shown to induce IFN-ß production in myeloid cells, but this IFN-ß is not always beneficial to the host. We examined whether Staphylococcus aureus induces IFN-ß from myeloid phagocytes, and if so, whether it is helpful or harmful to the host to do so. We found that S. aureus poorly induces IFN-ß production compared with other bacteria. S. aureus is highly resistant to degradation in the phagosome because it is resistant to lysozyme. Using a mutant that is more sensitive to lysozyme, we show that phagosomal degradation and release of intracellular ligands is essential for induction of IFN-ß and inflammatory chemokines downstream of IFN-ß. Further, we found that adding exogenous IFN-ß during S. aureus infection (in vitro and in vivo) was protective. Together, the data demonstrate that failure to induce IFN-ß production during S. aureus infection contributes to pathogenicity.

Inhibitory receptor paired Ig-like receptor B is exploited by Staphylococcus aureus for virulence.

The innate immune system has developed to acquire a wide variety of pattern-recognition receptors (PRRs) to identify potential pathogens, whereas pathogens have also developed to escape host innate immune responses. ITIM-bearing receptors are attractive targets for pathogens to attenuate immune responses against them; however, the in vivo role of the inhibitory PRRs in host-bacteria interactions remains unknown. We demonstrate in this article that Staphylococcus aureus, a major Gram-positive bacteria, exploits inhibitory PRR paired Ig-like receptor (PIR)-B on macrophages to suppress ERK1/2 and inflammasome activation, and subsequent IL-6 and IL-1ß secretion. Consequently, Pirb(-/-) mice infected with S. aureus showed enhanced inflammation and more effective bacterial clearance, resulting in resistance to the sepsis. Screening of S. aureus mutants identified lipoteichoic acid (LTA) as an essential bacterial cell wall component required for binding to PIR-B and modulating inflammatory responses. In vivo, however, an LTA-deficient S. aureus mutant was highly virulent and poorly recognized by macrophages in both wild-type and Pirb(-/-) mice, demonstrating that LTA recognition by PRRs other than PIR-B mediates effective bacterial elimination. These results provide direct evidence that bacteria exploit the inhibitory receptor for virulence, and host immune system counterbalances the infection.

The intestinal microbiota contributes to the development of immune-mediated cardiovascular inflammation and vasculitis in mice.

Alterations in the intestinal microbiota contribute to the pathogenesis of various cardiovascular disorders, but how they affect the development of Kawasaki disease (KD), an acute pediatric vasculitis, remains unclear. We report that depleting the gut microbiota reduces the development of cardiovascular inflammation in a murine model mimicking KD vasculitis. The development of cardiovascular lesions was associated with alterations in the intestinal microbiota composition and, notably, a decreased abundance of Akkermansia muciniphila and Faecalibacterium prausnitzii. Oral supplementation with either of these live or pasteurized individual bacteria, or with short-chain fatty acids (SCFAs) produced by them, attenuated cardiovascular inflammation. Treatment with Amuc_1100, the TLR-2 signaling outer membrane protein from A. muciniphila , also decreased the severity of vascular inflammation. This study reveals an underappreciated gut microbiota-cardiovascular inflammation axis in KD vasculitis pathogenesis and identifies specific intestinal commensals that regulate vasculitis in mice by producing metabolites or via extracellular proteins acting on gut barrier function. IN BRIEF: It remains unclear whether changes in the intestinal microbiota composition are involved in the development of cardiovascular lesions associated with Kawasaki disease (KD), an immune-mediated vasculitis. Jena et al. observe alterations in the intestinal microbiota composition of mice developing vasculitis, characterized by reduced A. muciniphila and F. prausnitzii . Oral supplementation with either of these bacteria, live or pasteurized, or with bacteria-produced short-chain fatty acids (SCFAs) or Amuc_1100, the TLR-2 signaling outer membrane protein of A. muciniphila , was sufficient to alleviate the development of cardiovascular lesions in mice by promoting intestinal barrier function. HIGHLIGHTS: Absence or depletion of the microbiota decreases the severity of vasculitis in a murine model mimicking KD vasculitis. Supplementation of B. wadsworthia and B. fragilis promotes murine KD vasculitis. Decreased abundances of F. prausnitzii and A. muciniphila are associated with the development of cardiovascular lesions in mice. Supplementation with either live or pasteurized A. muciniphila and F. prausnitzii, or the TLR-2 signaling Amuc_1100, reduces the severity of vasculitis by promoting gut barrier function.