Endothelial AHR activity prevents lung barrier disruption in viral infection.

Disruption of the lung endothelial-epithelial cell barrier following respiratory virus infection causes cell and fluid accumulation in the air spaces and compromises vital gas exchange function1. Endothelial dysfunction can exacerbate tissue damage2,3, yet it is unclear whether the lung endothelium promotes host resistance against viral pathogens. Here we show that the environmental sensor aryl hydrocarbon receptor (AHR) is highly active in lung endothelial cells and protects against influenza-induced lung vascular leakage. Loss of AHR in endothelia exacerbates lung damage and promotes the infiltration of red blood cells and leukocytes into alveolar air spaces. Moreover, barrier protection is compromised and host susceptibility to secondary bacterial infections is increased when endothelial AHR is missing. AHR engages tissue-protective transcriptional networks in endothelia, including the vasoactive apelin-APJ peptide system4, to prevent a dysplastic and apoptotic response in airway epithelial cells. Finally, we show that protective AHR signalling in lung endothelial cells is dampened by the infection itself. Maintenance of protective AHR function requires a diet enriched in naturally occurring AHR ligands, which activate disease tolerance pathways in lung endothelia to prevent tissue damage. Our findings demonstrate the importance of endothelial function in lung barrier immunity. We identify a gut-lung axis that affects lung damage following encounters with viral pathogens, linking dietary composition and intake to host fitness and inter-individual variations in disease outcome.

Interplay between CXCR4 and CCR2 regulates bone marrow exit of dendritic cell progenitors.

Conventional dendritic cells (cDCs) are found in most tissues and play a key role in initiation of immunity. cDCs require constant replenishment from progenitors called pre-cDCs that develop in the bone marrow (BM) and enter the blood circulation to seed all tissues. This process can be markedly accelerated in response to inflammation (emergency cDCpoiesis). Here, we identify two populations of BM pre-cDC marked by differential expression of CXCR4. We show that CXCR4lo cells constitute the migratory pool of BM pre-cDCs, which exits the BM and can be rapidly mobilized during challenge. We further show that exit of CXCR4lo pre-cDCs from BM at steady state is partially dependent on CCR2 and that CCR2 upregulation in response to type I IFN receptor signaling markedly increases efflux during infection with influenza A virus. Our results highlight a fine balance between retention and efflux chemokine cues that regulates steady-state and emergency cDCpoiesis.

Repair of airway epithelia requires metabolic rewiring towards fatty acid oxidation.

Epithelial tissues provide front-line barriers shielding the organism from invading pathogens and harmful substances. In the airway epithelium, the combined action of multiciliated and secretory cells sustains the mucociliary escalator required for clearance of microbes and particles from the airways. Defects in components of mucociliary clearance or barrier integrity are associated with recurring infections and chronic inflammation. The timely and balanced differentiation of basal cells into mature epithelial cell subsets is therefore tightly controlled. While different growth factors regulating progenitor cell proliferation have been described, little is known about the role of metabolism in these regenerative processes. Here we show that basal cell differentiation correlates with a shift in cellular metabolism from glycolysis to fatty acid oxidation (FAO). We demonstrate both in vitro and in vivo that pharmacological and genetic impairment of FAO blocks the development of fully differentiated airway epithelial cells, compromising the repair of airway epithelia. Mechanistically, FAO links to the hexosamine biosynthesis pathway to support protein glycosylation in airway epithelial cells. Our findings unveil the metabolic network underpinning the differentiation of airway epithelia and identify novel targets for intervention to promote lung repair.

A family of conserved bacterial virulence factors dampens interferon responses by blocking calcium signaling.

Interferons (IFNs) induce an antimicrobial state, protecting tissues from infection. Many viruses inhibit IFN signaling, but whether bacterial pathogens evade IFN responses remains unclear. Here, we demonstrate that the Shigella OspC family of type-III-secreted effectors blocks IFN signaling independently of its cell death inhibitory activity. Rather, IFN inhibition was mediated by the binding of OspC1 and OspC3 to the Ca2+ sensor calmodulin (CaM), blocking CaM kinase II and downstream JAK/STAT signaling. The growth of Shigella lacking OspC1 and OspC3 was attenuated in epithelial cells and in a murine model of infection. This phenotype was rescued in both models by the depletion of IFN receptors. OspC homologs conserved in additional pathogens not only bound CaM but also inhibited IFN, suggesting a widespread virulence strategy. These findings reveal a conserved but previously undescribed molecular mechanism of IFN inhibition and demonstrate the critical role of Ca2+ and IFN targeting in bacterial pathogenesis.

The interferon landscape along the respiratory tract impacts the severity of COVID-19.

Severe coronavirus disease 2019 (COVID-19) is characterized by overproduction of immune mediators, but the role of interferons (IFNs) of the type I (IFN-I) or type III (IFN-III) families remains debated. We scrutinized the production of IFNs along the respiratory tract of COVID-19 patients and found that high levels of IFN-III, and to a lesser extent IFN-I, characterize the upper airways of patients with high viral burden but reduced disease risk or severity. Production of specific IFN-III, but not IFN-I, members denotes patients with a mild pathology and efficiently drives the transcription of genes that protect against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In contrast, compared to subjects with other infectious or noninfectious lung pathologies, IFNs are overrepresented in the lower airways of patients with severe COVID-19 that exhibit gene pathways associated with increased apoptosis and decreased proliferation. Our data demonstrate a dynamic production of IFNs in SARS-CoV-2-infected patients and show IFNs play opposing roles at distinct anatomical sites.

A TLR7 antagonist restricts interferon-dependent and -independent immunopathology in a mouse model of severe influenza.

Cytokine-mediated immune-cell recruitment and inflammation contribute to protection in respiratory virus infection. However, uncontrolled inflammation and the "cytokine storm" are hallmarks of immunopathology in severe infection. Cytokine storm is a broad term for a phenomenon with diverse characteristics and drivers, depending on host genetics, age, and other factors. Taking advantage of the differential use of virus-sensing systems by different cell types, we test the hypothesis that specifically blocking TLR7-dependent, immune cell-produced cytokines reduces influenza-related immunopathology. In a mouse model of severe influenza characterized by a type I interferon (IFN-I)-driven cytokine storm, TLR7 antagonist treatment leaves epithelial antiviral responses unaltered but acts through pDCs and monocytes to reduce IFN-I and other cytokines in the lung, thus ameliorating inflammation and severity. Moreover, even in the absence of IFN-I signaling, TLR7 antagonism reduces inflammation and mortality driven by monocyte-produced chemoattractants and neutrophil recruitment into the infected lung. Hence, TLR7 antagonism reduces diverse types of cytokine storm in severe influenza.

Selective Janus kinase inhibition preserves interferon-λ-mediated antiviral responses.

Inflammatory diseases are frequently treated with Janus kinase (JAK) inhibitors to diminish cytokine signaling. These treatments can lead to inadvertent immune suppression and may increase the risk of viral infection. Tyrosine kinase 2 (TYK2) is a JAK family member required for efficient type I interferon (IFN-α/ß) signaling. We report here that selective TYK2 inhibition preferentially blocked potentially detrimental type I IFN signaling, whereas IFN-λ-mediated responses were largely preserved. In contrast, the clinically used JAK1/2 inhibitor baricitinib was equally potent in blocking IFN-α/ß- or IFN-λ-driven responses. Mechanistically, we showed that epithelial cells did not require TYK2 for IFN-λ-mediated signaling or antiviral protection. TYK2 deficiency diminished IFN-α-induced protection against lethal influenza virus infection in mice but did not impair IFN-λ-mediated antiviral protection. Our findings suggest that selective TYK2 inhibitors used in place of broadly acting JAK1/2 inhibitors may represent a superior treatment option for type I interferonopathies to counteract inflammatory responses while preserving antiviral protection mediated by IFN-λ.

Critical requirement for BCR, BAFF, and BAFFR in memory B cell survival.

Memory B cells (MBCs) are long-lived cells that form a critical part of immunological memory, providing rapid antibody responses to recurring infections. However, very little is known about signals controlling MBC survival. Previous work has shown that antigen is not required for MBC survival, but a requirement for the B cell antigen receptor (BCR) has not been tested. Other studies have shown that, unlike naive B cells, MBCs do not express BAFFR and their survival is independent of BAFF, the ligand for BAFFR. Here, using inducible genetic ablation, we show that survival of MBCs is critically dependent on the BCR and on signaling through the associated CD79A protein. Unexpectedly, we found that MBCs express BAFFR and that their survival requires BAFF and BAFFR; hence, loss of BAFF or BAFFR impairs recall responses. Finally, we show that MBC survival requires IKK2, a kinase that transduces BAFFR signals. Thus, MBC survival is critically dependent on signaling from BCR and BAFFR.

Teaching Old Dogs New Tricks? The Plasticity of Lung Alveolar Macrophage Subsets.

Alveolar macrophages (AMs) are highly abundant lung cells with important roles in homeostasis and immunity. Their function influences the outcome of lung infections, lung cancer, and chronic inflammatory disease. Recent findings reveal functional heterogeneity of AMs. Following lung insult, resident AMs can either remain unchanged, acquire new functionality, or be replaced by monocyte-derived AMs. Evidence from mouse models correlates AM function with their embryonic or monocyte origin. We hypothesize that resident AMs are terminally differentiated cells with low responsiveness and limited plasticity, while recruited, monocyte-derived AMs are initially highly immunoreactive but more plastic, able to change their function in response to environmental cues. Understanding cell-intrinsic and -extrinsic mechanisms determining AM function may provide opportunities for intervention in lung disease.

Type I and III interferons disrupt lung epithelial repair during recovery from viral infection.

Excessive cytokine signaling frequently exacerbates lung tissue damage during respiratory viral infection. Type I (IFN-α and IFN-ß) and III (IFN-λ) interferons are host-produced antiviral cytokines. Prolonged IFN-α and IFN-ß responses can lead to harmful proinflammatory effects, whereas IFN-λ mainly signals in epithelia, thereby inducing localized antiviral immunity. In this work, we show that IFN signaling interferes with lung repair during influenza recovery in mice, with IFN-λ driving these effects most potently. IFN-induced protein p53 directly reduces epithelial proliferation and differentiation, which increases disease severity and susceptibility to bacterial superinfections. Thus, excessive or prolonged IFN production aggravates viral infection by impairing lung epithelial regeneration. Timing and duration are therefore critical parameters of endogenous IFN action and should be considered carefully for IFN therapeutic strategies against viral infections such as influenza and coronavirus disease 2019 (COVID-19).

Microbiota-Driven Tonic Interferon Signals in Lung Stromal Cells Protect from Influenza Virus Infection.

Type I interferon (IFNα/ß) pathways are fine-tuned to elicit antiviral protection while minimizing immunopathology; however, the initiating stimuli, target tissues, and underlying mechanisms are unclear. Using models of physiological and dysregulated IFNα/ß receptor (IFNAR1) surface expression, we show here that IFNAR1-dependent signals set the steady-state IFN signature in both hematopoietic and stromal cells. Increased IFNAR1 levels promote a lung environment refractory to early influenza virus replication by elevating the baseline interferon signature. Commensal microbiota drive the IFN signature specifically in lung stroma, as shown by antibiotic treatment and fecal transplantation. Bone marrow chimera experiments identify lung stromal cells as crucially important for early antiviral immunity and stroma-immune cell interaction for late antiviral resistance. We propose that the microbiota-driven interferon signature in lung epithelia impedes early virus replication and that IFNAR1 surface levels fine-tune this signature. Our findings highlight the interplay between bacterial and viral exposure, with important implications for antibiotic use.

Natural amines inhibit activation of human plasmacytoid dendritic cells through CXCR4 engagement.

Plasmacytoid dendritic cells (pDC) are specialized in secretion of type I interferon in response to pathogens. Here we show that natural monoamines and synthetic amines inhibit pDC activation by RNA viruses. Furthermore, a synthetic analogue of histamine reduces type I interferon production in a mouse model of influenza infection. We identify CXC chemokine receptor 4 (CXCR4) as a receptor used by amines to inhibit pDC. Our study establishes a functional link between natural amines and the innate immune system and identifies CXCR4 as a potential 'on-off' switch of pDC activity with therapeutic potential.

IFNλ is a potent anti-influenza therapeutic without the inflammatory side effects of IFNα treatment.

Influenza A virus (IAV)-induced severe disease is characterized by infected lung epithelia, robust inflammatory responses and acute lung injury. Since type I interferon (IFNαß) and type III interferon (IFNλ) are potent antiviral cytokines with immunomodulatory potential, we assessed their efficacy as IAV treatments. IFNλ treatment of IAV-infected Mx1-positive mice lowered viral load and protected from disease. IFNα treatment also restricted IAV replication but exacerbated disease. IFNα treatment increased pulmonary proinflammatory cytokine secretion, innate cell recruitment and epithelial cell death, unlike IFNλ-treatment. IFNλ lacked the direct stimulatory activity of IFNα on immune cells. In epithelia, both IFNs induced antiviral genes but no inflammatory cytokines. Similarly, human airway epithelia responded to both IFNα and IFNλ by induction of antiviral genes but not of cytokines, while hPBMCs responded only to IFNα. The restriction of both IFNλ responsiveness and productive IAV replication to pulmonary epithelia allows IFNλ to limit IAV spread through antiviral gene induction in relevant cells without overstimulating the immune system and driving immunopathology. We propose IFNλ as a non-inflammatory and hence superior treatment option for human IAV infection.

TRAIL+ monocytes and monocyte-related cells cause lung damage and thereby increase susceptibility to influenza-Streptococcus pneumoniae coinfection.

Streptococcus pneumoniae coinfection is a major cause of influenza-associated mortality; however, the mechanisms underlying pathogenesis or protection remain unclear. Using a clinically relevant mouse model, we identify immune-mediated damage early during coinfection as a new mechanism causing susceptibility. Coinfected CCR2(-/-) mice lacking monocytes and monocyte-derived cells control bacterial invasion better, show reduced epithelial damage and are overall more resistant than wild-type controls. In influenza-infected wild-type lungs, monocytes and monocyte-derived cells are the major cell populations expressing the apoptosis-inducing ligand TRAIL. Accordingly, anti-TRAIL treatment reduces bacterial load and protects against coinfection if administered during viral infection, but not following bacterial exposure. Post-influenza bacterial outgrowth induces a strong proinflammatory cytokine response and massive inflammatory cell infiltrate. Depletion of neutrophils or blockade of TNF-α facilitate bacterial outgrowth, leading to increased mortality, demonstrating that these factors aid bacterial control. We conclude that inflammatory monocytes recruited early, during the viral phase of coinfection, induce TRAIL-mediated lung damage, which facilitates bacterial invasion, while TNF-α and neutrophil responses help control subsequent bacterial outgrowth. We thus identify novel determinants of protection versus pathology in influenza-Streptococcus pneumoniae coinfection.

Themis2 is not required for B cell development, activation, and antibody responses.

Themis1 is a protein implicated in transducing signals from the TCR. Mice deficient in Themis1 show a strong impairment in T cell selection in the thymus and defective T cell activation. The related Themis2 protein is expressed in B cells where it associates with signaling proteins Grb2 and Vav1, and is tyrosine phosphorylated after BCR stimulation. Thus, it has been proposed that Themis2 may transduce BCR signals, and hence play important roles in B cell development and activation. In this article, we show that Themis2 is expressed in all developing subsets of B cells, in mature follicular and marginal zone B cells, and in activated B cells, including germinal center B cells and plasma cells. In contrast, B lineage cells express no other Themis-family genes. Activation of B cells leads to reduced Themis2 expression, although it remains the only Themis-family protein expressed. To analyze the physiological function of Themis2, we generated a Themis2-deficient mouse strain. Surprisingly, we found that Themis2 is not required for B cell development, for activation, or for Ab responses either to model Ags or to influenza viral infection.

Pathogenic potential of interferon αß in acute influenza infection.

Influenza symptoms vary from mild disease to death; however, determinants of severity are unclear. Type I interferons (IFNαß) are recognized as key antiviral cytokines. Here we show that, surprisingly, influenza-infected 129 mice have increased lung damage, morbidity and mortality, yet higher levels of IFNαß, than C57BL/6 mice. Consistently, IFNα treatment of influenza-infected C57BL/6 mice increases morbidity. IFNαß receptor deficiency in 129 mice decreases morbidity, lung damage, proinflammatory cytokines and lung-infiltrating inflammatory cells, and reduces expression of the death-inducing receptor DR5 on lung epithelia and its ligand TRAIL on inflammatory monocytes. Depletion of PDCA-1+ cells or interruption of TRAIL-DR5 interaction protects infected 129 mice. Selective lack of IFNαß signalling in stromal cells abolishes epithelial DR5 upregulation and apoptosis, reducing host susceptibility. Hence, excessive IFNαß signalling in response to acute influenza infection can result in uncontrolled inflammation and TRAIL-DR5-mediated epithelial cell death, which may explain morbidity and has important implications for treatment of severe disease.

The transcription factor E4BP4 is not required for extramedullary pathways of NK cell development.

NK cells contribute to antitumor and antiviral immunosurveillance. Their development in the bone marrow (BM) requires the transcription factor E4BP4/NFIL3, but requirements in other organs are less well defined. In this study, we show that CD3(-)NK1.1(+)NKp46(+)CD122(+) NK cells of immature phenotype and expressing low eomesodermin levels are found in thymus, spleen, and liver of E4BP4-deficient mice, whereas numbers of mature, eomesodermin(high) conventional NK cells are drastically reduced. E4BP4-deficient CD44(+)CD25(-) double-negative 1 thymocytes efficiently develop in vitro into NK cells with kinetics, phenotype, and functionality similar to wild-type controls, whereas no NK cells develop from E4BP4-deficient BM precursors. In E4BP4/Rag-1 double-deficient (DKO) mice, NK cells resembling those in Rag-1-deficient controls are found in similar numbers in the thymus and liver. However, NK precursors are reduced in DKO BM, and no NK cells develop from DKO BM progenitors in vitro. DKO thymocyte precursors readily develop into NK cells, but DKO BM transfers into nude recipients and NK cells in E4BP4/Rag-1/IL-7 triple-KO mice indicated thymus-independent NK cell development. In the presence of T cells or E4BP4-sufficient NK cells, DKO NK cells have a selective disadvantage, and thymic and hepatic DKO NK cells show reduced survival when adoptively transferred into lymphopenic hosts. This correlates with higher apoptosis rates and lower responsiveness to IL-15 in vitro. In conclusion, we demonstrate E4BP4-independent development of NK cells of immature phenotype, reduced fitness, short t1/2, and potential extramedullary origin. Our data identify E4BP4-independent NK cell developmental pathways and a role for E4BP4 in NK cell homeostasis.

Type I and type III interferons drive redundant amplification loops to induce a transcriptional signature in influenza-infected airway epithelia.

Interferons (IFNs) are a group of cytokines with a well-established antiviral function. They can be induced by viral infection, are secreted and bind to specific receptors on the same or neighbouring cells to activate the expression of hundreds of IFN stimulated genes (ISGs) with antiviral function. Type I IFN has been known for more than half a century. However, more recently, type III IFN (IFNλ, IL-28/29) was shown to play a similar role and to be particularly important at epithelial surfaces. Here we show that airway epithelia, the primary target of influenza A virus, produce both IFN I and III upon infection, and that induction of both depends on the RIG-I/MAVS pathway. While IRF3 is generally regarded as the transcription factor required for initiation of IFN transcription and the so-called "priming loop", we find that IRF3 deficiency has little impact on IFN expression. In contrast, lack of IRF7 reduced IFN production significantly, and only IRF3(-/-)IRF7(-/-) double deficiency completely abolished it. The transcriptional response to influenza infection was largely dependent on IFNs, as it was reduced to a few upregulated genes in epithelia lacking receptors for both type I and III IFN (IFNAR1(-/-)IL-28Rα(-/-)). Wild-type epithelia and epithelia deficient in either the type I IFN receptor or the type III IFN receptor exhibit similar transcriptional profiles in response to virus, indicating that none of the induced genes depends selectively on only one IFN system. In chimeric mice, the lack of both IFN I and III signalling in the stromal compartment alone significantly increased the susceptibility to influenza infection. In conclusion, virus infection of airway epithelia induces, via a RIG-I/MAVS/IRF7 dependent pathway, both type I and III IFNs which drive two completely overlapping and redundant amplification loops to upregulate ISGs and protect from influenza infection.

Adjuvanticity of the oil-in-water emulsion MF59 is independent of Nlrp3 inflammasome but requires the adaptor protein MyD88.

Oil-in-water emulsions have been successfully used to increase the efficacy, immunogenicity, and cross-protection of human vaccines; however, their mechanism of action is still largely unknown. Nlrp3 inflammasome has been previously associated to the activity of alum, another adjuvant broadly used in human vaccines, and MyD88 adaptor protein is required for the adjuvanticity of most Toll-like receptor agonists. We compared the contribution of Nlrp3 and MyD88 to the adjuvanticity of alum, the oil-in-water emulsion MF59, and complete Freund's adjuvant in mice using a three-component vaccine against serogroup B Neisseria meningitidis (rMenB). Although the basal antibody responses to the nonadjuvanted rMenB vaccine were largely dependent on Nlrp3, the high-level antibody responses induced by alum, MF59, or complete Freund's adjuvant did not require Nlrp3. Surprisingly, we found that MF59 requires MyD88 to enhance bactericidal antibody responses to the rMenB vaccine. Because MF59 did not activate any of the Toll-like receptors in vitro, we propose that MF59 requires MyD88 for a Toll-like receptor-independent signaling pathway.