Lung Resident Cells Research Articles

Autophagy prevents early proinflammatory responses and neutrophil recruitment during Mycobacterium tuberculosis infection without affecting pathogen burden in macrophages.

The immune response to Mycobacterium tuberculosis infection determines tuberculosis disease outcomes, yet we have an incomplete understanding of what immune factors contribute to a protective immune response. Neutrophilic inflammation has been associated with poor disease prognosis in humans and in animal models during M. tuberculosis infection and, therefore, must be tightly regulated. ATG5 is an essential autophagy protein that is required in innate immune cells to control neutrophil-dominated inflammation and promote survival during M. tuberculosis infection; however, the mechanistic basis for how ATG5 regulates neutrophil recruitment is unknown. To interrogate what innate immune cells require ATG5 to control neutrophil recruitment during M. tuberculosis infection, we used different mouse strains that conditionally delete Atg5 in specific cell types. We found that ATG5 is required in CD11c+ cells (lung macrophages and dendritic cells) to control the production of proinflammatory cytokines and chemokines during M. tuberculosis infection, which would otherwise promote neutrophil recruitment. This role for ATG5 is autophagy dependent, but independent of mitophagy, LC3-associated phagocytosis, and inflammasome activation, which are the most well-characterized ways that autophagy proteins regulate inflammation. In addition to the increased proinflammatory cytokine production from macrophages during M. tuberculosis infection, loss of ATG5 in innate immune cells also results in an early induction of TH17 responses. Despite prior published in vitro cell culture experiments supporting a role for autophagy in controlling M. tuberculosis replication in macrophages, the effects of autophagy on inflammatory responses occur without changes in M. tuberculosis burden in macrophages. These findings reveal new roles for autophagy proteins in lung resident macrophages and dendritic cells that are required to suppress inflammatory responses that are associated with poor control of M. tuberculosis infection.

A new tractable method for generating human alveolar macrophage-like cells in vitro to study lung inflammatory processes and diseases.

Alveolar macrophages (AMs) are unique lung resident cells that contact airborne pathogens and environmental particulates. The contribution of human AMs (HAMs) to pulmonary diseases remains poorly understood due to the difficulty in accessing them from human donors and their rapid phenotypic change during in vitro culture. Thus, there remains an unmet need for cost-effective methods for generating and/or differentiating primary cells into a HAM phenotype, particularly important for translational and clinical studies. We developed cell culture conditions that mimic the lung alveolar environment in humans using lung lipids, that is, Infasurf (calfactant, natural bovine surfactant) and lung-associated cytokines (granulocyte macrophage colony-stimulating factor, transforming growth factor-β, and interleukin 10) that facilitate the conversion of blood-obtained monocytes to an AM-like (AML) phenotype and function in tissue culture. Similar to HAM, AML cells are particularly susceptible to both Mycobacterium tuberculosis and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. This study reveals the importance of alveolar space components in the development and maintenance of HAM phenotype and function and provides a readily accessible model to study HAM in infectious and inflammatory disease processes, as well as therapies and vaccines. IMPORTANCE Millions die annually from respiratory disorders. Lower respiratory track gas-exchanging alveoli maintain a precarious balance between fighting invaders and minimizing tissue damage. Key players herein are resident AMs. However, there are no easily accessible in vitro models of HAMs, presenting a huge scientific challenge. Here, we present a novel model for generating AML cells based on differentiating blood monocytes in a defined lung component cocktail. This model is non-invasive, significantly less costly than performing a bronchoalveolar lavage, yields more AML cells than HAMs per donor, and retains their phenotype in culture. We have applied this model to early studies of M. tuberculosis and SARS-CoV-2. This model will significantly advance respiratory biology research.

WS14.06 Single cell RNA sequencing of lung biopsies reveals altered immune-structural cell crosstalk in cystic fibrosis

Background: Cystic Fibrosis (CF) is a life-limiting genetic disease that involves chronic airway inflammation and progressive lung tissue remodeling, which are the main causes of mortality. A continuous crosstalk between immune cells and structural cells within the lung is essential for mounting effective immune responses and maintaining homeostasis. Many studies have investigated how individual cell types or classes are deregulated in CF, but few have studied the complete spectrum of lung resident cells. Therefore, we lack a complete overview of immune-structural cell crosstalk in CF lungs, which may be relevant for developing therapies aimed at restoring homeostasis in the airways of CF patients.

Mechanisms of lung resident memory B cell activation during secondary influenza infection

Abstract B cells and the antibodies they produce are essential components of immunity to respiratory viruses, including influenza virus (IAV) and SARS-CoV-2. B cells responding to viral infection can differentiate into antibody secreting cells (ASCs), or resting memory B cells, some of which are tissue-resident (BRM cells). ASCs provide protection by secreting virus-specific antibodies, whereas BRM cells rapidly differentiate into ASCs after secondary infection. We previously published that IAV-specific BRM cells are retained in the lungs and do not recirculate. We now show that many lung-resident BRM cells reside in the airways and can be collected by bronchoalveolar lavage (BAL). BRM cells in the BAL are phenotypically and functionally different than those in the lung tissue and lymph nodes and express markers at various levels that possibly effect function. There is also a distinction in the role of the ASCs between the lungs and the BAL that will secrete certain antibodies at different time points during the IAV challenge. To understand BRM cell homing, differentiation, and function, we are using single cell approaches to define the transcriptional and epigenetic programs of airway-resident BRM cells at resting memory and during the response to IAV challenge. We are also using this technique to identify the changes among the entire immune system during a secondary IAV challenge to gain an understanding of how the entire immune system plays a role in protection. By understanding the programs that regulate BRM placement in the lung and their response to challenge infection, we expect to identify ways to improve vaccines against respiratory viruses and ameliorate B cell-dependent lung pathology in the context of autoimmunity or allergy. Supported by grants from NIH (R01 AI152476)

25-Hydroxycholesterol exacerbates vascular leak during acute lung injury.

Cholesterol-25-hydroxylase (CH25H), the biosynthetic enzyme for 25-hydroxycholesterol (25HC), is most highly expressed in the lung, but its role in lung biology is poorly defined. Recently, we reported that Ch25h is induced in monocyte-derived macrophages recruited to the airspace during resolution of lung inflammation and that 25HC promotes liver X receptor-dependent (LXR-dependent) clearance of apoptotic neutrophils by these cells. Ch25h and 25HC are, however, also robustly induced by lung-resident cells during the early hours of lung inflammation, suggesting additional cellular sources and targets. Here, using Ch25h-/- mice and exogenous 25HC in lung injury models, we provide evidence that 25HC sustains proinflammatory cytokines in the airspace and augments lung injury, at least in part, by inducing LXR-independent endoplasmic reticulum stress and endothelial leak. Suggesting an autocrine effect in endothelium, inhaled LPS upregulates pulmonary endothelial Ch25h, and non-hematopoietic Ch25h deletion is sufficient to confer lung protection. In patients with acute respiratory distress syndrome, airspace 25HC and alveolar macrophage CH25H were associated with markers of microvascular leak, endothelial activation, endoplasmic reticulum stress, inflammation, and clinical severity. Taken together, our findings suggest that 25HC deriving from and acting on different cell types in the lung communicates distinct, temporal LXR-independent and -dependent signals to regulate inflammatory homeostasis.

The inflammatory profiles of pulmonary alveolar macrophages and alveolar type 2 cells in SCD.

The lung microenvironment plays a crucial role in maintaining lung homeostasis as well as the initiation and resolution of both acute and chronic lung injury. Acute chest syndrome (ACS) is a complication of sickle cell disease (SCD) like acute lung injury. Both the endothelial cells and peripheral blood mononuclear cells are known to secrete proinflammatory cytokines elevated during ACS episodes. However, in SCD, the lung microenvironment that may favor excessive production of proinflammatory cytokines and the contribution of other lung resident cells, such as alveolar macrophages and alveolar type 2 epithelial (AT-2) cells, to ACS pathogenesis is not completely understood. Here, we sought to understand the pulmonary microenvironment and the proinflammatory profile of lung alveolar macrophages (LAMs) and AT-2 cells at steady state in Townes sickle cell (SS) mice compared to control mice (AA). In addition, we examined lung function and micromechanics molecules essential for pulmonary epithelial barrier function in these mice. Our results showed that bronchoalveolar lavage (BAL) fluid in SS mice had elevated protein levels of pro-inflammatory cytokines interleukin (IL)-1β and IL-12 (p ⩽ 0.05) compared to AA controls. We showed for the first time, significantly increased protein levels of inflammatory mediators (Human antigen R (HuR), Toll-like receptor 4 (TLR4), MyD88, and PU.1) in AT-2 cells (1.4 to 2.2-fold) and LAM (17-21%) isolated from SS mice compared to AA control mice at steady state. There were also low levels of anti-inflammatory transcription factors (Nrf2 and PPARy) in SS mice compared to AA controls (p ⩽ 0.05). Finally, we found impaired lung function and a dysregulated composition of surfactant proteins (B and C). Our results demonstrate that SS mice at steady state had a compromised lung microenvironment with elevated expression of proinflammatory cytokines by AT-2 cells and LAM, as well as dysregulated expression of surfactant proteins necessary for maintaining the alveolar barrier integrity and lung function.

Type 2 Dendritic Cells Orchestrate a Local Immune Circuit to Confer Antimetastatic Immunity.

The progression of transformed primary tumors to metastatic colonization is a lethal determinant of disease outcome. Although circulating adaptive and innate lymphocyte effector responses are required for effective antimetastatic immunity, whether tissue-resident immune circuits confer initial immunity at sites of metastatic dissemination remains ill defined. Here we examine the nature of local immune cell responses during early metastatic seeding in the lung using intracardiac injection to mimic monodispersed metastatic spread. Using syngeneic murine melanoma and colon cancer models, we demonstrate that lung-resident conventional type 2 dendritic cells (DC2) orchestrate a local immune circuit to confer host antimetastatic immunity. Tissue-specific ablation of lung DC2, and not peripheral DC populations, led to increased metastatic burden in the presence of an intact T cell and NK cell compartment. We demonstrate that DC nucleic acid sensing and transcription factors IRF3 and IRF7 signaling are required for early metastatic control and that DC2 serve as a robust source of proinflammatory cytokines in the lung. Critically, DC2 direct the local production of IFN-γ by lung-resident NK cells, which limits the initial metastatic burden. Collectively, our results highlight, to our knowledge, a novel DC2-NK cell axis that colocalizes around pioneering metastatic cells to orchestrate an early innate immune response program to limit initial metastatic burden in the lung.

Abstract B008: The role of the androgen receptor in reciprocal activation of breast cancer metastases and the lung epithelium during metastatic outgrowth

Abstract The lung is one of the most common sites of breast cancer (BC) metastasis. A major gap in our ability to effectively treat BC patients with established lung metastases lies in our lack of knowledge regarding how the lung microenvironment is remodeled during metastasis and how this contributes to disease progression. Our data suggest that the lung epithelium, in particular lung type II alveolar epithelial (AT2) cells, contribute to metastatic outgrowth. We hypothesize that BC lung micrometastases activate surrounding lung epithelial cells which, in turn, support the survival and outgrowth of BC metastases within the lung. The overall goal of these studies is to identify factors secreted by resident lung cells that could be used as metastasis-specific therapeutic targets/agents. We utilized immunocompetent mouse metastasis models to study metastatic progression within the lung. Multispectral fluorescent imagining of metastases demonstrated that a wound repair-related phenotype, characterized by chronic inflammation, develops within the lung microenvironment during metastatic outgrowth. We observed an increase in the number and activation of AT2 cells surrounding metastases as they grow. To gain mechanistic insight into how growing metastases may impact AT2 cells, we conducted single-cell RNA-sequencing (scRNAseq) on mouse lungs with high vs. low metastatic burden. This investigation showed that metastatic outgrowth significantly changes AT2 gene expression resulting in a modified secretome. Cell culture experiments, using multiple AT2 cell models, indicated that no contact co-culture with TNBC also caused significant changes in AT2 gene expression. Upstream regulator analysis of these datasets predicted that the androgen receptor (AR) may contribute to AT2 activation during metastatic outgrowth. Compared to non-adjacent tumor-free MMTV-PyMT lung tissue, there was an increased percentage of AR+ cells in the lung adjacent to metastases, which was further associated with large metastases only. Co-immunofluorescence experiments demonstrated that these lung-specific AR+ cells were primarily AT2 cells and fibroblasts, while in human lungs the majority of AR+ cells were AT2 cells and macrophages. Interestingly, co-culturing of TNBC and AT2 cells enhanced BC proliferation, suggesting that following activation, AT2 secreted factors may reciprocally impact metastatic growth. In conclusion, these data indicate that a tumor supportive microenvironment develops as lung metastases grow and suggests that by the time patients present with established/detectable lung metastases, the lung microenvironment has already undergone fundamental alterations that promote metastatic progression. Future studies will elucidate how activated lung cells support metastatic progression and could lead to the development or repurposing of therapeutic strategies to prevent destructive metastatic outgrowth. Citation Format: Jessica L. Christenson, Nicole S. Spoelstra, G. Devon Trahan, Kathleen I. O'Neill, Michelle M. Williams, Jennifer K. Richer. The role of the androgen receptor in reciprocal activation of breast cancer metastases and the lung epithelium during metastatic outgrowth [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B008.

The role of Notch-dependent differentiation of resident fibroblasts in the development of pulmonary fibrosis

Background. Molecular mechanisms of the development of pulmonary fibrosis are poorly understood. It is known that differentiation of resident lung cells into myofibroblasts plays a key role in this disease. The search for factors capable of regulating such differentiation is an urgent task. Objective. To evaluate the effect of Notch signaling pathway activation on resident lung fibroblasts by introducing the intracellular domains of each of the 4 Notch receptors (N1-4ICD). Design and methods. Primary cultures of pulmonary fibroblasts from donors (n = 4) were used. Notch-dependent activation of fibroblasts was carried out by introducing lentiviral vectors with/ without sequences of N1-4ICD domains. After 8 days, immunocytochemical staining was performed and the relative expression levels of the PDPN, HOPX, SLUG, SNAIL and ACTA2 genes were evaluated by qPCR. Results. Activation of the Notch signaling pathway by N1-4CD resulted in increased expression of SLUG, SNAIL and ACTA2. The most pronounced effect was observed with the introduction of Notch4-activating sequence. Induction of signaling by the introduction of N1-3ICD activating components contributed to an increase in PDPN expression, with the introduction of N4ICD, an increase in the level of HOPX expression was noted. Conclusion. Activation of each of the 4 intracellular Notch receptor domains is able to trigger the differentiation of resident alveolar fibroblasts into myofibroblasts, which are key players in the development of pulmonary fibrosis.

Lipid-laden lung mesenchymal cells foster breast cancer metastasis via metabolic reprogramming of tumor cells and natural killer cells

While the distant organ environment is known to support metastasis of primary tumors, its metabolic roles in this process remain underdetermined. Here, in breast cancer models, we found lung-resident mesenchymal cells (MCs) accumulating neutral lipids at the pre-metastatic stage. This was partially mediated by interleukin-1β (IL-1β)-induced hypoxia-inducible lipid droplet-associated (HILPDA) that subsequently represses adipose triglyceride lipase (ATGL) activity in lung MCs. MC-specific ablation of the ATGL or HILPDA genes in mice reinforced and reduced lung metastasis of breast cancer respectively, suggesting a metastasis-promoting effect of lipid-laden MCs. Mechanistically, lipid-laden MCs transported their lipids to tumor cells and natural killer (NK) cells via exosome-like vesicles, leading to heightened tumor cell survival and proliferation and NK cell dysfunction. Blockage of IL-1β, which was effective singly, improved the efficacy of adoptive NK cell immunotherapy in mitigating lung metastasis. Collectively, lung MCs metabolically regulate tumor cells and anti-tumor immunity to facilitate breast cancer lung metastasis.

S7.4a Vaccine-inducing lung resident CD4 + memory T cells are protective against Cryptococcus gattii infections

S7.4 Pathogenesis and host defense, September 23, 2022, 10:30 AM - 12:00 PM Cryptococccus gattii is a highly virulent fungal pathogen that can cause cryptococcosis in previously healthy individuals. It is not fully understood how innate and acquired immune responses cooperatively suppress C. gattii infection. Hitherto, we have reported the following findings, (1) Specific environment for exposure of dectin-1 and dectin-2 ligands in cryptococcal cells (PLOS ONE 2019, PMID 31398236), (2) CD11b-mediated immune recognition for C. gattii and capsule dependent immune evasion from CD11b recognition (Eur J Immunol 2021, PMID 33728652), (3) Dendritic cells (DCs)-based vaccine induced lung resident memory Th17 cells and ameliorates C. gattii infection (Mucosal Immunol 2018, PMID 30279512), and (4) Neutrophil and opsonization can mediate fungicidal activity for cryptococcal cells (Med Mycol 2019, PMID 30668754). Based on these findings, we have inferred that induction of lung resident memory T cells (lung TRM) and TRM-driven myeloid cells including granulocytes is necessary for controlling this infection. Currently, we are developing new intranasal (IN) vaccines using highly immunogenic whole cell antigens and characterizing protective lung TRM induced by the vaccines. Just as with component vaccines, whole cell vaccines are also featured for cryptococcal infection because these are highly cross-reactive and protective against each serotype (Biol Pharm Bull 2020, PMID 32009111). We developed two IN vaccines using whole cell antigen and adjuvant. Both vaccines induced IL-5-producing lung TRM and significantly reduced fungal burden in lungs and improved survival rates of mice following C. gattii infection, while not protective against C. neoformans infection. Although immune suppressive agent FTY720 can eliminate most circulating T cells and sustain tissue-resident subset, IN vaccines induced Th2-related immune response including eosinophils and IFN-γ independent granuloma, and reduced the fungal burden in lungs after the infection in the presence of FTY720. Vaccine-mediated protection was completely lost in the Rag-1 knockout mice deficient in T- and B-cells, while the fungal burden was reduced in the Rag-1 knockout mice after receiving the immunized CD4 + T cells in the adoptive transfer experiments. These results suggest that Th2-related lung TRM induced by IN vaccines are also protective against C. gattii infection as well as Th17-related counterparts induced by DC vaccine.

Lung fibroblasts facilitate pre-metastatic niche formation by remodeling the local immune microenvironment

Primary tumors are drivers of pre-metastatic niche formation, but the coordination by the secondary organ toward metastatic dissemination is underappreciated. Here, by single-cell RNA sequencing and immunofluorescence, we identified a population of cyclooxygenase 2 (COX-2)-expressing adventitial fibroblasts that remodeled the lung immune microenvironment. At steady state, fibroblasts in the lungs produced prostaglandin E2 (PGE2), which drove dysfunctional dendritic cells (DCs) and suppressive monocytes. This lung-intrinsic stromal program was propagated by tumor-associated inflammation, particularly the pro-inflammatory cytokine interleukin-1β, supporting a pre-metastatic niche. Genetic ablation of Ptgs2 (encoding COX-2) in fibroblasts was sufficient to reverse the immune-suppressive phenotypes of lung-resident myeloid cells, resulting in heightened immune activation and diminished lung metastasis in multiple breast cancer models. Moreover, the anti-metastatic activity of DC-based therapy and PD-1 blockade was improved by fibroblast-specific Ptgs2 deletion or dual inhibition of PGE2 receptors EP2 and EP4. Collectively, lung-resident fibroblasts reshape the local immune landscape to facilitate breast cancer metastasis.

Plasma cell but not CD20-mediated B-cell depletion protects from bleomycin-induced lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease associated with chronic inflammation and tissue remodelling leading to fibrosis, reduced pulmonary function, respiratory failure and death. Bleomycin (Blm)-induced lung fibrosis in mice replicates several clinical features of human IPF, including prominent lymphoid aggregates of predominantly B-cells that accumulate in the lung adjacent to areas of active fibrosis. We have shown previously a requirement for B-cells in the development of Blm-induced lung fibrosis in mice. To determine the therapeutic potential of inhibiting B-cell function in pulmonary fibrosis, we examined the effects of anti-CD20 B-cell ablation therapy to selectively remove mature B-cells from the immune system and inhibit Blm-induced lung fibrosis. Anti-CD20 B-cell ablation did not reduce fibrosis in this model; however, immune phenotyping of peripheral blood and lung resident cells revealed that anti-CD20-treated mice retained a high frequency of CD19+ CD138+ plasma cells. Interestingly, high levels of CD138+ cells were also identified in the lung tissue of patients with IPF, consistent with the mouse model. Treatment of mice with bortezomib, which depletes plasma cells, reduced the level of Blm-induced lung fibrosis, implicating plasma cells as important effector cells in the development and progression of pulmonary fibrosis.

PM2.5 Synergizes With Pseudomonas aeruginosa to Suppress Alveolar Macrophage Function in Mice Through the mTOR Pathway.

High concentrations of PM2.5 in enclosed broiler houses cause respiratory disorders in humans and animals. Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen that can induce severe respiratory disease in animals under stress or with abnormal immune functions. Alveolar macrophages are lung-resident immune cells that play important roles in lung host defence and immune balance. In this study, the mechanism by which PM2.5 synergizes with P. aeruginosa to damage alveolar macrophage function and induce inflammation was investigated. The results will provide a theoretical basis for improving the poultry breeding environment and preventing the recurrence of infection with P. aeruginosa. Alveolar macrophages were stimulated by PM2.5 collected in an enclosed broiler house and P. aeruginosa. Phagocytosis was determined by the neutral red test. The apoptosis rate and cytoskeleton changes were observed by flow cytometry assays and laser scanning confocal microscopy. Protein levels related to autophagy and the mTOR pathway were detected by Western blotting. The results indicated that PM2.5 in combination with P. aeruginosa could decrease phagocytosis, inhibit autophagy, increase apoptosis, and destroy the cytoskeleton in alveolar macrophages. In addition, alveolar macrophages had significantly increased expression of mTOR pathway-related proteins in response to the synergistic stimulation of PM2.5 and P. aeruginosa. The above results confirmed that PM2.5 in poultry houses synergized with P. aeruginosa to impede alveolar macrophage function and caused more severe respiratory system injuries through a process closely related to the activation of the mTOR signalling pathway.

Tissue resident lung natural killer (NK) cells demonstrate enhanced responses to IL-15 compared to peripheral populations

Abstract Tissue resident NK cells with unique phenotype and function have been identified in organs such as the lung and liver. We have shown that inhaled (IH) rhIL-15 can lead to durable clinical responses in dogs with naturally occurring osteosarcoma or melanoma lung metastases, suggesting that lung NK populations can be targeted therapeutically. Thus, we set out to evaluate the phenotype of resting and rhIL-15-stimulated lung NK cells isolated from murine and human lung. C57BL/6 and BALB/c mice were exposed to IH or systemic IL-15 at escalating doses, and NK cells were isolated by negative selection from lungs and spleen of wild type mice for co-culture. In human patients undergoing surgery for malignancy, non-tumor lung tissue was isolated for flow cytometry and ex vivo stimulation with IL-15. Using mouse spleen or human blood as controls, we observed a higher proportion of lung NK cells per live CD45+ cells in baseline mouse and human samples. Lung NK also had higher expression of activation markers CD69 and HLA-DR (human), but lower expression of the inhibitory receptor TIGIT. Ex vivo culture with IL-15 also resulted in greater upregulation of Ki67 on lung NK cells. Compared to systemic delivery of IL-15, IH IL-15 resulted in greater upregulation of Ki67 and lower upregulation of TIGIT in lung NK cells. Subset analysis of CD49a+ lung NK cells showed similar results in both mouse and human models for activation marker and Ki67 expression after IL-15. Taken together, our results suggest that lung NK cells possess a more active baseline phenotype and exhibit preferential stimulation in response to rhIL-15 compared to circulating NK cells. Ultimately, targeting of lung resident NK cells may be a viable therapeutic strategy in patients with lung metastases. Supported by NIH/ NCI 1R03CA252793 NIH/NCI U01 CA224166-01 NIH/NCI T32 CA251007-01

Time course scRNAseq analysis on host mucosal immune responses to influenza virus infection during aging

Abstract Aging has been shown to be one of the major risk factors for host morbidity and mortality following infections with respiratory viruses including influenza virus and SARS-CoV-2. The unique immune properties of the aged hosts not only influence pathogen clearance and tissue damage responses, but also modulate the pathogenesis of the long-term sequalae post acute viral clearance. Currently, the dynamics and features of mucosal immune responses following acute respiratory viral infections in the aged hosts remain to be fully elucidated. Here, we characterized the lung cellular and molecular profiles following influenza virus infection in young (~3-month-old) or aged (~24-month-old) mice with a chronological manner from acute to chronic phase. Using DNA-oligo-conjugated CD45 antibody and i.v. labeling, we were able to distinguish the unique responses of circulating vs lung-resident immune cells to influenza virus infection during aging. These responses were further validated by spectral flow cytometry analysis of the lung innate and adaptive immune cells. Further data analysis and integration are warranted for this unique large data set detailing the single cell molecular profiles of mucosal responses to influenza virus infection comparing young and aged hosts.

Establishment of influenza-specific lung tissue-resident helper memory CD4+ T cells relies on expression the eATP sensor P2RX7

Abstract Influenza remains an infection of high medical relevance. Development of universal anti-influenza vaccines will require a better knowledge of lung immune responses. That includes influenza-specific CD4+ T cells, which establish long-term lung residency and induce a diversified local protective response to subsequent infection. Lung-resident memory CD4+ T cells are heterogeneous, including Th1-like cells, and a subset that shares phenotypic and functional features with follicular helper (Tfh) cells – called tissue-resident helper (Trh). Notably, both Tfh and lung Trh cells express the extracellular ATP (eATP) sensor P2RX7 at high levels. Given its known deleterious role in Tfh cells, we investigated if P2RX7 could also limit the Trh pool in influenza-infected lungs. Contrary to our hypothesis, T cell-specific P2RX7 ablation led to a significant decrease in the Trh cell subset. Consequently, lungs of T cell-P2RX7 knockout mice harbor reduced B cell and CD8+ T cell responses. Initial Trh cell establishment is not affected by P2RX7 knockout; rather, P2RX7-deficient Trh cells gradually decay over time. This is associated with increased apoptosis and decreased in situ proliferation of P2RX7-knockout Trh cells. Our bulk RNA-seq analysis of lung CD4+ T cells revealed many cell cycle-associated genes poorly expressed in P2RX7-knockout cells. Some (but not all) Trh-fate genes are also decreased in P2RX7 deficiency. We are currently performing single-cell RNA-seq to define whether P2RX7 promotes the Trh fate in a particular subset of lung precursor cells. Our data shows that, in response to influenza, P2RX7 promotes lung-resident Trh cells, suggesting this eATP sensor has opposite roles in Tfh-like CD4+ T cells in a tissue-specific manner. Supported by grant from NIH (R00 AI139381)

Isthmin 1 is Expressed by Progenitor-Like Cells in the Lung: Phenotypical Analysis of Isthmin 1+ Hematopoietic Stem-Like Cells in Homeostasis and during Infection.

The process by which blood cells are generated has been widely studied in homeostasis and during pathogen-triggered inflammatory response. Recently, murine lungs have been shown to be a significant source of hematopoietic progenitors in a process known as extramedullary hematopoiesis. Using multiparametric flow cytometry, we have identified mesenchymal, endothelial, and hematopoietic progenitor cells that express the secreted small protein Isthmin 1 (ISM1). Further characterization of hematopoietic progenitor cells indicated that ISM1+ Lineage− Sca-1+ c-kit+ (ISM1+ LSK) cells are enriched in short-term hematopoietic stem cells (ST-HSCs). Moreover, most Sca-1+ ISM1+ cells express the residence marker CD49a, and this correlated with their localization in the extravascular region of the lung, indicating that ISM1+ cells are lung-resident cells. We also observed that ISM1+ cells express TLR4, TLR5, and TLR9, and, in a mouse model of sepsis induced by P. aeruginosa, we observed that all the LSK and ISM1+LSK cells were affected. We conclude that ISM1 is a novel biomarker associated with progenitor-like cells. ISM1+ cells are involved in the response to a bacterial challenge, suggesting an association between ISM1-producing cells and dangerous inflammatory responses like sepsis.

Clonotypic analysis of protective influenza M2e-specific lung resident Th17 memory cells reveals extensive functional diversity

The fate of tissue-resident memory CD4 T cells (Trm) has been incompletely investigated. Here we show that intranasal, but not parenteral, immunization with CTA1-3M2e-DD stimulated M2e-specific Th17 Trm cells, which conferred strong protection against influenza virus infection in the lung. These cells rapidly expanded upon infection and effectively restricted virus replication as determined by CD4 T cell depletion studies. Single-cell RNAseq transcriptomic and TCR VDJ-analysis of M2e-tetramer-sorted CD4 T cells on day 3 and 8 post infection revealed complete Th17-lineage dominance (no Th1 or Tregs) with extensive functional diversity and expression of gene markers signifying mature resident Trm cells (Cd69, Nfkbid, Brd2, FosB). Unexpectedly, the same TCR clonotype hosted cells with different Th17 subcluster functions (IL-17, IL-22), regulatory and cytotoxic cells, suggesting a tissue and context-dependent differentiation of reactivated Th17 Trm cells. A gene set enrichment analysis demonstrated up-regulation of regulatory genes (Lag3, Tigit, Ctla4, Pdcd1) in M2e-specific Trm cells on day 8, indicating a tissue damage preventing function. Thus, contrary to current thinking, lung M2e-specific Th17 Trm cells are sufficient for controlling infection and for protecting against tissue injury. These findings will have strong implications for vaccine development against respiratory virus infections and influenza virus infections, in particular.

Paracrine Regulation of Alveolar Epithelial Damage and Repair Responses by Human Lung-Resident Mesenchymal Stromal Cells.

COPD is characterized by irreversible lung tissue damage. We hypothesized that lung-derived mesenchymal stromal cells (LMSCs) reduce alveolar epithelial damage via paracrine processes, and may thus be suitable for cell-based strategies in COPD. We aimed to assess whether COPD-derived LMSCs display abnormalities. LMSCs were isolated from lung tissue of severe COPD patients and non-COPD controls. Effects of LMSC conditioned-medium (CM) on H2O2-induced, electric field- and scratch-injury were studied in A549 and NCI-H441 epithelial cells. In organoid models, LMSCs were co-cultured with NCI-H441 or primary lung cells. Organoid number, size and expression of alveolar type II markers were assessed. Pre-treatment with LMSC-CM significantly attenuated oxidative stress-induced necrosis and accelerated wound repair in A549. Co-culture with LMSCs supported organoid formation in NCI-H441 and primary epithelial cells, resulting in significantly larger organoids with lower type II-marker positivity in the presence of COPD-derived versus control LMSCs. Similar abnormalities developed in organoids from COPD compared to control-derived lung cells, with significantly larger organoids. Collectively, this indicates that LMSCs’ secretome attenuates alveolar epithelial injury and supports epithelial repair. Additionally, LMSCs promote generation of alveolar organoids, with abnormalities in the supportive effects of COPD-derived LMCS, reflective of impaired regenerative responses of COPD distal lung cells.