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Early-life (EL) respiratory infections increase pulmonary disease risk, especially EL-Respiratory Syncytial Virus (EL-RSV) infections linked to asthma. Mechanisms underlying asthma predisposition remain unknown. In this study, we examined the long-term effects on the lung after four weeks post EL-RSV infection. We identified alterations in the lung epithelial cell, with a rise in the percentage of alveolar type 2 epithelial cells (AT2) and a decreased percentage of cells in the AT1 and AT2-AT1 subclusters, as well as upregulation of Bmp2 and Krt8 genes that are associated with AT2-AT1 trans-differentiation, suggesting potential defects in lung repair processes. We identified persistent upregulation of asthma-associated genes, including Il33. EL-RSV-infected mice allergen-challenged exhibited exacerbated allergic response, with significant upregulation of Il33 in the lung and AT2 cells. Similar long-term effects were observed in mice exposed to EL-IL-1β. Notably, treatment with IL-1ra during acute EL-RSV infection mitigated the long-term alveolar alterations and the allergen-exacerbated response. Finally, epigenetic modifications in the promoter of the Il33 gene were detected in AT2 cells harvested from EL-RSV and EL-IL1β groups, suggesting that long-term alteration in the epithelium after RSV infection is dependent on the IL-1β pathway. This study provides insight into the molecular mechanisms of asthma predisposition after RSV infection., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The aryl hydrocarbon receptor (AHR) is a receptor/transcription factor widely expressed in the lung. The physiological roles of AHR expressed in the alveolar epithelium remain unclear. In this study, we tested the hypothesis that alveolar epithelial AHR activity plays an important role in modulating inflammatory responses and maintaining alveolar integrity during lung injury and repair. AHR is expressed in alveolar epithelial cells (AECs) and is active. AHR activation with the endogenous AHR ligand, FICZ (5,11-dihydroindolo[3,2-b] carbazole-6-carboxaldehyde), significantly suppressed inflammatory cytokine expression in response to inflammatory stimuli in primary murine AECs and in the MLE-15 epithelial cell line. In an LPS model of acute lung injury in mice, coadministration of FICZ with LPS suppressed protein leak, reduced neutrophil accumulation in BAL fluid, and suppressed inflammatory cytokine expression in lung tissue and BAL fluid. Relevant to healing following inflammatory injury, AHR activation suppressed TGF-β-induced expression of genes associated with epithelial-mesenchymal transition. Knockdown of AHR in primary AECs with shRNA or in CRISPR-Cas-9-induced MLE-15 cells resulted in upregulation of α-smooth muscle actin (αSma), Col1a1, and Fn1 and reduced expression of epithelial genes Col4a1 and Sdc1. MLE-15 clones lacking AHR demonstrated accelerated wound closure in a scratch model. AHR activation with FICZ enhanced barrier function (transepithelial electrical resistance) in primary murine AECs and limited decline of transepithelial electrical resistance following inflammatory injury. AHR activation in AECs preserves alveolar integrity by modulating inflammatory cytokine expression while enhancing barrier function and limiting stress-induced expression of mesenchymal genes., (Copyright © 2024 by The American Association of Immunologists, Inc.)

Recruitment of immune cells to the injury site plays a pivotal role in the pathology of radiation-associated diseases. In this study, we investigated the impact of the chemokine CCL22 released from alveolar type II epithelial (AT2) cells after irradiation on the recruitment and functional changes of dendritic cells (DCs) in the development of radiation-induced lung injury (RILI). By examining changes in CCL22 protein levels in lung tissue of C57BL/6N mice with RILI, we discovered that ionizing radiation increased CCL22 expression in irradiated alveolar AT2 cells, as did MLE-12 cells after irradiation. A transwell migration assay revealed that CCL22 promoted the migration of CCR4-positive DCs to the injury site, which explained the migration of pulmonary CCR4-positive DCs in RILI mice in vivo. Coculture experiments demonstrated that, consistent with the response of regulatory T cells in the lung tissue of RILI mice, exogenous CCL22-induced DCs promoted regulatory T cell proliferation. Mechanistically, we demonstrated that Dectin2 and Nr4a2 are key targets in the CCL22 signaling pathway, which was confirmed in pulmonary DCs of RILI mice. As a result, CCL22 upregulated the expression of PD-L1, IL-6, and IL-10 in DCs. Consequently, we identified a mechanism in which CCL22 induced DC tolerance through the CCR4-Dectin2-PLC-γ2-NFATC2-Nr4a2-PD-L1 pathway. Collectively, these findings demonstrated that ionizing radiation stimulates the expression of CCL22 in AT2 cells to recruit DCs to the injury site and further polarizes them into a tolerant subgroup of CCL22 DCs to regulate lung immunity, ultimately providing potential therapeutic targets for DC-mediated RILI., (Copyright © 2024 by The American Association of Immunologists, Inc.)

CD4 + T cells play a key role in γ-herpesvirus infection control. However, the mechanisms involved are unclear. Murine herpesvirus type 4 (MuHV-4) allows relevant immune pathways to be dissected experimentally in mice. In the lungs, it colonizes myeloid cells, which can express MHC class II (MHCII), and type 1 alveolar epithelial cells (AEC1), which lack it. Nevertheless, CD4 + T cells can control AEC1 infection, and this control depends on MHCII expression in myeloid cells. Interferon-gamma (IFNγ) is a major component of CD4 + T cell-dependent MuHV-4 control. Here, we show that the action of IFNγ is also indirect, as CD4 + T cell-mediated control of AEC1 infection depended on IFNγ receptor (IFNγR1) expression in CD11c + cells. Indirect control also depended on natural killer (NK) cells. Together, the data suggest that the activation of MHCII + CD11c + antigen-presenting cells is key to the CD4 + T cell/NK cell protection axis. By contrast, CD8 + T cell control of AEC1 infection appeared to operate independently., Importance: CD4 + T cells are critical for the control of gamma-herpesvirus infection; they act indirectly, by recruiting natural killer (NK) cells to attack infected target cells. Here, we report that the CD4 + T cell/NK cell axis of gamma-herpesvirus control requires interferon-γ engagement of CD11c + dendritic cells. This mechanism of CD4 + T cell control releases the need for the direct engagement of CD4 + T cells with virus-infected cells and may be a common strategy for host control of immune-evasive pathogens., Competing Interests: The authors declare no conflict of interest.

Background: The role of autoreactive T cells on the course of Coronavirus disease-19 (COVID-19) remains elusive. Type II pneumocytes represent the main target cells of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Autoimmune responses against antigens highly expressed in type II pneumocytes may influence the severity of COVID-19 disease., Objective: The aim of this study was to investigate autoreactive T cell responses against self-antigens highly expressed in type II pneumocytes in the blood of COVID-19 patients with severe and non-severe disease., Methods: We collected blood samples of COVID-19 patients with varying degrees of disease severity and of pre-pandemic controls. T cell stimulation assays with peptide pools of type II pneumocyte antigens were performed in two independent cohorts to analyze the autoimmune T cell responses in patients with non-severe and severe COVID-19 disease. Target cell lysis assays were performed with lung cancer cell lines to determine the extent of cell killing by type II PAA-specific T cells., Results: We identified autoreactive T cell responses against four recently described self-antigens highly expressed in type II pneumocytes, known as surfactant protein A, surfactant protein B, surfactant protein C and napsin A, in the blood of COVID-19 patients. These antigens were termed type II pneumocyte-associated antigens (type II PAAs). We found that patients with non-severe COVID-19 disease showed a significantly higher frequency of type II PAA-specific autoreactive T cells in the blood when compared to severely ill patients. The presence of high frequencies of type II PAA-specific T cells in the blood of non-severe COVID-19 patients was independent of their age. We also found that napsin A-specific T cells from convalescent COVID-19 patients could kill lung cancer cells, demonstrating the functional and cytotoxic role of these T cells., Conclusions: Our data suggest that autoreactive type II PAA-specific T cells have a protective role in SARS-CoV-2 infections and the presence of high frequencies of these autoreactive T cells indicates effective viral control in COVID-19 patients. Type II-PAA-specific T cells may therefore promote the killing of infected type II pneumocytes and viral clearance., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Immunosurveillance of cancer requires the presentation of peptide antigens on major histocompatibility complex class I (MHC-I) molecules 1-5 . Current approaches to profiling of MHC-I-associated peptides, collectively known as the immunopeptidome, are limited to in vitro investigation or bulk tumour lysates, which limits our understanding of cancer-specific patterns of antigen presentation in vivo 6 . To overcome these limitations, we engineered an inducible affinity tag into the mouse MHC-I gene (H2-K1) and targeted this allele to the Kras LSL-G12D/+ Trp53 fl/fl mouse model (KP/K b Strep) 7 . This approach enabled us to precisely isolate MHC-I peptides from autochthonous pancreatic ductal adenocarcinoma and from lung adenocarcinoma (LUAD) in vivo. In addition, we profiled the LUAD immunopeptidome from the alveolar type 2 cell of origin up to late-stage disease. Differential peptide presentation in LUAD was not predictable by mRNA expression or translation efficiency and is probably driven by post-translational mechanisms. Vaccination with peptides presented by LUAD in vivo induced CD8 + T cell responses in naive mice and tumour-bearing mice. Many peptides specific to LUAD, including immunogenic peptides, exhibited minimal expression of the cognate mRNA, which prompts the reconsideration of antigen prediction pipelines that triage peptides according to transcript abundance 8 . Beyond cancer, the K b Strep allele is compatible with other Cre-driver lines to explore antigen presentation in vivo in the pursuit of understanding basic immunology, infectious disease and autoimmunity., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Lung infections are a perennial leading cause of death worldwide. The lung epithelium comprises three main cell types: alveolar type I (AT1), alveolar type II (AT2), and bronchiolar cells. Constitutively, these three cell types express extremely low amounts of surface MHC class I (MHC I) molecules, that is, <1% of levels found on medullary thymic epithelial cells (ECs). We report that inhalation of the TLR4 ligand LPS upregulates cell surface MHC I by ∼25-fold on the three subtypes of mouse lung ECs. This upregulation is dependent on Nlrc5 , Stat1 , and Stat2 and caused by a concerted production of the three IFN families. It is nevertheless hampered, particularly in AT1 cells, by the limited expression of genes instrumental in the peptide loading of MHC I molecules. Genes involved in production and response to cytokines and chemokines were selectively induced in AT1 cells. However, discrete gene subsets were selectively downregulated in AT2 or bronchiolar cells following LPS inhalation. Genes downregulated in AT2 cells were linked to cell differentiation and cell proliferation, and those repressed in bronchiolar cells were primarily involved in cilium function. Our study shows a delicate balance between the expression of transcripts maintaining lung epithelium integrity and transcripts involved in Ag presentation in primary lung ECs., (Copyright © 2022 by The American Association of Immunologists, Inc.)

In epigenome-wide association studies analysing DNA methylation from samples containing multiple cell types, it is essential to adjust the analysis for cell type composition. One well established strategy for achieving this is reference-based cell type deconvolution, which relies on knowledge of the DNA methylation profiles of purified constituent cell types. These are then used to estimate the cell type proportions of each sample, which can then be incorporated to adjust the association analysis. Bronchoalveolar lavage is commonly used to sample the lung in clinical practice and contains a mixture of different cell types that can vary in proportion across samples, affecting the overall methylation profile. A current barrier to the use of bronchoalveolar lavage in DNA methylation-based research is the lack of reference DNA methylation profiles for each of the constituent cell types, thus making reference-based cell composition estimation difficult. Herein, we use bronchoalveolar lavage samples collected from children with cystic fibrosis to define DNA methylation profiles for the four most common and clinically relevant cell types: alveolar macrophages, granulocytes, lymphocytes and alveolar epithelial cells. We then demonstrate the use of these methylation profiles in conjunction with an established reference-based methylation deconvolution method to estimate the cell type composition of two different tissue types; a publicly available dataset derived from artificial blood-based cell mixtures and further bronchoalveolar lavage samples. The reference DNA methylation profiles developed in this work can be used for future reference-based cell type composition estimation of bronchoalveolar lavage. This will facilitate the use of this tissue in studies examining the role of DNA methylation in lung health and disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Shanthikumar, Neeland, Saffery, Ranganathan, Oshlack and Maksimovic.)

In this review, we discuss the role of pulmonary surfactant in the host defense against respiratory pathogens, including novel coronavirus SARS-CoV-2. In the lower respiratory system, the virus uses angiotensin-converting enzyme 2 (ACE2) receptor in conjunction with serine protease TMPRSS2, expressed by alveolar type II (ATII) cells as one of the SARS-CoV-2 target cells, to enter. ATII cells are the main source of surfactant. After their infection and the resulting damage, the consequences may be severe and may include injury to the alveolar-capillary barrier, lung edema, inflammation, ineffective gas exchange, impaired lung mechanics and reduced oxygenation, which resembles acute respiratory distress syndrome (ARDS) of other etiology. The aim of this review is to highlight the key role of ATII cells and reduced surfactant in the pathogenesis of the respiratory form of COVID-19 and to emphasize the rational basis for exogenous surfactant therapy in COVID-19 ARDS patients.

Aspergillus fumigatus is an important human fungal pathogen and its conidia are constantly inhaled by humans. In immunocompromised individuals, conidia can grow out as hyphae that damage lung epithelium. The resulting invasive aspergillosis is associated with devastating mortality rates. Since infection is a race between the innate immune system and the outgrowth of A. fumigatus conidia, we use dynamic optimization to obtain insight into the recruitment and depletion of alveolar macrophages and neutrophils. Using this model, we obtain key insights into major determinants of infection outcome on host and pathogen side. On the pathogen side, we predict in silico and confirm in vitro that germination speed is an important virulence trait of fungal pathogens due to the vulnerability of conidia against host defense. On the host side, we found that epithelial cells, which have been underappreciated, play a role in fungal clearance and are potent mediators of cytokine release. Both predictions were confirmed by in vitro experiments on established cell lines as well as primary lung cells. Further, our model affirms the importance of neutrophils in invasive aspergillosis and underlines that the role of macrophages remains elusive. We expect that our model will contribute to improvement of treatment protocols by focusing on the critical components of immune response to fungi but also fungal virulence traits., Competing Interests: The authors have declared that no competing interests exist.

Complement receptor 3 (CD11b/CD18) is an important receptor that mediates adhesion, phagocytosis and chemotaxis in various immunocytes. The conidia of the medically-important pathogenic fungus, Aspergillus fumigatus can be internalized into alveolar epithelial cells to disseminate its infection in immunocompromised host; however, the role of CR3 in this process is poorly understood. In the present study, we investigated the potential role of CR3 on A. fumigatus internalization into type II alveolar epithelial cells and its effect on host intracellular PA content induced by A. fumigatus . We found that CR3 is expressed in alveolar epithelial cells and that human serum and bronchoalveolar lavage fluid (BALF) could improve A. fumigatus conidial internalization into A549 type II alveolar epithelial cell line and mouse primary alveolar epithelial cells, which were significantly inhibited by the complement C3 quencher and CD11b-blocking antibody. Serum-opsonization of swollen conidia, but not resting conidia led to the increase of cellular phosphatidic acid (PA) in A549 cells during infection. Moreover, both conidial internalization and induced PA production were interfered by CD11b-blocking antibody and dependent on FAK activity, but not Syk in alveolar epithelial cells. Overall, our results revealed that CR3 is a critical modulator of Aspergillus fumigatus internalization into alveolar epithelial cells.

Mycoplasma pneumoniae-induced pneumonia (MPP) is a common cause of community-acquired respiratory tract infections, increasing risk of morbidity and mortality, in children. However, diagnosing early-stage MPP is difficult owing to the lack of good diagnostic methods. Here, we examined the protein profile of bronchoalveolar lavage fluid (BALF) and found that S100A8/A9 was highly expressed. Enzyme-linked immunosorbent assays used to assess protein levels in serum samples indicated that S100A8/A9 concentrations were also increased in serum obtained from children with MPP, with no change in S100A8/A9 levels in children with viral or bacterial pneumonia. In vitro, S100A8/A9 treatment significantly increased apoptosis in a human alveolar basal epithelial cell line (A549 cells). Bioinformatics analyses indicated that up-regulated S100A8/A9 proteins participated in the interleukin (IL)-17 signaling pathway. The origin of the increased S100A8/A9 was investigated in A549 cells and in neutrophils obtained from children with MPP. Treatment of neutrophils, but not of A549 cells, with IL-17A released S100A8/A9 into the culture medium. In summary, we demonstrated that S100A8/A9, possibly released from neutrophils, is a new potential biomarker for the clinical diagnosis of children MPP and involved in the development of this disease through enhancing apoptosis of alveolar basal epithelial cells., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The occurrence of allergic diseases induced by aeroallergens has increased in the past decades. Among inhalant allergens, mites remain the important causal agent of allergic diseases. Storage mites- Tyrophagus putrescentiae are found in stored products or domestic environments. Major allergen Tyr-p3 plays a significant role in triggering IgE-mediated hypersensitivity. However, its effects on pulmonary inflammation, internalization, and activation in human epithelium remain elusive. Protease-activated receptors (PARs) are activated upon cleavage by proteases. A549 cells were used as an epithelial model to examine the PAR activation by Tyr-p3 and therapeutic potential of PAR-2 antagonist (GB88) in allergic responses. Enzymatic properties and allergen localization of Tyr-p3 were performed. The release of inflammatory mediators, phosphorylation of mitogen-activated protein kinase (MAPK), and cell junction disruptions were evaluated after Tyr-p3 challenge. Enzymatic properties determined by substrate digestion and protease inhibitors indicated that Tyr-p3 processes a trypsin-like serine protease activity. The PAR-2 mRNA levels were significantly increased by nTyr-p3 but inhibited by protease inhibitors or GB88. Protease allergen of nTyr-p3 significantly increased the levels of pro-inflammatory cytokines (IL-6 and TNF-α), chemokine (IL-8), and IL-1β in epithelial cells. nTyr-p3 markedly increased phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and MAP kinase. When cells were pretreated with GB88 then added nTyr-p3, the phosphorylated ERK1/2 did not inhibit by GB88. GB88 increased ERK1/2 phosphorylation in human epithelium cells. GB88 is able to block PAR-2-mediated calcium signaling which inhibits the nTyr-p3-induced Ca 2+ release. Among the pharmacologic inhibitors, the most effective inhibitor of the nTyr-p3 in the induction of IL-8 or IL-1β levels was GB88 followed by SBTI, MAPK/ERK, ERK, and p38 inhibitors. Levels of inflammatory mediators, including GM-CSF, VEGF, COX-2, TSLP, and IL-33 were reduced by treatment of GB88 or SBTI. Further, GB88 treatment down-regulated the nTyr-p3-induced PAR-2 expression in allergic patients with asthma or rhinitis. Tight junction and adherens junction were disrupted in epithelial cells by nTyr-p3 exposure; however, this effect was avoided by GB88. Immunostaining with frozen sections of the mite body showed the presence of Tyr-p3 throughout the intestinal digestive system, especially in the hindgut around the excretion site. In conclusion, our findings suggest that Tyr-p3 from domestic mites leads to disruption of the airway epithelial barrier after inhalation. Proteolytic activity of Tyr-p3 causes the PAR-2 mRNA expression, thus leading to the release of numerous inflammatory mediators. Antagonism of PAR2 activity suggests GB88 as the therapeutic potential for anti-inflammation medicine, especially in allergy development triggered by protease allergens., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wang, Yu, Tsai, Yu and Liao.)

Perturbation of lung homeostasis is frequently associated with progressive and fatal respiratory diseases, such as pulmonary fibrosis. Leucine-rich repeat kinase 2 (LRRK2) is highly expressed in healthy lungs, but its functions in lung homeostasis and diseases remain elusive. Herein, we showed that LRRK2 expression was clearly reduced in mammalian fibrotic lungs, and LRRK2-deficient mice exhibited aggravated bleomycin-induced pulmonary fibrosis. Furthermore, we demonstrated that in bleomycin-treated mice, LRRK2 expression was dramatically decreased in alveolar type II epithelial (AT2) cells, and its deficiency resulted in profound dysfunction of AT2 cells, characterized by impaired autophagy and accelerated cellular senescence. Additionally, LRRK2-deficient AT2 cells showed a higher capacity of recruiting profibrotic macrophages via the CCL2/CCR2 signaling, leading to extensive macrophage-associated profibrotic responses and progressive pulmonary fibrosis. Taken together, our study demonstrates that LRRK2 plays a crucial role in preventing AT2 cell dysfunction and orchestrating the innate immune responses to protect against pulmonary fibrosis., Competing Interests: The authors declare no competing interest.

Aluminum hydroxide salts (alum) have been added to inactivated vaccines as safe and effective adjuvants to increase the effectiveness of vaccination. However, the exact cell types and immunological factors that initiate mucosal immune responses to alum adjuvants are unclear. In this study, the mechanism of action of alum adjuvant in nasal vaccination was investigated. Alum has been shown to act as a powerful and unique adjuvant when added to a nasal influenza split vaccine in mice. Alum is cytotoxic in the alveoli and stimulates the release of damage-associated molecular patterns, such as dsDNA, interleukin (IL)-1α, and IL-33. We found that Ag-specific IgA antibody (Ab) production was markedly reduced in IL-33-deficient mice. However, no decrease was observed in Ag-specific IgA Ab production with DNase I treatment, and no decrease was observed in IL-1α/β or IL-6 production in IL-33-deficient mice. From the experimental results of primary cultured cells and immunofluorescence staining, although IL-1α was secreted by alveolar macrophage necroptosis, IL-33 release was observed in alveolar epithelial cell necroptosis but not in alveolar macrophages. Alum- or IL-33-dependent Ag uptake enhancement and elevation of OX40L expression were not observed. By stimulating the release of IL-33, alum induced Th2 immunity via IL-5 and IL-13 production in group 2 innate lymphoid cells (ILC2s) and increased MHC class II expression in antigen-presenting cells (APCs) in the lung. Our results suggest that IL-33 secretion by epithelial cell necroptosis initiates APC- and ILC2-mediated T cell activation, which is important for the enhancement of Ag-specific IgA Ab production by alum., Competing Interests: The authors have declared that no competing interests exist.

The respiratory tract is considered the main port of entry of Mycobacterium leprae , the causative agent of leprosy. However, the great majority of individuals exposed to the leprosy bacillus will never manifest the disease due to their capacity to develop protective immunity. Besides acting as a physical barrier, airway epithelium cells are recognized as key players by initiating a local innate immune response that orchestrates subsequent adaptive immunity to control airborne infections. However, to date, studies exploring the interaction of M. leprae with the respiratory epithelium have been scarce. In this work, the capacity of  M. leprae  to immune activate human alveolar epithelial cells was investigated, demonstrating that M. leprae -infected A549 cells secrete significantly increased IL-8 that is dependent on NF-κB activation. M. leprae was also able to induce IL-8 production in human primary nasal epithelial cells. M. leprae -treated A549 cells also showed higher expression levels of human β-defensin-2 (hβD-2), MCP-1, MHC-II and the co-stimulatory molecule CD80. Furthermore, the TLR-9 antagonist inhibited both the secretion of IL-8 and NF-κB activation in response to M. leprae , indicating that bacterial DNA sensing by this Toll-like receptor constitutes an important innate immune pathway activated by the pathogen. Finally, evidence is presented suggesting that extracellular DNA molecules anchored to Hlp, a histone-like protein present on the M. leprae surface, constitute major TLR-9 ligands triggering this pathway. The ability of M. leprae to immune activate respiratory epithelial cells herein demonstrated may represent a very early event during infection that could possibly be essential to the generation of a protective response., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Dias, Silva, Silva, Linhares, Santos, Vivarini, Marques, Rosa, Lopes, Berrêdo-Pinho and Pessolani.)

In COVID-19, immune responses are key in determining disease severity. However, cellular mechanisms at the onset of inflammatory lung injury in SARS-CoV-2 infection, particularly involving endothelial cells, remain ill-defined. Using Syrian hamsters as a model for moderate COVID-19, we conduct a detailed longitudinal analysis of systemic and pulmonary cellular responses, and corroborate it with datasets from COVID-19 patients. Monocyte-derived macrophages in lungs exert the earliest and strongest transcriptional response to infection, including induction of pro-inflammatory genes, while epithelial cells show weak alterations. Without evidence for productive infection, endothelial cells react, depending on cell subtypes, by strong and early expression of anti-viral, pro-inflammatory, and T cell recruiting genes. Recruitment of cytotoxic T cells as well as emergence of IgM antibodies precede viral clearance at day 5 post infection. Investigating SARS-CoV-2 infected Syrian hamsters thus identifies cell type-specific effector functions, providing detailed insights into pathomechanisms of COVID-19 and informing therapeutic strategies., (© 2021. The Author(s).)

The small GTPase RhoA and its downstream effectors are critical regulators in the pathophysiological processes of asthma. The underlying mechanism, however, remains undetermined. Here, we generated an asthma mouse model with RhoA-conditional KO mice (Sftpc-cre;RhoAfl/fl) in type II alveolar epithelial cells (AT2) and demonstrated that AT2 cell-specific deletion of RhoA leads to exacerbation of allergen-induced airway hyperresponsiveness and airway inflammation with elevated Th2 cytokines in bronchoalveolar lavage fluid (BALF). Notably, Sftpc-cre;RhoAfl/fl mice showed a significant reduction in Tgf-β1 levels in BALF and lung tissues, and administration of recombinant Tgf-β1 to the mice rescued Tgf-β1 and alleviated the increased allergic airway inflammation observed in Sftpc-cre;RhoAfl/fl mice. Using RNA sequencing technology, we identified Slc26a4 (pendrin), a transmembrane anion exchange, as the most upregulated gene in RhoA-deficient AT2 cells. The upregulation of SLC26A4 was further confirmed in AT2 cells of asthmatic patients and mouse models and in human airway epithelial cells expressing dominant-negative RHOA (RHOA-N19). SLA26A4 was also elevated in serum from asthmatic patients and negatively associated with the percentage of forced expiratory volume in 1 second (FEV1%). Furthermore, SLC26A4 inhibition promoted epithelial TGF-β1 release and attenuated allergic airway inflammation. Our study reveals a RhoA/SLC26A4 axis in AT2 cells that functions as a protective mechanism against allergic airway inflammation.

Type II alveolar cells (AT2s) are critical for basic respiratory homeostasis and tissue repair after lung injury. Prior studies indicate that AT2s also express major histocompatibility complex class II (MHCII) molecules, but how MHCII expression by AT2s is regulated and how it contributes to host defense remain unclear. Here we show that AT2s express high levels of MHCII independent of conventional inflammatory stimuli, and that selective loss of MHCII from AT2s in mice results in modest worsening of respiratory virus disease following influenza and Sendai virus infections. We also find that AT2s exhibit MHCII presentation capacity that is substantially limited compared to professional antigen presenting cells. The combination of constitutive MHCII expression and restrained antigen presentation may position AT2s to contribute to lung adaptive immune responses in a measured fashion, without over-amplifying damaging inflammation.

Ethnopharmacological Relevance: Since the occurrence of coronavirus disease 2019 (COVID-19) in Wuhan, China in December 2019, COVID-19 has been quickly spreading out to other provinces and countries. Considering that traditional Chinese medicine (TCM) played an important role during outbreak of SARS and H1N1, finding potential alternative approaches for COVID-19 treatment is necessary before vaccines are developed. According to previous studies, Maxing Shigan decoction (MXSGD) present a prominent antivirus effect and is often used to treat pulmonary diseases. Furthermore, we collected 115 open prescriptions for COVID-19 therapy from the National Health Commission, State Administration of TCM and other organizations, MXSGD was identified as the key formula. However, the underlying molecular mechanism of MXSGD against COVID-19 is still unknown., Aim of the Study: The present study aimed to evaluate the therapeutic mechanism of MXSGD against COVID-19 by network pharmacology and in vitro experiment verification, and screen the potential components which could bind to key targets of COVID-19 via molecular docking method., Materials and Methods: Multiple open-source databases related to TCM or compounds were employed to screen active ingredients and potential targets of MXSGD. Network pharmacology analysis methods were used to initially predict the antivirus and anti-inflammatory effects of MXSGD against COVID-19. IL-6 induced rat lung epithelial type Ⅱ cells (RLE-6TN) damage was established to explore the anti-inflammatory damage activity of MXSGD. After MXSGD intervention, the expression level of related proteins and their phosphorylation in the IL-6 mediated JAK-STAT signaling pathway were detected by Western blot. Molecular docking technique was used to further identify the potential substances which could bind to three key targets (ACE2, Mpro and RdRp) of COVID-19., Results: In this study, 105 active ingredients and 1025 candidate targets were selected for MXSGD, 83 overlapping targets related to MXSGD and COVID-19 were identified, and the protein-protein interaction (PPI) network of MXSGD against COVID-19 was constructed. According to the results of biological enrichment analysis, 63 significant KEGG pathways were enriched, and most of them were related to signal transduction, immune system and virus infection. Furthermore, according the relationship between signal pathways, we confirmed MXSGD could effectively inhibit IL-6 mediated JAK-STAT signal pathway related protein expression level, decreased the protein expression levels of p-JAK2, p-STAT3, Bax and Caspase 3, and increased the protein expression level of Bcl-2, thereby inhibiting RLE-6TN cells damage. In addition, according to the LibDock scores screening results, the components with strong potential affinity (Top 10) with ACE2, Mpro and RdRp are mainly from glycyrrhiza uralensis (Chinese name: Gancao) and semen armeniacae amarum (Chinese name: Kuxingren). Among them, amygdalin was selected as the optimal candidate component bind to all three key targets, and euchrenone, glycyrrhizin, and glycyrol also exhibited superior affinity interactions with ACE2, Mpro and RdRp, respectively., Conclusion: This work explained the positive characteristics of multi-component, multi-target, and multi-approach intervention with MXSGD in combating COVID-19, and preliminary revealed the antiviral and anti-inflammatory pharmacodynamic substances and mechanism of MXSGD, which might provide insights into the vital role of TCM in the prevention and treatment of COVID-19., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Microvesicles (MVs) are cell-derived extracellular vesicles that have emerged as markers and mediators of acute lung injury (ALI). One of the most common pathogens in pneumonia-induced ALI is Streptococcus pneumoniae (Spn), but the role of MVs during Spn lung infection is largely unknown. In the first line of defense against Spn and its major virulence factor, pneumolysin (PLY), are the alveolar epithelial cells (AEC). In this study, we aim to characterize MVs shed from PLY-stimulated AEC and explore their contribution in mediating crosstalk with neutrophils. Using in vitro cell and ex vivo (human lung tissue) models, we demonstrated that Spn in a PLY-dependent manner stimulates AEC to release increased numbers of MVs. Spn infected mice also had higher levels of epithelial-derived MVs in their alveolar compartment compared to control. Furthermore, MVs released from PLY-stimulated AEC contain mitochondrial content and can be taken up by neutrophils. These MVs then suppress the ability of neutrophils to produce reactive oxygen species, a critical host-defense mechanism. Taken together, our results demonstrate that AEC in response to pneumococcal PLY release MVs that carry mitochondrial cargo and suggest that these MVs regulate innate immune responses during lung injury.

Idiopathic pulmonary fibrosis (IPF) is a lethal and agnogenic interstitial lung disease, which has limited therapeutic options. Recently, the NOD-, LRR- and pyrin domain-containing 3 (NLRP3) inflammasome has been demonstrated as an important contributor to various fibrotic diseases following its persistent activation. However, the role of NLRP3 inflammasome in pulmonary fibrogenesis still needs to be further clarified. Here, we found that the activation of the NLRP3 inflammasome was raised in fibrotic lungs. In addition, the NLRP3 inflammasome was found to be activated in alveolar epithelial cells (AECs) in the lung tissue of both IPF patients and pulmonary fibrosis mouse models. Further research revealed that epithelial cells, following activation of the NLRP3 inflammasome, could induce the myofibroblast differentiation of lung-resident mesenchymal stem cells (LR-MSCs). In addition, inhibiting the activation of the NLRP3 inflammasome in epithelial cells promoted the expression of dickkopf-1 (DKK1), a secreted Wnt antagonist. DKK1 was capable of suppressing the profibrogenic differentiation of LR-MSCs and bleomycin-induced pulmonary fibrosis. In conclusion, this study not only provides a further in-depth understanding of the pathogenesis of pulmonary fibrosis, but also reveals a potential therapeutic strategy for disorders associated with pulmonary fibrosis., (Copyright © 2021 Elsevier B.V. All rights reserved.)

DNA methyltransferase (Dnmt)3b mediates de novo DNA methylation and modulation of Dnmt3b in respiratory epithelial cells has been shown to affect the expression of multiple genes. Respiratory epithelial cells provide a first line of defense against pulmonary pathogens and play a crucial role in the immune response during pneumonia caused by Pseudomonas (P.) aeruginosa, a gram-negative bacterium that expresses flagellin as an important virulence factor. We here sought to determine the role of Dntm3b in respiratory epithelial cells in immune responses elicited by P. aeruginosa. DNMT3B expression was reduced in human bronchial epithelial (BEAS-2B) cells as well as in primary human and mouse bronchial epithelial cells grown in air liquid interface upon exposure to P. aeruginosa (PAK). Dnmt3b deficient human bronchial epithelial (BEAS-2B) cells produced more CXCL1, CXCL8 and CCL20 than control cells when stimulated with PAK, flagellin-deficient PAK (PAKflic) or flagellin. Dnmt3b deficiency reduced DNA methylation at exon 1 of CXCL1 and enhanced NF-ĸB p65 binding to the CXCL1 promoter. Mice with bronchial epithelial Dntm3b deficiency showed increased Cxcl1 mRNA expression in bronchial epithelium and CXCL1 protein release in the airways during pneumonia caused by PAK, which was associated with enhanced neutrophil recruitment and accelerated bacterial clearance; bronchial epithelial Dnmt3b deficiency did not modify responses during pneumonia caused by PAKflic or Klebsiella pneumoniae (an un-flagellated gram-negative bacterium). Dnmt3b deficiency in type II alveolar epithelial cells did not affect mouse pulmonary defense against PAK infection. These results suggest that bronchial epithelial Dnmt3b impairs host defense during Pseudomonas induced pneumonia, at least in part, by dampening mucosal responses to flagellin., Competing Interests: The authors have declared that no competing interests exist.

Respiratory Syncytial Virus (RSV) causes severe inflammation and airway pathology in children and the elderly by infecting the epithelial cells of the upper and lower respiratory tract. RSV replication is sensed by intracellular pattern recognition receptors upstream of the IRF and NF-κB transcription factors. These proteins coordinate an innate inflammatory response via Bromodomain-containing protein 4 (BRD4), a protein that functions as a scaffold for unknown transcriptional regulators. To better understand the pleiotropic regulatory function of BRD4, we examine the BRD4 interactome and identify how RSV infection dynamically alters it. To accomplish these goals, we leverage native immunoprecipitation and Parallel Accumulation-Serial Fragmentation (PASEF) mass spectrometry to examine BRD4 complexes isolated from human alveolar epithelial cells in the absence or presence of RSV infection. In addition, we explore the role of BRD4's acetyl-lysine binding bromodomains in mediating these interactions by using a highly selective competitive bromodomain inhibitor. We identify 101 proteins that are significantly enriched in the BRD4 complex and are responsive to both RSV-infection and BRD4 inhibition. These proteins are highly enriched in transcription factors and transcriptional coactivators. Among them, we identify members of the AP1 transcription factor complex, a complex important in innate signaling and cell stress responses. We independently confirm the BRD4/AP1 interaction in primary human small airway epithelial cells. We conclude that BRD4 recruits multiple transcription factors during RSV infection in a manner dependent on acetyl-lysine binding domain interactions. This data suggests that BRD4 recruits transcription factors to target its RNA processing complex to regulate gene expression in innate immunity and inflammation.

All coronaviruses known to have recently emerged as human pathogens probably originated in bats 1 . Here we use a single experimental platform based on immunodeficient mice implanted with human lung tissue (hereafter, human lung-only mice (LoM)) to demonstrate the efficient in vivo replication of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as well as two endogenous SARS-like bat coronaviruses that show potential for emergence as human pathogens. Virus replication in this model occurs in bona fide human lung tissue and does not require any type of adaptation of the virus or the host. Our results indicate that bats contain endogenous coronaviruses that are capable of direct transmission to humans. Our detailed analysis of in vivo infection with SARS-CoV-2 in human lung tissue from LoM showed a predominant infection of human lung epithelial cells, including type-2 pneumocytes that are present in alveoli and ciliated airway cells. Acute infection with SARS-CoV-2 was highly cytopathic and induced a robust and sustained type-I interferon and inflammatory cytokine and chemokine response. Finally, we evaluated a therapeutic and pre-exposure prophylaxis strategy for SARS-CoV-2 infection. Our results show that therapeutic and prophylactic administration of EIDD-2801-an oral broad-spectrum antiviral agent that is currently in phase II/III clinical trials-markedly inhibited SARS-CoV-2 replication in vivo, and thus has considerable potential for the prevention and treatment of COVID-19.

The Corona Virus Disease (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) requires a rapid solution and global collaborative efforts in order to define preventive and treatment strategies. One of the major challenges of this disease is the high number of patients needing advanced respiratory support due to the Acute Respiratory Distress Syndrome (ARDS) as the lung is the major - although not exclusive - target of the virus. The molecular mechanisms, pathogenic drivers and the target cell type(s) in SARS-CoV-2 infection are still poorly understood, but the development of a "hyperactive" immune response is proposed to play a role in the evolution of the disease and it is envisioned as a major cause of morbidity and mortality. Here we propose a theory by which the main targets for SARS-CoV-2 are the Type II Alveolar Epithelial Cells and the clinical manifestations of the syndrome are a direct consequence of their involvement. We propose the existence of a vicious cycle by which once alveolar damage starts in AEC II cells, the inflammatory state is supported by macrophage pro-inflammatory polarization (M1), cytokines release and by the activation of the NF-κB pathway. If this theory is confirmed, future therapeutic efforts can be directed to target Type 2 alveolar cells and the molecular pathogenic drivers associated with their dysfunction with currently available therapeutic strategies., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Preclinical mouse models that recapitulate some characteristics of coronavirus disease (COVID-19) will facilitate focused study of pathogenesis and virus-host responses. Human agniotensin-converting enzyme 2 (hACE2) serves as an entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to infect people via binding to envelope spike proteins. Herein we report development and characterization of a rapidly deployable COVID-19 mouse model. C57BL/6J (B6) mice expressing hACE2 in the lung were transduced by oropharyngeal delivery of the recombinant human adenovirus type 5 that expresses hACE2 (Ad5-hACE2). Mice were infected with SARS-CoV-2 at Day 4 after transduction and developed interstitial pneumonia associated with perivascular inflammation, accompanied by significantly higher viral load in lungs at Days 3, 6, and 12 after infection compared with Ad5-empty control group. SARS-CoV-2 was detected in pneumocytes in alveolar septa. Transcriptomic analysis of lungs demonstrated that the infected Ad5-hACE mice had a significant increase in IFN-dependent chemokines Cxcl9 and Cxcl10 , and genes associated with effector T-cell populations including C d3 g, Cd8a, and Gzmb . Pathway analysis showed that several Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were enriched in the data set, including cytokine-cytokine receptor interaction, the chemokine signaling pathway, the NOD-like receptor signaling pathway, the measles pathway, and the IL-17 signaling pathway. This response is correlative to clinical response in lungs of patients with COVID-19. These results demonstrate that expression of hACE2 via adenovirus delivery system sensitized the mouse to SARS-CoV-2 infection and resulted in the development of a mild COVID-19 phenotype, highlighting the immune and inflammatory host responses to SARS-CoV-2 infection. This rapidly deployable COVID-19 mouse model is useful for preclinical and pathogenesis studies of COVID-19.

Alveolar macrophages are among the first immune cells that respond to inhaled pathogens. However, numerous pathogens block macrophage-intrinsic immune responses, making it unclear how robust antimicrobial responses are generated. The intracellular bacterium Legionella pneumophila inhibits host translation, thereby impairing cytokine production by infected macrophages. Nevertheless, Legionella-infected macrophages induce an interleukin-1 (IL-1)-dependent inflammatory cytokine response by recruited monocytes and other cells that controls infection. How IL-1 directs these cells to produce inflammatory cytokines is unknown. Here, we show that collaboration with the alveolar epithelium is critical for controlling infection. IL-1 induces the alveolar epithelium to produce granulocyte-macrophage colony-stimulating factor (GM-CSF). Intriguingly, GM-CSF signaling amplifies inflammatory cytokine production in recruited monocytes by enhancing Toll-like receptor (TLR)-induced glycolysis. Our findings reveal that alveolar macrophages engage alveolar epithelial signals to metabolically reprogram monocytes for antibacterial inflammation., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Respiratory diseases adversely affect infants and are the focus of efforts to develop vaccinations and other modalities to prevent disease. The infant immune system differs from that of older children and adults in many ways that are as yet ill understood. We have used a C57BL/6 mouse model of infection with a laboratory- adapted strain of influenza (PR8) to delineate the importance of the cytokine IL-6 in the innate response to primary infection and in the development of protective immunity in adult mice. Herein, we used this same model in infant (14 days of age) mice to determine the effect of IL-6 deficiency. Infant wild type mice are more susceptible than older mice to infection, similar to the findings in humans. IL-6 is expressed in the lung in the early response to PR8 infection. While intramuscular immunization does not protect against lethal challenge, intranasal administration of heat inactivated virus is protective and correlates with expression of IL-6 in the lung, activation of lung CD8 cells, and development of an influenza-specific antibody response. In IL-6 deficient mice, this response is abrogated, and deficient mice are not protected against lethal challenge. These studies support the importance of the role of the tissue environment in infant immunity, and further suggest that IL-6 may be helpful in the generation of protective immune responses in infants., (Copyright © 2020 Bonney, Krebs, Kim, Prakash, Torrance, Haynes and Rincon.)

The main function of the lung is to perform gas exchange while maintaining lung homeostasis despite environmental pathogenic and non-pathogenic elements contained in inhaled air. Resident cells must keep lung homeostasis and eliminate pathogens by inducing protective immune response and silently remove innocuous particles. Which lung cell type is crucial for this function is still subject to debate, with reports favoring either alveolar macrophages (AMs) or lung epithelial cells (ECs) including airway and alveolar ECs. AMs are the main immune cells in the lung in steady-state and their function is mainly to dampen inflammatory responses. In addition, they phagocytose inhaled particles and apoptotic cells and can initiate and resolve inflammatory responses to pathogens. Although AMs release a plethora of mediators that modulate immune responses, ECs also play an essential role as they are more than just a physical barrier. They produce anti-microbial peptides and can secrete a variety of mediators that can modulate immune responses and AM functions. Furthermore, ECs can maintain AMs in a quiescent state by expressing anti-inflammatory membrane proteins such as CD200. Thus, AMs and ECs are both very important to maintain lung homeostasis and have to coordinate their action to protect the organism against infection. Thus, AMs and lung ECs communicate with each other using different mechanisms including mediators, membrane glycoproteins and their receptors, gap junction channels, and extracellular vesicles. This review will revisit characteristics and functions of AMs and lung ECs as well as different communication mechanisms these cells utilize to maintain lung immune balance and response to pathogens. A better understanding of the cross-talk between AMs and lung ECs may help develop new therapeutic strategies for lung pathogenesis., (Copyright © 2020 Bissonnette, Lauzon-Joset, Debley and Ziegler.)

Pulmonary fibrosis arises from the repeated epithelial mild injuries and insufficient repair lead to over activation of fibroblasts and excessive deposition of extracellular matrix, which result in a mechanical stretched niche. However, increasing mechanical stress likely exists before the establishment of fibrosis since early micro injuries increase local vascular permeability and prompt cytoskeletal remodeling which alter cellular mechanical forces. It is noteworthy that COVID-19 patients with severe hypoxemia will receive mechanical ventilation as supportive treatment and subsequent pathology studies indicate lung fibrosis pattern. At advanced stages, mechanical stress originates mainly from the stiff matrix since boundaries between stiff and compliant parts of the tissue could generate mechanical stress. Therefore, mechanical stress has a significant role in the whole development process of pulmonary fibrosis. The alveoli are covered by abundant capillaries and function as the main gas exchange unit. Constantly subject to variety of damages, the alveolar epithelium injuries were recently recognized to play a vital role in the onset and development of idiopathic pulmonary fibrosis. In this review, we summarize the literature regarding the effects of mechanical stress on the fundamental cells constituting the alveoli in the process of pulmonary fibrosis, particularly on epithelial cells, capillary endothelial cells, fibroblasts, mast cells, macrophages and stem cells. Finally, we briefly review this issue from a more comprehensive perspective: the metabolic and epigenetic regulation.

Tuberculosis kills more than 1 million people every year, and its control depends on the effective mechanisms of innate immunity, with or without induction of adaptive immune response. We investigated the interaction of type II alveolar epithelial cells (AEC-II) infected by Mycobacterium tuberculosis with dendritic cells (DCs). We hypothesized that the microenvironment generated by this interaction is critical for the early innate response against mycobacteria. We found that AEC-II infected by M. tuberculosis induced DC maturation, which was negatively regulated by HIF-1α-inducible NOS2 axis, and switched DC metabolism from an early and short peak of glycolysis to a low energetic status. However, the infection of DCs by M. tuberculosis up-regulated NOS2 expression and inhibited AEC-II-induced DC maturation. Our study demonstrated, for the first time, that HIF-1α-NOS2 axis plays a negative role in the maturation of DCs during M. tuberculosis infection. Such modulation might be useful for the exploitation of molecular targets to develop new therapeutic strategies against tuberculosis., (©2020 Society for Leukocyte Biology.)

In this paper, a model is proposed of the pathophysiological processes of COVID-19 starting from the infection of human type II alveolar epithelial cells (pneumocytes) by SARS-CoV-2 and culminating in the development of ARDS. The innate immune response to infection of type II alveolar epithelial cells leads both to their death by apoptosis and pyroptosis and to alveolar macrophage activation. Activated macrophages secrete proinflammatory cytokines and chemokines and tend to polarise into the inflammatory M1 phenotype. These changes are associated with activation of vascular endothelial cells and thence the recruitment of highly toxic neutrophils and inflammatory activated platelets into the alveolar space. Activated vascular endothelial cells become a source of proinflammatory cytokines and reactive oxygen species (ROS) and contribute to the development of coagulopathy, systemic sepsis, a cytokine storm and ARDS. Pulmonary activated platelets are also an important source of proinflammatory cytokines and ROS, as well as exacerbating pulmonary neutrophil-mediated inflammatory responses and contributing to systemic sepsis by binding to neutrophils to form platelet-neutrophil complexes (PNCs). PNC formation increases neutrophil recruitment, activation priming and extraversion of these immune cells into inflamed pulmonary tissue, thereby contributing to ARDS. Sequestered PNCs cause the development of a procoagulant and proinflammatory environment. The contribution to ARDS of increased extracellular histone levels, circulating mitochondrial DNA, the chromatin protein HMGB1, decreased neutrophil apoptosis, impaired macrophage efferocytosis, the cytokine storm, the toll-like receptor radical cycle, pyroptosis, necroinflammation, lymphopenia and a high Th17 to regulatory T lymphocyte ratio are detailed., Competing Interests: Declaration of competing interest MB is supported by a NHMRC Senior Principal Research Fellowship (1059660 and 1156072). MB has received Grant/Research Support from the NIH, Cooperative Research Centre, Simons Autism Foundation, Cancer Council of Victoria, Stanley Medical Research Foundation, Medical Benefits Fund, National Health and Medical Research Council, Medical Research Futures Fund, Beyond Blue, Rotary Health, A2 milk company, Meat and Livestock Board, Woolworths, Avant and the Harry Windsor Foundation, has been a speaker for Astra Zeneca, Lundbeck, Merck, Pfizer, and served as a consultant to Allergan, Astra Zeneca, Bioadvantex, Bionomics, Collaborative Medicinal Development, Lundbeck Merck, Pfizer and Servier – all unrelated to this work. LO is supported by a NHMRC Early Career Fellowship (1158487). WM is currently funded by an Alfred Deakin Postdoctoral Research Fellowship and a Multiple Sclerosis Research Australia early-career fellowship. WM has previously received funding from the Cancer Council Queensland and university grants/fellowships from La Trobe University, Deakin University, University of Queensland, and Bond University. WM has received industry funding and has attended events funded by Cobram Estate Pty. Ltd. WM has received travel funding from Nutrition Society of Australia. WM has received consultancy funding from Nutrition Research Australia. WM has received speaker honoraria from The Cancer Council Queensland and the Princess Alexandra Research Foundation. The Food & Mood Centre has received Grant/Research support from Fernwood Foundation, Wilson Foundation, the A2 Milk Company, and Be Fit Foods. AO is supported by a Future Leader Fellowship (#101160) from the Heart Foundation Australia and Wilson Foundation. She has received research funding from National Health & Medical Research Council, Australian Research Council, University of Melbourne, Deakin University, Sanofi, Meat and Livestock Australia and Woolworths Limited and Honoraria from Novartis., (Copyright © 2020 Elsevier Inc. All rights reserved.)

By enhancing tissue repair and modulating immune responses, Foxp3 + regulatory T cells (Tregs) play essential roles in resolution from lung injury. The current study investigated the effects that Tregs exert directly or indirectly on the transcriptional profiles of type 2 alveolar epithelial (AT2) cells during resolution in an experimental model of acute lung injury. Purified AT2 cells were isolated from uninjured mice or mice recovering from LPS-induced lung injury, either in the presence of Tregs or in Treg-depleted mice, and transcriptome profiling identified differentially expressed genes. Depletion of Tregs resulted in altered expression of 49 genes within AT2 cells during resolution, suggesting that Tregs present in this microenvironment influence AT2-cell function. Biological processes from Gene Ontology enriched in the absence of Tregs included those describing responses to IFN. Neutralizing IFN-γ in Treg-depleted mice reversed the effect of Treg depletion on inflammatory macrophages and B cells by preventing the increase in inflammatory macrophages and the decrease in B cells. Our results provide insight into the effects of Tregs on AT2 cells. Tregs directly or indirectly impact many AT2-cell functions, including IFN type I and II-mediated signaling pathways. Inhibition of IFN-γ expression and/or function may be one mechanism through which Tregs accelerate resolution after acute lung injury.

The innate response plays a crucial role in the protection against tuberculosis development. Moreover, the initial steps that drive the host-pathogen interaction following Mycobacterium tuberculosis infection are critical for the development of adaptive immune response. As alveolar Mϕs, airway epithelial cells, and dendritic cells can sense the presence of M. tuberculosis and are the first infected cells. These cells secrete mediators, which generate inflammatory signals that drive the differentiation and activation of the T lymphocytes necessary to clear the infection. Throughout this review article, we addressed the interaction between epithelial cells and M. tuberculosis, as well as the interaction between dendritic cells and M. tuberculosis. The understanding of the mechanisms that modulate those interactions is critical to have a complete view of the onset of an infection and may be useful for the development of dendritic cell-based vaccine or immunotherapies., (©2020 Society for Leukocyte Biology.)

Suppressor of cytokine signaling 3 (SOCS3) is a negative regulator of TBK1 and interferon pathway and the expression of SOCS3 is closely correlated with symptoms of influenza patients. However, whether deletion of Socs3 in the lung epithelial cells would affect influenza lung replication and inflammation in vivo is unknown. To test this, we approached the influenza infected Socs3 f/f and SpcCre.Socs3 f/f mice. We first found that knockdown of Socs3 in lung epithelial cells reduced influenza replication. However, in the in vivo study, there was a reduction of SOCS3 in the influenza-infected neutrophils coincided with an increase of SOCS3 in the CD45 - CD326 + lung epithelial cells in PR8-infected SpcCre.Socs3 f/f mice. SOCS3-deficient neutrophils expressed higher levels of IL-17 that enhanced chemokine expression in the lung epithelial cells. Lung SOCS3-dificient epithelial cells increased expression of GM-CSF and PGE 2 which promoted SpcCre.Socs3 f/f neutrophils to yield SOCS3. SpcCre.Socs3 f/f lung epithelial cells internalized SOCS3 released from GM-CSF + PGE 2 -stimulated SpcCre.Socs3 f/f neutrophils, which could boost influenza replication in the lung epithelial cells. Thus, in the in vivo study, deletion of SOCS3 from lung epithelium could be nullified by the uptake from SOCS3 from infiltrated neutrophils. In addition, deletion of Socs3 from myeloid cells reduced lung influenza infection, but increased lung inflammation. Taken together, deletion of SOCS3 could suppress influenza replication, but intracellular SOCS3 communication between neutrophils and lung epithelial cells confounds this effect., Competing Interests: Declaration of Competing Interest None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

MHC-II on alveolar type-II (AT-II) cells is associated with immune tolerance in an inflammatory microenvironment. Recently, we found TNF-α upregulated MHC-II in AT-II in vitro. In this study, we explored whether TNF-α-mediated inflammation upregulates MHC-II on AT-II cells to trigger Treg expansion in inflammation-driven lung adenocarcinoma (IDLA). Using urethane-induced mice IDLA model, we found that IDLA cells mainly arise from AT-II cells, which are the major source of MHC-II. Blocking urethane-induced inflammation by TNF-α neutralization inhibited tumorigenesis and reversed MHC-II upregulation on tumor cells of AT-II cellular origin in IDLA. MHC-II-dependent AT-II cells were isolated from IDLA-induced Treg expansion. In human LA samples, we found high expression of MHC-II in tumor cells of AT-II cellular origin, which was correlated with increased Foxp3 + T cells infiltration as well as CXCR-2 expression. CXCR-2 and MHC-II colocalization was observed in inflamed lung tissue and IDLA cells of AT-II cellular origin. Furthermore, at the pro-IDLA inflammatory stage, TNF-α-neutralization or CXCR-2 deficiency inhibited the upregulation of MHC-II on AT-II cells in inflamed lung tissue. Thus, tumor cells of AT-II cellular origin contribute to Treg expansion in an MHC-II-dependent manner in TNF-α-mediated IDLA. At the pro-tumor inflammatory stage, TNF-α-dependent lung inflammation plays an important role in MHC-II upregulation on AT-II cells., (© 2020 Federation of American Societies for Experimental Biology.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an RNA virus, is responsible for the coronavirus disease 2019 (COVID-19) pandemic of 2020. Experimental evidence suggests that microRNA can mediate an intracellular defence mechanism against some RNA viruses. The purpose of this study was to identify microRNA with predicted binding sites in the SARS-CoV-2 genome, compare these to their microRNA expression profiles in lung epithelial tissue and make inference towards possible roles for microRNA in mitigating coronavirus infection. We hypothesize that high expression of specific coronavirus-targeting microRNA in lung epithelia may protect against infection and viral propagation, conversely, low expression may confer susceptibility to infection. We have identified 128 human microRNA with potential to target the SARS-CoV-2 genome, most of which have very low expression in lung epithelia. Six of these 128 microRNA are differentially expressed upon in vitro infection of SARS-CoV-2. Additionally, 28 microRNA also target the SARS-CoV genome while 23 microRNA target the MERS-CoV genome. We also found that a number of microRNA are commonly identified in two other studies. Further research into identifying bona fide coronavirus targeting microRNA will be useful in understanding the importance of microRNA as a cellular defence mechanism against pathogenic coronavirus infections.

Excessive cytokine signaling frequently exacerbates lung tissue damage during respiratory viral infection. Type I (IFN-α and IFN-β) and III (IFN-λ) interferons are host-produced antiviral cytokines. Prolonged IFN-α and IFN-β 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)., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

A novel coronavirus, SARS-CoV-2, has recently emerged in China and spread internationally, posing a health emergency to the global community. COVID-19 caused by SARS-CoV-2 is associated with an acute respiratory illness that varies from mild to the life-threatening acute respiratory distress syndrome (ARDS). The complement system is part of the innate immune arsenal against pathogens, in which many viruses can evade or employ to mediate cell entry. The immunopathology and acute lung injury orchestrated through the influx of pro-inflammatory macrophages and neutrophils can be directly activated by complement components to prime an overzealous cytokine storm. The manifestations of severe COVID-19 such as the ARDS, sepsis and multiorgan failure have an established relationship with activation of the complement cascade. We have collected evidence from all the current studies we are aware of on SARS-CoV-2 immunopathogenesis and the preceding literature on SARS-CoV-1 and MERS-CoV infection linking severe COVID-19 disease directly with dysfunction of the complement pathways. This information lends support for a therapeutic anti-inflammatory strategy against complement, where a number of clinically ready potential therapeutic agents are available., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

Acute lung injury (ALI) is a pathologic condition responsible for incurable human chronic respiratory diseases. Recent studies have shown the involvement of the glycoprotein, IL17A secreted by IL-17 producing cells in chronic inflammation. The current investigation was carried out to study the IL-17A mediated activation of SMAD and non- SMAD signaling in alveolar epithelial cells and to assess the putative modulatory role of curcumin. C57BL/6 mice were exposed to IL-17A and curcumin was administered as an intervention to modulate the IL-17A-induced alveolar damage. Techniques like Immunofluorescence and real-time PCR were used. We found elevated expression of IL-17A and IL-17A-associated signaling pathways to be activated in mice lung tissues. Curcumin intervention in vivo promoted the resolution of IL-17A-induced ALI and attenuated pulmonary damage. Increase phosphorylation of non- SMAD proteins like P-EGFR, P-STAT-1, STAT-3, P-JAK-1/2, P-JNK, and also SMAD proteins like P- SMAD 2/3 and TGF-β1 was encountered upon IL-17A exposure, while curcumin intervention reversed the protein expression levels. Curcumin was found to block mRNA expressions of non- SMAD genes EGFR, JNK-1, JAK1, JAK2, STAT-1, STAT-3, MAPK14, also of TGF-β1 and SMAD genes like SMAD 2, SMAD 3. However, mRNA expressions of SMAD 6 and SMAD 7 were increased upon curcumin intervention. Our study indicates that IL-17A participates in the development of ALI in both SMAD dependent and independent manner and the IL-17A signaling components were effectively controlled by curcumin, suggesting probable anti-inflammatory use of curcumin during ALI.

Monoclonal antibodies (mAbs) that specifically target podoplanin (PDPN), a marker for type I alveolar cells, are needed for immunohistochemical analyses. Anti-PDPN mAbs are available for many species, including human, mouse, rat, rabbit, dog, cat, bovine, pig, Tasmanian devil, alpaca, tiger, whale, goat, horse, bear, and sheep PDPNs. However, no antilion PDPN (lioPDPN) antibody has been developed. In this study, possible cross-reaction between available anti-PDPN mAbs and lioPDPN was examined. Immunohistochemical analysis showed that antitiger PDPN mAb PMab-231 (IgG 2a , kappa) reacted with type I alveolar cells from lion lung, indicating that PMab-231 is useful for the detection of lioPDPN.

There is pressing urgency to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2), which causes the disease COVID-19. SARS-CoV-2 spike (S) protein binds angiotensin-converting enzyme 2 (ACE2), and in concert with host proteases, principally transmembrane serine protease 2 (TMPRSS2), promotes cellular entry. The cell subsets targeted by SARS-CoV-2 in host tissues and the factors that regulate ACE2 expression remain unknown. Here, we leverage human, non-human primate, and mouse single-cell RNA-sequencing (scRNA-seq) datasets across health and disease to uncover putative targets of SARS-CoV-2 among tissue-resident cell subsets. We identify ACE2 and TMPRSS2 co-expressing cells within lung type II pneumocytes, ileal absorptive enterocytes, and nasal goblet secretory cells. Strikingly, we discovered that ACE2 is a human interferon-stimulated gene (ISG) in vitro using airway epithelial cells and extend our findings to in vivo viral infections. Our data suggest that SARS-CoV-2 could exploit species-specific interferon-driven upregulation of ACE2, a tissue-protective mediator during lung injury, to enhance infection., Competing Interests: Declaration of Interests A.R. is an SAB member of ThermoFisher Scientific, Neogene Therapeutics, Asimov, and Syros Pharmaceuticals; a co-founder of and equity holder in Celsius Therapeutics; and an equity holder in Immunitas Therapeutics. A.K.S. reports compensation for consulting and/or SAB membership from Merck, Honeycomb Biotechnologies, Cellarity, Cogen Therapeutics, Orche Bio, and Dahlia Biosciences. L.S.K. is on the SAB for HiFiBio; she reports research funding from Kymab Limited, Bristol Meyers Squibb, Magenta Therapeutics, BlueBird Bio, and Regeneron Pharmaceuticals and consulting fees from Equillium, FortySeven, Inc, Novartis, Inc, EMD Serono, Gilead Sciences, and Takeda Pharmaceuticals. A.S. is an employee of Johnson and Johnson. N.K. is an inventor on a patent using thyroid hormone mimetics in acute lung injury that is now being considered for intervention in COVID-19 patients. J.L. is a scientific consultant for 10X Genomics, Inc. O.R.R, is a co-inventor on patent applications filed by the Broad Institute to inventions relating to single-cell genomics applications, such as in PCT/US2018/060860 and US Provisional Application No. 62/745,259. S.T. in the last three years was a consultant at Genentech, Biogen, and Roche and is a member of the SAB of Foresite Labs. M.H.W. is now an employee of Pfizer. F.J.T. reports receiving consulting fees from Roche Diagnostics GmbH and ownership interest in Cellarity, Inc. P.H. is a co-inventor on a patent using artificial intelligence and high-resolution microscopy for COVID-19 infection testing based on serology., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Impact Statement: We suggest a novel modality in terms of IL-25/IL-33/TSLP's pro-fibrotic role in IPF. First, IL-25/IL-33/TSLP fully activates (myo)fibroblasts in fibroblastic foci (FF) in a paracrine-dependent manner. (IL-25/IL-33/TSLP) + alveolar epithelial cells-(IL-25R/IL-33R/TSLPR) + (myo)fibroblasts axis may contribute greatly to the abnormal epithelial-mesenchymal crosstalk and lung fibrosis. Second, IL-25/IL-33/TSLP causes significant injury and phenotypic changes of alveolar epithelial cells in an autocrine-dependent manner. By acting directly on the two most important cells in the fibrotic process, i.e. alveolar epithelial cells and (myo)fibroblasts, we support the notion that biological therapies targeting IL-25/IL-33/TSLP will shed new light on the cure of IPF patients.

Background: Silica nanoparticles (SiNPs) are among the most widely manufactured and used nanoparticles. Concerns about potential health effects of SiNPs have therefore risen. Using a 3D tri-culture model of the alveolar lung barrier we examined effects of exposure to SiNPs (Si10) and crystalline silica (quartz; Min-U-Sil) in the apical compartment consisting of human alveolar epithelial A549 cells and THP-1-derived macrophages, as well as in the basolateral compartment with Ea.hy926 endothelial cells. Inflammation-related responses were measured by ELISA and gene expression., Results: Exposure to both Si10 and Min-U-Sil induced gene expression and release of CXCL8, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α) and interleukin-1β (IL-1β) in a concentration-dependent manner. Cytokine/chemokine expression and protein levels were highest in the apical compartment. Si10 and Min-U-Sil also induced expression of adhesion molecules ICAM-1 and E-selectin in the apical compartment. In the basolateral endothelial compartment we observed marked, but postponed effects on expression of all these genes, but only at the highest particle concentrations. Geneexpressions of heme oxygenase-1 (HO-1) and the metalloproteases (MMP-1 and MMP-9) were less affected. The IL-1 receptor antagonist (IL-1RA), markedly reduced effects of Si10 and Min-U-Sil exposures on gene expression of cytokines and adhesion molecules, as well as cytokine-release in both compartments., Conclusions: Si10 and Min-U-Sil induced gene expression and release of pro-inflammatory cytokines/adhesion molecules at both the epithelial/macrophage and endothelial side of a 3D tri-culture. Responses in the basolateral endothelial cells were only induced at high concentrations, and seemed to be mediated by IL-1α/β released from the apical epithelial cells and macrophages.

Chronic obstructive pulmonary fibrosis (COPD) is a chronic and fatal lung disease with few treatment options. Sodium hydrosulfide (NaHS), a donor of hydrogen sulfide (H 2 S), was found to alleviate cigarette smoke (CS)-induced emphysema in mice, however, the underlying mechanisms have not yet been clarified. In this study, we investigated its effects on COPD in a CS-induced mouse model in vivo and in cigarette smoke extract (CSE)-stimulated alveolar epithelial A549 cells in vitro. The results showed that NaHS not only relieved emphysema, but also improved pulmonary function in CS-exposed mice. NaHS significantly increased the expressions of tight junction proteins (i.e., ZO-1, Occludin and claudin-1), and reduced apoptosis and secretion of pro-inflammatory cytokines (i.e., TNF-α, IL-6 and IL-1β) in CS-exposed mouse lungs and CSE-incubated A549 cells, indicating H 2 S inhibits CS-induced inflammation, injury and apoptosis in alveolar epithelial cells. NaHS also upregulated prolyl hydroxylase (PHD)2, and suppressed hypoxia-inducible factor (HIF)-1α expression in vivo and in vitro, suggesting H 2 S inhibits CS-induced activation of PHD2/HIF-1α axis. Moreover, NaHS inhibited CS-induced phosphorylation of ERK, JNK and p38 MAPK in vivo and in vitro, and treatment with their inhibitors reversed CSE-induced ZO-1 expression and inflammation in A549 cells. These results suggest that NaHS may prevent emphysema via the suppression of PHD2/HIF-1α/MAPK signaling pathway, and subsequently inhibition of inflammation, epithelial cell injury and apoptosis, and may be a novel strategy for the treatment of COPD., (Copyright © 2019. Published by Elsevier B.V.)

Objective: Probiotics are fed to improve enteric health, and they may also affect respiratory immunity through their exposure to the upper respiratory tract upon ingestion. However, their effect on the respiratory system is not known. Our aim was to determine how probiotics affect functions and markers of bronchoalveolar lung lavage cells (BAL) isolated from lungs of calves at slaughter., Results: Treatments consisted of ten probiotic species and one control treatment. Probiotics and BAL were incubated 1:1 for 2 h at 37 °C and 5% CO 2 . The cell surface markers measured included CD14, CD205, and CD18, and E. coli bioparticles were used to measure phagocytosis and oxidative burst. Differences were considered significant at P ≤ 0.05 and were noted for percent cells fluorescing and mean fluorescence intensity for CD14 and CD205. Additionally, oxidative burst was different as measured by both percentage of cells fluorescing and mean fluorescence intensity, and phagocytosis differed among species as measured by mean fluorescence intensity. Overall, probiotic species differed in their ability to suppress or increase leukocyte function showing that probiotic bacteria differentially modulate BAL.

Introduction: Long-term pulmonary complications are one of the major long-term consequences of sulfur mustard (SM) exposure. Toll-like receptor 4 (TLR4) involves in the pathogenesis of several pulmonary disorders. Surfactant protein-A (SP-A) regulates LPS-induced TLR4 localization and activation responses. However, the intensity and significance of TLR4 and SP-A expression by lung cells in SM-exposed patients is not clear., Methods: The gene expression of TLR4 (through real-time PCR) and TLR4 and SP-A positive cells and alveolar type II cells, as SP-A producers, (using IHC) were assessed in formalin fixed paraffin embedded (FFPE) specimens from SM-exposed (n = 17), and non-SM exposed individuals (n = 12)., Results: TLR4 gene expression did not change between study groups. However, its cell surface presentation was significantly reduced in SM-exposed patients and particularly in which with constrictive bronchiolitis compared with the control group (P < 0.001 and P = 0.002, respectively). Frequency of alveolar type II cells was lower in the case group rather than the control group while the number of SP-A positive cells did not alter., Conclusions: These findings suggest that reduced TLR4 cell surface presentation may have anti-inflammatory function and SP-A may have a critical role in regulation of inflammatory responses in SM-exposed patients. Further investigation on other possible mechanisms involved in TLR4 internalization maybe help to illustrate the modulatory or inflammatory activity of TLR4 in these patients., Competing Interests: Declaration of Competing Interest All of the authors declare no conflict of interest., (Copyright © 2019. Published by Elsevier B.V.)

Smoking increases susceptibility to becoming infected with and developing tuberculosis. Among the components of cigarette smoke, nicotine has been identified as the main immunomodulatory molecule; however, its effect on the innate immune system is unknown. In the present study, the effect of nicotine on molecules of the innate immune system was evaluated. Lung epithelial cells and macrophages were infected with Mycobacterium tuberculosis (Mtb) and/or treated with nicotine. The results show that nicotine alone decreases the expression of the Toll-like receptors (TLR)-2, TLR-4 and NOD-2 in all three cell types, as well as the production of the SP-D surfactant protein in type II pneumocytes. Moreover, it was observed that nicotine decreases the production of interleukin (IL)-6 and C-C chemokine ligand (CCL)5 during Mtb infection in epithelial cells (EpCs), whereas in macrophages derived from human monocytes (MDMs) there is a decrease in IL-8, IL-6, tumor necrosis factor (TNF)-α, IL-10, CCL2, C-X-C chemokine ligand (CXCL)9 and CXCL10 only during infection with Mtb. Although modulation of the expression of cytokines and chemokines appears to be partially mediated by the nicotinic acetylcholine receptor α7, blocking this receptor found no effect on the expression of receptors and SP-D. In summary, it was found that nicotine modulates the expression of innate immunity molecules necessary for the defense against tuberculosis., (© 2019 British Society for Immunology.)

Background: Interleukin (IL)-22 is a cytokine involved in tissue protection and repair following lung pathologies. Interferon (IFN)-λ cytokines displayed similar properties during viral infection and a synergy of action between these two players has been documented in the intestine. We hypothesize that during Pseudomonas aeruginosa challenge, IL-22 up-regulates IFN-λ and that IFN-λ exhibits protective functions during Pseudomonas aeruginosa acute pneumonia model in mice., Methods: Using an in vitro human alveolar epithelial cell line A549, we assessed the ability of IL-22 to enhance IFN-λ expression during infection. IFN-λ protective function was evaluated in an acute mouse pneumonia model., Results: We first demonstrated in murine lungs that only type-II alveolar cells express IL-22 receptor and that IL-22 treatment of A549 cell line up-regulates IFN-λ expression. In a murine acute pneumonia model, IL-22 administration maintained significant IFN-λ levels in the broncho-alveolar fluids whereas IL-22 neutralization abolished IFN-λ up-regulation. In vivo administration of IFN-λ during Pseudomonas aeruginosa pneumonia improves mice outcome by dampening neutrophil recruitment and decreasing epithelium damages., Discussion: We show here that IL-22 regulates IFN-λ levels during Pseudomonas aeruginosa pneumonia., (Copyright © 2019. Published by Elsevier Ltd.)