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Outpatients with Covid-19 were followed serially with frequent PCR and viral-culture assessments. The SARS-CoV-2 omicron (BA.1) variant could be cultured a median of 8 days after symptom onset or the initial positive test.

Key Points Antigen processing and presentation is the mechanism by which whole antigens are degraded and loaded onto MHC molecules for display on the cell surface for recognition by T cells. Both macrophages and dendritic cells (DCs) are considered professional antigen-presenting cells, although DCs possess the unique capacity to activate naive T cells. DCs phagocytose antigens and whole microorganisms and place them into membrane-delimited compartments termed phagosomes. These structures are modified over time and ultimately fuse with lysosomes to form phagolysosomes. Cross-presentation allows DCs to take up antigens from the extracellular environment and present them on MHC class I molecules for CD8 + T-cell activation. Although the exact trafficking patterns of antigens are not known, many hypotheses have been generated, including the recruitment of the endoplasmic reticulum dislocation machinery to the phagosome and protein-independent passing of antigens from the phagosome or phagolysosome into the cytosol. The autophagy machinery is thought to have an important role in the generation of peptide antigens for MHC class II molecules. The pathways of antigen processing and presentation are well known, but how do antigens gain access to MHC molecules in dendritic cells and what is the role of autophagy, endoplasmic reticulum dislocation and Toll-like receptors in these pathways? The principal components of both MHC class I and class II antigen processing and presentation pathways are well known. In dendritic cells, these pathways are tightly regulated by Toll-like-receptor signalling and include features, such as cross-presentation, that are not seen in other cell types. However, the exact mechanisms involved in the subcellular trafficking of antigens remain poorly understood and in some cases are controversial. Recent data suggest that diverse cellular machineries, including autophagy, participate in antigen processing and presentation, although their relative contributions remain to be fully elucidated. Here, we highlight some emerging themes of antigen processing and presentation that we think merit further attention.

Microbial pathogens generate extracellular vesicles (EVs) for intercellular communication and quorum sensing. Microbial EVs also induce inflammatory pathways within host innate immune cells. We previously demonstrated that EVs secreted by Candida albicans trigger type I interferon signaling in host cells specifically via the cGAS-STING innate immune signaling pathway. Here, we show that despite sharing similar properties of morphology and internal DNA content, the interactions between EVs and the innate immune system differ according to the parental fungal species. EVs secreted by C. albicans , Saccharomyces cerevisiae, Cryptococcus neoformans, and Aspergillus fumigatus are differentially endocytosed by murine macrophages triggering varied cytokine responses, innate immune signaling, and subsequent immune cell recruitment. Notably, polysaccharide and hydrophobic protein structures on the outer layers of C. neoformans and A. fumigatus EVs inhibit efficient internalization by macrophages and dampen innate immune activation. Our data uncover the functional consequences of the internalization of diverse fungal EVs by immune cells and reveal novel insights into the early innate immune response to distinct clinically significant fungal pathogens.

[This corrects the article DOI: 10.1371/journal.ppat.1012879.].

Natural killer (NK) cells use the activating receptor NKp30 as a microbial pattern-recognition receptor to recognize, activate cytolytic pathways, and directly kill the fungi Cryptococcus neoformans and Candida albicans . However, the fungal pathogen-associated molecular pattern (PAMP) that triggers NKp30-mediated killing remains to be identified. Here we show that β-1,3-glucan, a component of the fungal cell wall, binds to NKp30. We further demonstrate that β-1,3-glucan stimulates granule convergence and polarization, as shown by live cell imaging. Through Src Family Kinase signaling, β-1,3-glucan increases expression and clustering of NKp30 at the microbial and NK cell synapse to induce perforin release for fungal cytotoxicity. Rather than blocking the interaction between fungi and NK cells, soluble β-1,3-glucan enhances fungal killing and restores defective cryptococcal killing by NK cells from HIV-positive individuals, implicating β-1,3-glucan to be both an activating ligand and a soluble PAMP that shapes NK cell host immunity. Natural killer (NK) cells has been show to mediate fungi killing via the activating receptor NKp30, but the fungal target for NKp30 is still unclear. Here the authors show, using atomic force microscopy and live cell imaging, that β-1,3-glucan is expressed by Cryptococcus neoformans and Candida albicans and responsible for NKp30-mediated NK killing.

The rate of invasive fungal infections has risen drastically over the last decade and continues to carry devastatingly high mortality rates. Currently, there are no licensed vaccines and limited antifungal agents in clinical trials for fungal-mediated diseases. The limited effectiveness of FDA-approved antifungal medications against invasive fungal infections and the lack of mechanistic understanding of how these infections manifest pose a significant burden on healthcare systems worldwide. Therefore, understanding the molecular details of the host-fungal interactions has never been more urgent. Here, we examine the role of fungal melanin as a virulence factor through its immunomodulatory effects during respiratory infections. Although previous literature on fungal pathogenicity has touched briefly on fungal pigments, they are incomplete in discussing how melanin dysregulates essential functions of the innate immune system. To provide a contemporary perspective, literature on melanized fungal species commonly associated with infections via the respiratory tract has been reviewed to detail holistic mechanisms by which melanin subverts the immune system and manipulates the respiratory epithelium.

Respiratory infections caused by the human fungal pathogen Aspergillus fumigatus are a major cause of mortality for immunocompromised patients. Exposure to these pathogens occurs through inhalation, although the role of the respiratory epithelium in disease pathogenesis has not been fully defined. Employing a primary human airway epithelial model, we demonstrate that fungal melanins potently block the post-translational secretion of the chemokines CXCL1 and CXCL8 independent of transcription or the requirement of melanin to be phagocytosed, leading to a significant reduction in neutrophil recruitment to the apical airway both in vitro and in vivo. Aspergillus -derived melanin, a major constituent of the fungal cell wall, dampened airway epithelial chemokine secretion in response to fungi, bacteria, and exogenous cytokines. Furthermore, melanin muted pathogen-mediated calcium fluxing and hindered actin filamentation. Taken together, our results reveal a critical role for melanin interaction with airway epithelium in shaping the host response to fungal and bacterial pathogens. Here, Reedy et al. use a human airway culture model to show that fungal melanin blocks the secretion of pro-inflammatory chemokines resulting in diminished immune responses to Aspergillus fumigatus and Pseudomonas aeruginosa .

Dendritic cells (DCs) and macrophages are critical to innate and adaptive immunity to the intestinal bacterial microbiota. Here, we identify a myeloid-derived mucosal DC in mice, which populates the entire lamina propria of the small intestine. Lamina propria DCs were found to depend on the chemokine receptor CX₃CR1 to form transepithelial dendrites, which enable the cells to directly sample luminal antigens. CX₃CR1 was also found to control the clearance of entero-invasive pathogens by DCs. Thus, CX₃CR1-dependent processes, which control host interactions of specialized DCs with commensal and pathogenic bacteria, may regulate immunological tolerance and inflammation.

Candida glabrata currently ranks as the second most frequent cause of invasive candidiasis. Our previous work has shown that C. glabrata is adapted to intracellular survival in macrophages and replicates within non-acidified late endosomal-stage phagosomes. In contrast, heat killed yeasts are found in acidified matured phagosomes. In the present study, we aimed at elucidating the processes leading to inhibition of phagosome acidification and maturation. We show that phagosomes containing viable C. glabrata cells do not fuse with pre-labeled lysosomes and possess low phagosomal hydrolase activity. Inhibition of acidification occurs independent of macrophage type (human/murine), differentiation (M1-/M2-type) or activation status (vitamin D3 stimulation). We observed no differential activation of macrophage MAPK or NFκB signaling cascades downstream of pattern recognition receptors after internalization of viable compared to heat killed yeasts, but Syk activation decayed faster in macrophages containing viable yeasts. Thus, delivery of viable yeasts to non-matured phagosomes is likely not triggered by initial recognition events via MAPK or NFκB signaling, but Syk activation may be involved. Although V-ATPase is abundant in C. glabrata phagosomes, the influence of this proton pump on intracellular survival is low since blocking V-ATPase activity with bafilomycin A1 has no influence on fungal viability. Active pH modulation is one possible fungal strategy to change phagosome pH. In fact, C. glabrata is able to alkalinize its extracellular environment, when growing on amino acids as the sole carbon source in vitro. By screening a C. glabrata mutant library we identified genes important for environmental alkalinization that were further tested for their impact on phagosome pH. We found that the lack of fungal mannosyltransferases resulted in severely reduced alkalinization in vitro and in the delivery of C. glabrata to acidified phagosomes. Therefore, protein mannosylation may play a key role in alterations of phagosomal properties caused by C. glabrata.

Streptococcus pneumoniae ( Sp ), a leading cause of pneumonia, can spread from the lung into the bloodstream to cause systemic disease. Limitations in vaccine efficacy and a rise in antimicrobial resistance have spurred searches for host-directed therapies that limit pathologic host immune responses to Sp . Excessive polymorphonuclear leukocyte (PMN) infiltration into Sp -infected airways promotes systemic disease. Using stem cell-derived respiratory cultures that reflect bona fide lung epithelium, we identified eicosanoid hepoxilin A3 as a critical pulmonary PMN chemoattractant that is sufficient to drive PMN-mediated epithelial damage by inducing the release of neutrophil elastase. Inhibition of the release or activity of this protease in mice limited epithelial barrier disruption and bacterial dissemination, suggesting a new host-directed treatment for Sp lung infection.