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Fungal infections have increased at an alarming rate as a result of increased usage of immunosuppressive therapies, growing resistance to antifungal drugs, and global warming. This recently prompted the World Health Organization to publish the first-ever fungal priority pathogens list, which focused on 19 organisms, ultimately deeming 4 pathogens of critical importance based on perceived public health importance. Among these four was the opportunistic mold Aspergillus fumigatus , the etiological agent of the most lethal fungal infection known to humans, IPA. Innate immunity is paramount for controlling IPA with protective roles identified for multiple myeloid cell types. In the current report, employing three complementary animal models, we show that cDC1s hinder the clearance of A. fumigatus from the lung. We further identified specific responses that are regulated by cDC1s. Overall, our study uncovers a new mechanism of immune regulation during IPA.

Pathogen interactions with the lungs are very dynamic processes. In biomedical research, it is paramount to model these processes in the laboratory as accurately as possible. Influenza A virus has been extensively studied in epithelial cell culture models, including advanced organoids and organ-on-a-chip systems. Here, we use ex vivo cultured precision cut lung slices (PCLS) and generate transcriptomic data to assess the global tissue resident host response to viral and bacterial challenges. Our data show (i) that murine PCLS faithfully reflect core responses to viral infection, while missing proinflammatory responses linked to infiltrating immune cells and (ii) that human PCLS show a highly diversified tissue resident immune response to viral infection due to previous exposures of the host to this pathogen. These responses are clearly distinct from antibacterial gene profiles. Our data advertise PCLS as a complex and realistic model to study tissue resident immune responses to microbes in a human system.

Klebsiella pneumoniae , particularly hypervirulent strains (hv Kp ), poses a critical public health threat due to its capacity to cause severe, rapidly progressing infections such as pneumonia and sepsis, often leading to acute lung injury (ALI) and high mortality. Despite the recognized role of excessive inflammation and cytokine storms in hv Kp pathogenesis, the precise mechanisms linking immune hyperactivation to fatal tissue damage remain poorly defined. This study reveals that hv Kp infection triggers a coordinated form of inflammatory cell death, PANoptosis, in AMs, the frontline immune defenders in the lungs. We identify the transcription factor STAT1 as a central regulator of this process, driven by a synergistic cytokine milieu, especially involving IFN-γ. Our findings establish a direct mechanistic pathway from hv Kp -induced cytokine release to STAT1-mediated PANoptosis, macrophage depletion, and subsequent lung failure. This work not only advances the understanding of hv Kp virulence but also highlights host signaling pathways and specific cytokines as potential therapeutic targets to modulate immunopathology and improve outcomes in severe Klebsiella infections.

Cryptococcal meningitis is the most common cause of meningitis among HIV/AIDS patients in sub-Saharan Africa, and worldwide causes over 223,000 cases leading to more than 181,000 annual deaths. Usually, the fungus gets inhaled into the lungs where the initial interactions occur with pulmonary phagocytes such as dendritic cells and macrophages. Following phagocytosis, the pathogen can be killed or can replicate intracellularly. Previous studies in mice showed that different subsets of these innate immune cells can either be antifungal or permissive for intracellular fungal growth. Our studies tested phagocytic antigen-presenting cell (APC) subsets from the human lung against C. neoformans . Human bronchoalveolar lavage was processed for phagocytic APCs and incubated with C. neoformans for two hours to analyze the initial interactions and fate of the fungus, living or killed. Results showed all subsets (3 macrophage and 3 dendritic cell subsets) interacted with the fungus, and both living and killed morphologies were discernable within the subsets using imaging flow cytometry. Single cell RNA-seq identified several different clusters of cells which more closely related to interactions with C. neoformans and its protective capacity against the pathogen rather than discrete cellular subsets. Differential gene expression analyses identified several changes in the innate immune cell’s transcriptome as it kills the fungus including increases of TNF-α ( TNF ) and the switch to using fatty acid metabolism by upregulation of the gene FABP4 . Also, increases of TNF-α correlated to cryptococcal interactions and uptake. Together, these analyses implicated signaling networks that regulate expression of many different genes – both metabolic and immune - as certain clusters of cells mount a protective response and kill the pathogen. Future studies will examine these genes and networks to understand the exact mechanism(s) these phagocytic APC subsets use to kill C. neoformans in order to develop immunotherapeutic strategies to combat this deadly disease.

Microbiota are integral regulators of host gene expression, utilizing diverse mechanisms that are shaped by the interplay between microbiome composition and inter-individual differences, i.e., host-specific factors. While previous studies have characterized inter-individual variation in microbiome composition and the effects of variable microbiome composition on the host, the extent to which host-specificity itself regulates host-microbe interactions remains poorly understood. In this study, we address this gap by characterizing changes in epithelial gene expression from six different human donors following colonization with one of three phylogenetically diverse bacteria. By systematically comparing donor-specific responses, we demonstrate that host specificity is a key determinant of the host transcriptional response to microbial colonization. Importantly, we demonstrate that the effects of host specificity are not uniform, but instead are dependent on the colonizing microbe. Our findings underscore the complexity of host-microbe relationships and establish host specificity as a significant factor shaping host-microbe interactions.

With over 220,000 cases and 180,000 deaths annually, Cryptococcus neoformans is the most common cause of fungal meningitis and a leading cause of death in HIV/AIDS patients in Sub-Saharan Africa. Either C. neoformans can be killed by innate airway phagocytes, or it can survive intracellularly. Pulmonary murine macrophage and dendritic cell (DC) subsets have been identified in the naïve lung, and we hypothesize that each subset has different interactions with C. neoformans . For these studies, we purified murine pulmonary macrophage and DC subsets from naïve mice – alveolar macrophages, Ly6c - and Ly6c + monocyte-like macrophages, interstitial macrophages, CD11b + and CD103 + DCs. With each subset, we examined cryptococcal association (binding/internalization), fungicidal activity, intracellular fungal morphology, cytokine secretion and transcriptional profiling in an ex vivo model using these pulmonary phagocyte subsets. Results showed that all subsets associate with C. neoformans , but only female Ly6c - monocyte-like macrophages significantly inhibited growth, while male CD11b + DCs significantly enhanced fungal growth. In addition, cytokine analysis revealed that some subsets from female mice produced increased amounts of cytokines compared to their counterparts in male mice following exposure to C. neoformans . In addition, although cells were analyzed ex vivo without the influence of the lung microenviroment, we did not find evidence of phagocyte polarization following incubation with C. neoformans . Imaging flow cytometry showed differing ratios of cryptococcal morphologies, c-shaped or budding, depending on phagocyte subset. RNA sequencing analysis revealed the up- and down-regulation of many genes, from immunological pathways (including differential regulation of MHC class I in the antigen processing pathway and the cell adhesion pathway) and pathways relating to relating to metabolic activity (genes in the Cytochrome P450 family, genes related to actin binding, calcium voltage channels, serine proteases, and phospholipases). Future studies gaining a more in-depth understanding on the functionality of individual genes and pathways specific to permissive and non-permissive pulmonary phagocytes will allow identification of key targets when developing therapeutic strategies to prevent cryptococcal meningitis.

Cystic fibrosis (CF) is a genetically inherited disease that leads to chronic lung infections. Pseudomonas aeruginosa is often implicated in the worsening of lung disease, and it evolves in the lung over time to resist eradication. One of the most commonly disrupted genes in P. aeruginosa isolates from chronically infected CF lungs is lasR , which encodes a transcription factor that regulates multiple virulence factors. What contributes to the apparent fitness of lasR mutants in the CF lung is not well known. Our study shows that lasR loss-of-function mutants resist phagocytosis by macrophages, one of the fundamental mechanisms of clearance by the immune system. We identify mechanisms promoting resistance to phagocytosis and explore the downstream consequences on inflammatory responses. Understanding why lasR mutations arise could inform strategies to eradicate them from the CF lung and improve outcomes.

Brucella abortus strains RB51 and RB51SOD are live attenuated vaccine strains which protect mice against virulent B. abortus strain 2308 intraperitoneal challenge. By comparison, limited information is available on how Brucella vaccines stimulate pulmonary immunity against respiratory infection, another route of exposure in humans. Therefore, in this study, we assessed the ability of intranasally delivered vaccine strains RB51 and RB51SOD to induce innate immunity. Based on parameters assessed, rough strain RB51 induces a better innate immune response in lung versus strain RB51SOD. Additional studies to further delineate strain RB51's ability to stimulate DC and adaptive immunity are warranted.

We treated a 27-year-old patient with Duchenne’s muscular dystrophy (DMD) with recombinant adeno-associated virus (rAAV) serotype 9 containing d Sa Cas9 (i.e., “dead” Staphylococcus aureus Cas9, in which the Cas9 nuclease activity has been inactivated) fused to VP64; this transgene was designed to up-regulate cortical dystrophin as a custom CRISPR–transactivator therapy. The dose of rAAV used was 1×10 14 vector genomes per kilogram of body weight. Mild cardiac dysfunction and pericardial effusion developed, followed by acute respiratory distress syndrome (ARDS) and cardiac arrest 6 days after transgene treatment; the patient died 2 days later. A postmortem examination showed severe diffuse alveolar damage. Expression of transgene in the liver was minimal, and there was no evidence of AAV serotype 9 antibodies or effector T-cell reactivity in the organs. These findings indicate that an innate immune reaction caused ARDS in a patient with advanced DMD treated with high-dose rAAV gene therapy. (Funded by Cure Rare Disease.) A 27-year-old man with Duchenne’s muscular dystrophy who was treated with a CRISPR-Cas9 transgene died from an innate immune response to the high dose of recombinant AAV used for delivery of the transgene.

ObjectiveThe gut microbiota is essential for the development of the intestinal immune system. Animal models have suggested that the gut microbiota also acts as a major modulator of systemic innate immunity during sepsis. Microbiota disruption by broad-spectrum antibiotics could thus have adverse effects on cellular responsiveness towards invading pathogens. As such, the use of antibiotics may attribute to immunosuppression as seen in sepsis. We aimed to test whether disruption of the gut microbiota affects systemic innate immune responses during endotoxemia in healthy subjects.DesignIn this proof-of-principle intervention trial, 16 healthy young men received either no treatment or broad-spectrum antibiotics (ciprofloxacin, vancomycin and metronidazole) for 7 days, after which all were administered lipopolysaccharide intravenously to induce a transient sepsis-like syndrome. At various time points, blood and faeces were sampled.ResultsGut microbiota diversity was significantly lowered by the antibiotic treatment in all subjects. Clinical parameters, neutrophil influx, cytokine production, coagulation activation and endothelial activation during endotoxemia were not different between antibiotic-pretreated and control individuals. Antibiotic treatment had no impact on blood leucocyte responsiveness to various Toll-like receptor ligands and clinically relevant causative agents of sepsis (Streptococcus pneumoniae, Klebsiella pneumoniae, Escherichia coli) during endotoxemia.ConclusionsThese findings suggest that gut microbiota disruption by broad-spectrum antibiotics does not affect systemic innate immune responses in healthy subjects during endotoxemia in humans, disproving our hypothesis. Further research is needed to test this hypothesis in critically ill patients. These data underline the importance of translating findings in mice to humans.Trial registration numberClinicalTrials.gov (NCT02127749; Pre-results).