Phagocytosis of Aspergillus fumigatus by Human Bronchial Epithelial Cells Is Mediated by the Arp2/3 Complex and WIPF2

Aspergillus fumigatus is an opportunistic fungal pathogen capable of causing severe infection in humans. One of the limitations in our understanding of how A. fumigatus causes infection concerns the initial stages of infection, notably the initial interaction between inhaled spores or conidia and the human airway. Using publicly-available datasets, we identified the Arp2/3 complex and the WAS-Interacting Protein Family Member 2 WIPF2 as being potentially responsible for internalization of conidia by airway epithelial cells. Using a cell culture model, we demonstrate that RNAi-mediated knockdown of WIPF2 significantly reduces internalization of conidia into airway epithelial cells. Furthermore, we demonstrate that inhibition of Arp2/3 by a small molecule inhibitor causes similar effects. Using super-resolution fluorescence microscopy, we demonstrate that WIPF2 is transiently localized to the site of bound conidia. Overall, we demonstrate the active role of the Arp2/3 complex and WIPF2 in mediating the internalization of A. fumigatus conidia into human airway epithelial cells

Human IRF1 governs macrophagic IFN-γ immunity to mycobacteria

Inborn errors of human IFN-γ-dependent macrophagic immunity underlie mycobacterial diseases, whereas inborn errors of IFN-α/β-dependent intrinsic immunity underlie viral diseases. Both types of IFNs induce the transcription factor IRF1. We describe unrelated children with inherited complete IRF1 deficiency and early-onset, multiple, life-threatening diseases caused by weakly virulent mycobacteria and related intramacrophagic pathogens. These children have no history of severe viral disease, despite exposure to many viruses, including SARS-CoV-2, which is life-threatening in individuals with impaired IFN-α/β immunity. In leukocytes or fibroblasts stimulated in vitro, IRF1-dependent responses to IFN-γ are, both quantitatively and qualitatively, much stronger than those to IFN-α/β. Moreover, IRF1-deficient mononuclear phagocytes do not control mycobacteria and related pathogens normally when stimulated with IFN-γ. By contrast, IFN-α/β-dependent intrinsic immunity to nine viruses, including SARS-CoV-2, is almost normal in IRF1-deficient fibroblasts. Human IRF1 is essential for IFN-γ-dependent macrophagic immunity to mycobacteria, but largely redundant for IFN-α/β-dependent antiviral immunity

Une dissection génétique du développement des cellules lymphoïdes innées et leur plasticité

Les cellules lymphoïdes innées (ILCs) sont une sous-lignée de lymphocytes n'exprimant pas les récepteurs à antigène à réarrangement mais qui ont une forte capacité de produire les cytokines. Ils inclurent les lymphocytes cytotoxiques naturels (NK), ainsi que les trois types des ILCs auxiliaires. Ils participent dans la défense immunitaire contre les pathogènes, les cancers et jouent des rôles essentiels dans l'homéostasie des tissues. Nous avons récemment identifié une population de précurseurs des ILCs (ILCP) qui peuvent générer les ILCs auxiliaires et cytotoxiques. Nous avons investigué les facteurs et les signaux d'importance dans le développement et différentiation des ILCs en analysant les ILCP provenant du sang des patients avec les maladies génétiques rares compromettant la défense immunitaire avec un focus particulier sur les gènes supposés comme importants dans le développement des ILC1 et ILC3, en particulier la signalisation IL-23 et les deux parties de son récepteur IL12RB1 et IL23R, ainsi que les facteurs de transcription RORC et TBX21. Avec un système de clonage de cellule unique modulable, nous avons démontré comment ces gènes dirigent et contribuent au développement des ILCs matures des ILCP. En employant des échantillons des patients avec les mutations dans des gènes spécifiques, la modulation de notre système de clonage et des approches transcriptomiques, nous avons montré des rôles pour la signalisation IL-23, RORC et la signalisation Notch dans la génération des ILC4 et la rétention de multipotence dans les ILCP chez l'homme. Nous avons démontré aussi que le facteur de transcription TBX21 est important pour l'expression de la cytokine IFN¿ par les NK matures, mais n'est pas essentiel pour la génération des cellules exprimant IFN¿, soit par le développement des ILCP ou la plasticité des ILC2. Enfin ce travail précise notre savoir sur le développement des ILCs humains ainsi que notre capacité de produire ces cellules rares in vitro afin d'étudier leur biologie, et les rôles elles jouent dans la défense immunitaire.Innate lymphoid cells (ILCs) are a recently described sub-lineage of lymphocytes lacking recombined antigen receptors with potent cytokine producing capacity. They include natural killer (NK) cells and three subsets of helper ILCs that participate in immune defense against a variety of pathogens and cancers and play important roles in steady-state tissue homeostasis. We have recently identified a population of circulating ILC precursors (ILCP) in humans that are able to generate all known subtypes of ILCs. We investigated key pathways, signals, and factors in the development of innate lymphoid cells through the analysis of blood ILCP from patients with rare genetic mutations which compromise host defense. This work focusses on genes thought to be involved in ILC1 and ILC3 development, including cytokine pathways (IL23/IL12RB1/IL23R) and transcription factors (RORC and TBX21). Using an efficient and modular single cell cloning assay, we have demonstrated how these different genes direct the development of ILCP into mature ILC subsets. Through a combination of genetic screening, modulation of our culture system and transcriptomic approaches we have been able to demonstrate roles for IL-23 signalling, RORC and Notch signalling in the generation of ILC3 and retention of multipotency in human ILCP. In addition, we have demonstrated that the transcription factor TBX21 important in the function of mature NK cells, particularly the expression of IFN¿, but is not essential for the generation of IFN¿ expressing helper ILC, either through development from ILCP or plasticity from ILC2. This work ultimately expands our knowledge of the development and differentiation of ILCP, our ability to produce these rare subsets in vitro and subsequently study their biology and roles in host defense and response to infection

Interactions of Aspergillus fumigatus conidia with airway epithelial cells: a critical review

Aspergillus fumigatus is an environmental filamentous fungus which also acts as an opportunistic pathogen able to cause a variety of symptoms, from an allergic response to a life-threatening disseminated fungal infection. The infectious agents are inhaled conidia whose first point of contact is most likely to be an airway epithelial cell. The interaction between epithelial cells and conidia is multifaceted and complex, and has implications for later steps in pathogenesis. Increasing evidence has demonstrated a key role for the airway epithelium in the response to respiratory pathogens, particularly at early stages of infection; therefore, elucidating the early stages of interaction of conidia with airway epithelial cells is essential to understand the establishment of infection in cohorts of at-risk patients. Here, we present a comprehensive review of the early interactions between A. fumigatus and airway epithelial cells, including bronchial and alveolar epithelial cells. We describe mechanisms of adhesion, internalization of conidia by airway epithelial cells, the immune response of airway epithelial cells, as well as the role of fungal virulence factors and patterns of fungal gene expression characteristic of early infection. A clear understanding of the mechanisms involved in the early establishment of infection by A. fumigatus could point to novel targets for therapy and prophylaxis

Notch, RORC and IL-23 signals cooperate to promote multi-lineage human innate lymphoid cell differentiation

International audienceAbstract Innate lymphoid cells (ILCs) include cytotoxic natural killer cells and distinct groups of cytokine-producing innate helper cells which participate in immune defense and promote tissue homeostasis. Circulating human ILC precursors (ILCP) able to generate all canonical ILC subsets via multi-potent or uni-potent intermediates according to our previous work. Here we show potential cooperative roles for the Notch and IL-23 signaling pathways for human ILC differentiation from blood ILCP using single cell cloning analyses and validate these findings in patient samples with rare genetic deficiencies in IL12RB1 and RORC . Mechanistically, Notch signaling promotes upregulation of the transcription factor RORC , enabling acquisition of Group 1 (IFN-γ) and Group 3 (IL-17A, IL-22) effector functions in multi-potent and uni-potent ILCP. Interfering with RORC or signaling through its target IL-23R compromises ILC3 effector functions but also generally suppresses ILC production from multi-potent ILCP. Our results identify a Notch->RORC- > IL-23R pathway which operates during human ILC differentiation. These observations may help guide protocols to expand functional ILC subsets in vitro with an aim towards novel ILC therapies for human disease

Polarized mitochondria as guardians of NK cell fitness

International audienceDistinct metabolic demands accompany lymphocyte differentiation into short-lived effector and long-lived memory cells. How bioenergetics processes are structured in innate natural killer (NK) cells remains unclear. We demonstrate that circulating human CD56Dim (NKDim) cells have fused mitochondria and enhanced metabolism compared with CD56Br (NKBr) cells. Upon activation, these 2 subsets showed a dichotomous response, with further mitochondrial potentiation in NKBr cells vs paradoxical mitochondrial fission and depolarization in NKDim cells. The latter effect impaired interferon-γ production, but rescue was possible by inhibiting mitochondrial fragmentation, implicating mitochondrial polarization as a central regulator of NK cell function. NKDim cells are heterogeneous, and mitochondrial polarization was associated with enhanced survival and function in mature NKDim cells, including memory-like human cytomegalovirus-dependent CD57+NKG2C+ subsets. In contrast, patients with genetic defects in mitochondrial fusion had a deficiency in adaptive NK cells, which had poor survival in culture. These results support mitochondrial polarization as a central regulator of mature NK cell fitness

Dichotomous metabolic networks govern human ILC2 proliferation and function

International audienceGroup 2 innate lymphoid cells (ILC2s) represent innate homologs of type 2 helper T cells (T H 2) that participate in immune defense and tissue homeostasis through production of type 2 cytokines. While T lymphocytes metabolically adapt to microenvironmental changes, knowledge of human ILC2 metabolism is limited, and its key regulators are unknown. Here, we show that circulating ‘naive’ ILC2s have an unexpected metabolic profile with a higher level of oxidative phosphorylation (OXPHOS) than natural killer (NK) cells. Accordingly, ILC2s are severely reduced in individuals with mitochondrial disease (MD) and impaired OXPHOS. Metabolomic and nutrient receptor analysis revealed ILC2 uptake of amino acids to sustain OXPHOS at steady state. Following activation with interleukin-33 (IL-33), ILC2s became highly proliferative, relying on glycolysis and mammalian target of rapamycin (mTOR) to produce IL-13 while continuing to fuel OXPHOS with amino acids to maintain cellular fitness and proliferation. Our results suggest that proliferation and function are metabolically uncoupled in human ILC2s, offering new strategies to target ILC2s in disease settings

STING Gain-of-Function Disrupts Lymph Node Organogenesis and Innate Lymphoid Cell Development in Mice

International audienceSTING gain-of-function causes autoimmunity and immunodeficiency in mice and STING-associated vasculopathy with onset in infancy (SAVI) in humans. Here, we report that STING gain-of-function in mice prevents development of lymph nodes and Peyer's patches. We show that the absence of secondary lymphoid organs is associated with diminished numbers of innate lymphoid cells (ILCs), including lymphoid tissue inducer (LTi) cells. Although wild-type (WT) α4β7+ progenitors differentiate efficiently into LTi cells, STING gain-of-function progenitors do not. Furthermore, STING gain-of-function impairs development of all types of ILCs. Patients with STING gain-of-function mutations have fewer ILCs, although they still have lymph nodes. In mice, expression of the STING mutant in RORγT-positive lineages prevents development of lymph nodes and reduces numbers of LTi cells. RORγT lineage-specific expression of STING gain-of-function also causes lung disease. Since RORγT is expressed exclusively in LTi cells during fetal development, our findings suggest that STING gain-of-function prevents lymph node organogenesis by reducing LTi cell numbers in mice

Human T-bet Governs Innate and Innate-like Adaptive IFN-γ Immunity against Mycobacteria

International audienceInborn errors of human interferon gamma (IFN-γ) immunity underlie mycobacterial disease. We report a patient with mycobacterial disease due to inherited deficiency of the transcription factor T-bet. The patient has extremely low counts of circulating Mycobacterium-reactive natural killer (NK), invariant NKT (iNKT), mucosal-associated invariant T (MAIT), and Vδ2+ γδ T lymphocytes, and of Mycobacterium-non reactive classic TH1 lymphocytes, with the residual populations of these cells also producing abnormally small amounts of IFN-γ. Other lymphocyte subsets develop normally but produce low levels of IFN-γ, with the exception of CD8+ αβ T and non-classic CD4+ αβ TH1∗ lymphocytes, which produce IFN-γ normally in response to mycobacterial antigens. Human T-bet deficiency thus underlies mycobacterial disease by preventing the development of innate (NK) and innate-like adaptive lymphocytes (iNKT, MAIT, and Vδ2+ γδ T cells) and IFN-γ production by them, with mycobacterium-specific, IFN-γ-producing, purely adaptive CD8+ αβ T, and CD4+ αβ TH1∗ cells unable to compensate for this deficit