Rationale: Oncostatin M (OSM), an interleukin-6 family cytokine, is involved in various inflammatory diseases. Currently, the role of OSM during lung infection is not well understood. The goal of this study was to understand OSM-mediated protection during lung infection. Methods: We intratracheally instilled E.coli or influenza A virus A/Puerto Rico/8/34 (PR8) into the lungs of 6-12 week-old OSM knockout (OSM-/-) or OSM receptor knockout (OSMrβ-/-) mice and wild type (WT) controls. Outcomes measured include survival/weight loss, lung leukocyte count, pathogen burden and downstream transcription factor analyses. To investigate lung cell-type-specific responses to OSM, we performed single-cell RNA sequencing of WT and OSMrβ-/- lungs intratracheally instilled with recombinant mouse OSM or a vehicle control. Results: During lung infection, OSM was almost exclusively produced by myeloid cells, yet OSMrβ was expressed by lung structural cells, suggesting that OSM signals in a paracrine fashion. Loss of OSM increased morbidity and mortality, especially in female mice. We observed changes in lung STAT3 and ERK activation in OSM-/- female mice, suggesting that immune dysregulation due to loss of OSM may contribute to mortality in OSM-/- female mice. Interestingly, we observed no phenotype in OSMrβ-/- compared to WT mice indicating that OSMrβ is dispensable for OSM function. Single-cell RNA sequencing revealed that OSM-stimulated general capillaries (gCaps) activate pathways important for vascular repair both in the presence and absence of OSMrβ. Thus, OSMrβ-independent OSM signaling in gCaps may explain why pneumonia outcomes were unchanged in OSMrβ-/- mice. In accordance with this, we observed similar STAT3 activation in alveolar blood vessels upon OSM stimulation in both WT and OSMrβ-/- lungs. Since the related IL-6 family receptor, leukemia inhibitory factor receptor (LIFrβ), is highly expressed by gCaps, OSM may be signaling through this receptor in OSMrβ-/- gCaps. Conclusion: Our study indicates that OSM is an important component of the host response during lung infection. Female mice show greater morbidity/mortality with loss of OSM, suggesting that OSM-mediated protection is sex-specific. Furthermore, loss of OSM-OSMrβ regulated protection is rescued by OSM signaling through an alternative receptor, most likely LIFrβ, on gCaps. Further understanding of OSM in the lungs will aid in development of new and effective treatment strategies during lung infection.

The global incidence of respiratory infectious diseases caused by bacteria continues to increase, with acute lower respiratory tract infections contributing to significant morbidity and mortality. Preclinical models designed to investigate such respiratory bacterial diseases are of utmost importance to decipher their pathogenesis and develop novel targets for intervention and treatment. Animal models offer the powerful ability to investigate different pneumonia types at varying stages of infection and disease. However, the same models can promote important variations in outcome, potentially confounding scientific understanding in the field. Therefore, an expert panel was convened to deliberate best practices in animal models of bacterial pneumonia to identify validated methodologies and acknowledge limitations in the use of animal and non-animal models in this field of study. Herein, we summarize this American Thoracic Society workshop on animal models of bacterial pneumonia. This workshop further includes review of non-animal complementary or alternative models for studying bacterial pneumonia. Emphasis was placed on discussion of bacterial pathogens that frequently cause community- and hospital-acquired pneumonia, highlighting key aspects in modeling infection. Animal models discussed included small and large animals, based on their strengths. Finally and most importantly, the ethical considerations in the use of animal modeling for the study of bacterial lung infections was discussed. This workshop report is intended to provide insights to investigators in the field and may serve as a starting point for formal recommendations in the future.

Neutrophils have historically been envisioned as a homogenous population of short-lived innate immune cells that migrate to sites of infection, kill pathogens, and die. Recent work, including studies in pneumonia models, has shown that neutrophil transcriptomes reflect the environment from which they were isolated. We used high-parameter spectral flow cytometry to compare and contrast a wide array of surface proteins on neutrophils from different tissues, infections, host age, pathogen virulence, and across multiple time-points of pneumonia. Circulating and airspace neutrophils consistently differed, and surface protein phenotypes unique to each infection setting were identified, revealing tissue-specific and microbe-specific neutrophil plasticity. Phenotypic differences in circulating neutrophils from mice infected with different pathogens ( E. coli , S. pneumoniae , S. aureus , and P. aeruginosa ) identified, even in the absence of bacteremia. Neutrophil activation state was diminished with less virulent pathogens and host age. In the airspace, VISTA, CD200R, and PD-L1 were selectively high on BAL neutrophils (BALN) during S. pneumoniae infection, and we identified pro-degranulation-like (CD88High VISTAHigh PD-L1+ CD101-) neutrophils in S. pneumoniae and pro-phagocytosis-like (CD101+ CD18Low PD-L1-) neutrophils in E. coli infections. Stimulation of VISTA with its ligand VISG-3 enhanced the neutrophil respiratory burst, degranulation, and killing of S. pneumoniae but not E. coli . We conclude that neutrophil cell surface protein expression depends on anatomic location and infection type, resulting in pathogen-specific neutrophil-mediated immune defense in discreet areas of the pneumonic lung. Graphical Abstract In brief, Pihl et al. have found that neutrophil cell surface phenotype varies drastically based on tissue, time post-infection, and infection. BALN from early infections have higher activation and maturation statuses, while blood neutrophils are more ‘migration primed,’ and late infections have more immune-suppressive and altered pathogen killing statuses. Neutrophil phenotype is skewed towards pro-phagocytosis associated marker expression on BALN from E. coli -infected mice while S. pneumoniae results in a pro-degranulation phenotype. In vitro BMN stimulation of VISTA with VSIG-3 results in degranulation, respiratory burst, and pathogen specific killing of S. pneumoniae but not E. coli . ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. NIH, , T32 HL 7035-48, R01HL158732, K08130582, F32 HL120551, T32 HL703547, KL2 TR001411 [1]: pending:yes

Rationale: Pneumonia is a worldwide public health concern, demanding a better understanding of the host response to pulmonary pathogens. We recently reported that lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is enriched on alveolar macrophages (AMs), wherein it may limit inflammatory injury during pneumonia. However, the mechanisms that govern protection from injury via LOX-1 are not known. Oxidized low-density lipoprotein (oxLDL), the primary ligand for LOX-1, elicits inflammation in extrapulmonary macrophages, contrasting our observations in AMs. Here, our goal was to determine the AM-specific function of LOX-1 during pneumonia and identify whether LOX-1-dependent outcomes rely on canonical LOX-1/oxLDL interactions. Methods: Primary AMs and RAW 264.7 cells were cultured ex vivo with either oxLDL alone or E. coli following pre-treatment with anti-LOX-1 and control BALF. Wild-type mice were treated with oxLDL +/- anti-LOX-1 antibody alone or pretreated with oxLDL for one week and infected with E. coli for 24h. Mice with a conditional deletion of LOX-1 on AMs (Olr1LoxP/LoxP/Cd11c-Cre+/-; here called Mac-Lox1-/-) were generated and infected with E. coli or S. pneumoniae for 24-48h. Results: We previously showed that LOX-1 blockade increases lung injury in association with inflammatory dysregulation of AMs in WT mice, however the ligands responsible for LOX-1-dependent lung protection are not known. The canonical LOX-1 ligand, oxLDL, markedly upregulated cytokine expression in RAW 264.7 macrophages, yet this effect was absent in AMs. Moreover, neutralizing LOX-1 in the presence of BALF ex vivo caused a trend towards opposite cytokine responses in E. coli-stimulated AMs (increased) vs RAW 264.7 cells (decreased), consistent with a non-traditional role for LOX-1 in AMs. LOX-1 neutralization in vivo increased neutrophil recruitment with oxLDL alone, while pre-treatment of mice with oxLDL skewed AMs towards a more M2-like phenotype during E. coli infection, an effect that was abolished with LOX-1 neutralization. Finally, Mac-Lox1-/- mice exhibit modestly less lung injury and inflammation following E. coli infection, inflammatory cell recruitment is increased with S. pneumoniae infection. Mac-Lox1-/- mice also have an altered AM phenotype, whereby, these cells lose expression of markers associated with AM identity and markedly gain Marco expression, suggesting a compensatory mechanism to limit lung injury. Conclusion: While LOX-1 has a well-established pro-inflammatory role during vascular injury, our data implicate a uniquely immunoregulatory role in AMs, possibly through interactions with oxLDL. These results pave the way for future studies focused on the molecular mechanisms connecting LOX-1 to AM identity and response to infection.

The role of mesenchymal cells during respiratory infection is not well defined, including whether, which, and how the different types of mesenchymal cells respond. We collected all mesenchymal cells from lung single-cell suspensions of mice that were naive (after receiving only saline vehicle), pneumonic (after intratracheal instillation of pneumococcus 24 hours previously), or resolved from infection (after nonlethal pneumococcal infections 6 weeks previously) and performed single-cell RNA sequencing. Cells clustered into 5 well-separated groups based on their transcriptomes: matrix fibroblasts, myofibroblasts, pericytes, smooth muscle cells, and mesothelial cells. Fibroblasts were the most abundant and could be further segregated into Pdgfra+Npnt+Ces1d+Col13a1+ alveolar fibroblasts and Cd9+Pi16+Sca1+Col14a1+ adventitial fibroblasts. The cells from naive and resolved groups overlapped in dimension reduction plots, suggesting the mesenchymal cells returned to baseline transcriptomes after resolution. During pneumonia, all mesenchymal cells responded with altered transcriptomes, revealing a core response that had been conserved across cell types as well as distinct mesenchymal cell type-specific responses. The different subsets of fibroblasts induced similargene sets, but the alveolar fibroblasts responded more strongly than the adventitial fibroblasts. These data demonstrated diverse and specialized immune activities of lung mesenchymal cells during pneumonia.

RATIONALE: Neutrophilic asthma is an aggravating disease diagnosed predominantly in adult humans. Though good models for eosinophilic asthma exist, few models for neutrophilic asthma are available. Here, we hypothesized that repeated and intermittent exposures of sensitized mice to inhaled ovalbumin (OVA) over extended durations would better model adult human experience leading to neutrophilic asthma. As such, our model differs from the conventional OVA-based model of eosinophilic asthma by introducing intermittent and recurrent inhalation of aerosolized OVA in pre-sensitized mice. METHODS: We used diverse transgenic mice, flow cytometry, and an array of standard analytic assays for evaluating disease outcomes in our model. Human relevance of our murine findings were confirmed by mining publicly available scRNA-Seq datasets. RESULTS: Acute allergic challenge in mice with inhaled OVA experience presented with accelerated, steroid-resistant neutrophilia accompanied by airway hyperreactivity and unrestrained lung injury. We observed that OVA-experienced lungs exhibited rapid IL-17A production which augmented CXCL5 secretion by lung epithelial cells leading to robust airway neutrophilia on allergic exacerbation. Consistent with its extensive experience, we found that such murine lungs possessed diverse subsets of CD4+ tissue-resident memory (TRM) cells including TH1, TH2, and Treg TRM cells; a feature lacking in naïve lungs. Interestingly, despite rapid IL-17A accumulation, experienced lungs were starkly devoid of conventional IL-17A-producing TH17 cells (identified by their RORγt positivity). Instead, we discovered that a novel unconventional RORγtnegative/low TH17 subset was the predominant producer of IL-17A that kickstarted the allergic neutrophilia. Among the lung intrinsic regulators, we found that epithelial MHC-II was key. Specifically, MHC-II expressed by lung epithelia enhanced TH1 TRM cell abundance which negatively correlated with CXCL5 accumulation, neutrophilia, and airway edema. As a result of this serendipitous observation, we found that IFN-γ muted IL-17A-induced CXCL5 production by lung epithelial cells in vitro and by airways in vivo to mediate this immunoregulatory effect. Indeed, prophylactic or therapeutic delivery of IFN-γ was sufficient to prevent neutrophilia, and mice lacking IFN-γ displayed worse disease outcomes in our model. Of note, all the CD4+ TRM and epithelial signatures discovered in our mouse model were conserved in adult asthmatic human airways via scRNA-Seq datasets. CONCLUSIONS: Thus, using a tractable murine model for allergic airway neutrophilic disease that mimics adult human airway biology, we identify CD4+ TRM – epithelial crosstalk as an important regulator of this disease. Our findings suggest that IFN-γ and the downstream pathways it activates may represent promising avenues to mitigate allergic airway neutrophilic disease.

Rationale: Pneumonia is the largest cause of infectious disease-related deaths worldwide. We recently reported that lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) prevents inflammatory injury during pneumonia, potentially due to elevated expression on subset of airspace recruited neutrophils. Here, we examined the influence of LOX-1 activity on neutrophils during bacterial pneumonia to test the hypothesis that LOX-1+ neutrophils limit inflammatory injury. Methods: Single-cell sequencing datasets were interrogated to identify LOX-1-expressing lung cells. Wild-type mice, LOX-1 KO mice, and conditional mutant mice lacking LOX-1 on neutrophils (Olr1LoxP/LoxP/Mrp8-Cre+/-; here called PMN-Lox1-/-) were infected with S. pneumoniae or E. coli for 12-48h. Spectral flow cytometry was used to determine neutrophil phenotype. Bone-marrow neutrophils were isolated from WT and LOX-1 KO mice and stimulated ex vivo with bronchoalveolar lavage fluid from infected and uninfected mice. Results: We previously observed that intratracheal LOX-1 blockade increases pneumonia-induced injury. A comparison of single-cell sequencing datasets showed substantial enrichment of LOX-1 (Olr1) mRNA in airspace neutrophils during pneumonia; however, surface LOX-1 is virtually absent in neutrophils recovered from blood and bone marrow. Interestingly, LOX-1+ is inducible on bone marrow neutrophils when exposed to bronchoalveolar lavage fluid ex vivo, indicating that airway fluid is sufficient to activate its expression. Intriguingly, LOX-1 is only expressed on a subset of neutrophils during lung infection, and LOX-1+ neutrophils have a markedly altered phenotype. We previously reported gene expression profiles in LOX-1+ neutrophils consistent with changes in cholesterol metabolism, insinuating PPAR-g and RXR/LXR activation. We now observe that LOX-1+ neutrophils have elevated CXCR3/4, MARCO, and CD200R expression, together suggesting these cells as a subtype of pro-resolving neutrophils. LOX-1+ neutrophils also have higher levels of apoptosis, which may promote efferocytic uptake. In line with our hypothesis, PMN-LOX-1-/- mice exhibit elevated lung injury, cellular recruitment, and complement receptor expression during S. pneumoniae infection. Conclusion: Our data implicate a unique, immunoregulatory role for LOX-1 on recruited neutrophils, possibly taming their responses to infection. These results pave the way for future research studying the mechanisms connecting neutrophil LOX-1 to pneumonia outcomes.

Recovery from respiratory pneumococcal infections generates lung-localized protection against heterotypic bacteria, mediated by resident memory lymphocytes. Optimal protection in mice requires re-exposure to pneumococcus within days of initial infection. Serial surface marker phenotyping of B cell populations in a model of pneumococcal heterotypic immunity revealed that bacterial re-exposure stimulates the immediate accumulation of dynamic and heterogeneous populations of B cells in the lung, and is essential for the establishment of lung resident memory B (BRM) cells. The B cells in the early wave were activated, proliferating locally, and associated with both CD4+ T cells and CXCL13. Antagonist- and antibody-mediated interventions were implemented during this early timeframe to demonstrate that lymphocyte recirculation, CD4+ cells, and CD40 ligand (CD40L) signaling were all needed for lung BRM cell establishment, whereas CXCL13 signaling was not. While most prominent as aggregates in the loose connective tissue of bronchovascular bundles, morphometry and live lung imaging analyses showed that lung BRM cells were equally numerous as single cells dispersed throughout the alveolar septae. We propose that CD40L signaling from antigen-stimulated CD4+ T cells in the infected lung is critical to establishment of local BRM cells, which subsequently protect the airways and parenchyma against future potential infections.

Neutrophils were once considered a homogenous population of transcriptionally static, pathogen-killing cells, however, recent models have demonstrated neutrophil functional and transcriptional plasticity. We performed transcriptomic analyses in a murine model of pneumococcal pneumonia to investigate neutrophil plasticity and demonstrate that neutrophils are highly dynamic, leading to three distinct alveolar neutrophil populations, one immature (early bronchoalveolar lavage neutrophils [BALN]) and two mature (late BALN). Early BALNs produce high levels of inflammatory cytokine transcripts, maturing into late BALNs, including a pro-degranulation and phagocytosis population (late-degranulating BALN) or a population specializing in translation machinery and inflammatory cytokine production (late-cytokine producing BALN). Neutrophil metabolism is also regulated in a stepwise manner, tricarboxylic acid (TCA) cycle and respiratory electron transport chain (ETC) genes are downregulated as neutrophils migrate from the vasculature to the interstitium, lipid and carbohydrate metabolism genes are downregulated during migration from interstitium to the airspace. These transitions may be regulated by aspects of the integrated stress response (ISR), as key regulators including Eif2ak2 are upregulated in interstitial neutrophils. Overall, we demonstrate that pneumonic neutrophils are transcriptionally plastic, developing through two distinct transcriptional phenotypes in the airspace, and are metabolically and transcriptionally rewired with potential points of regulation occurring in the interstitial space. ### Competing Interest Statement Sponsored research agreement from Johnson and Johnson to J.D.C for the study of lung cancer. The remaining authors have declared that no conflict of interest exists.

Streptococcus pneumoniae is the most common etiology of bacterial pneumonia, one of the leading causes of death in children and the elderly worldwide. During non-lethal infections with S. pneumoniae, lymphocytes accumulate in the lungs and protect against reinfection with serotype-mismatched strains. Cluster of differentiation CD4+ resident memory T (TRM) cells are known to be crucial for this protection, but the diversity of lung CD4+ TRM cells has yet to be fully delineated. We aimed to identify unique subsets and their contributions to lung immunity. After recovery from pneumococcal infections, we identified a distinct subset of CD4+ T cells defined by the phenotype CD11ahiCD69+GL7+ in mouse lungs. Phenotypic analyses for markers of lymphocyte memory and residence demonstrated that GL7+ T cells are a subset of CD4+ TRM cells. Functional studies revealed that unlike GL7- TRM subsets that were mostly (RAR-related Orphan Receptor gamma T) ROR gamma T+, GL7+ TRM cells exhibited higher levels of (T-box expressed in T cells) T-bet and Gata-3, corresponding with increased synthesis of interferon-gamma, interleukin-13, and interleukin-5, inherent to both T helper 1 (TH1) and TH2 functions. Thus, we propose that these cells provide novel contributions during pneumococcal pneumonia, serving as important determinants of lung immunity.

Recovery from pneumococcal pneumonia remodels the pool of alveolar macrophages so that they exhibit new surface marker profiles, transcriptomes, metabolomes, and responses to infection. Mechanisms mediating alveolar macrophage phenotypes after pneumococcal pneumonia have not been delineated. IFN-γ and its receptor on alveolar macrophages were essential for certain, but not all, aspects of the remodeled alveolar macrophage phenotype. IFN-γ was produced by CD4+ T cells plus other cells, and CD4+ cell depletion did not prevent alveolar macrophage remodeling. In mice infected or recovering from pneumococcus, monocytes were recruited to the lungs, and the monocyte-derived macrophages developed characteristics of alveolar macrophages. CCR2 mediated the early monocyte recruitment but was not essential to the development of the remodeled alveolar macrophage phenotype. Lineage tracing demonstrated that recovery from pneumococcal pneumonias converted the pool of alveolar macrophages from being primarily of embryonic origin to being primarily of adult hematopoietic stem cell origin. Alveolar macrophages of either origin demonstrated similar remodeled phenotypes, suggesting that ontogeny did not dictate phenotype. Our data reveal that the remodeled alveolar macrophage phenotype in lungs recovered from pneumococcal pneumonia results from a combination of new recruitment plus training of both the original cells and the new recruits.

While childhood-onset asthma is extensively modeled in labs and includes TH2 cell driven-, steroid-responsive, eosinophilic etiology, little is understood about late-onset asthma which presents as severe, steroid-resistant, and destructive neutrophilic disease with poor outcomes. Lack of appropriate disease models widens this knowledge gap leading to fewer therapies and poorer quality of life for neutrophilic asthma patients. Here, we discover that while conventional models of allergic asthma (relying on naïve sensitized mice acutely challenged with aerosolized ovalbumin) induces eosinophilic asthma, transient and recurrent aeroallergenic exposure over extended durations (as would occur in humans progressing into adulthood) reprograms the lung myeloid and lymphoid landscape to instigate neutrophilic asthma. Mice with such lung history harbor diverse clusters of lung-resident CD4+ TRM cells including a novel RORγt-negative-IL17A+ TH17 subset; the latter detectable in asthmatic adult human lungs. On allergen reencounter, these RORγt-negative TH17 cells, rapidly secrete IL-17A which signals into lung epithelial and stromal cells to express CXCL5 and induce neutrophilic asthma including peribronchial neutrophilia, vascular leakage and lung damage. We find that lung epithelial antigen presentation is crucial to regulate disease severity by skewing CD4+ TRM phenotypes in asthmatic lungs. Specifically, epithelial MHC-II supports TH1 TRM cells and IFNγ secretion; the latter being a potent suppressor of IL-17A-induced CXCL5 and airway neutrophilia. Thus, using a relevant model of the disease, we identify an ‘epithelium-lymphocyte-neutrophil’ circuitry as a critical regulator of late-onset neutrophilic asthma. Supported by NIH grants including HL147461 to F.T.K., HL142199 to K.A.B., HL136725 to M.R.J., GM120060 and HL111449 to L.J.Q., AI115053, HL135756, and HL137081 to J.P.M. and T32 HL007035 for support of trainees.

Identifying host factors that contribute to pneumonia incidence and severity are of utmost importance to guiding the development of more effective therapies. Lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1, encoded by OLR1) is a scavenger receptor known to promote vascular injury and inflammation, but whether and how LOX-1 functions in the lung are unknown. Here, we provide evidence of substantial accumulation of LOX-1 in the lungs of patients with acute respiratory distress syndrome and in mice with pneumonia. Unlike previously described injurious contributions of LOX-1, we found that LOX-1 is uniquely protective in the pulmonary airspaces, limiting proteinaceous edema and inflammation. We also identified alveolar macrophages and recruited neutrophils as 2 prominent sites of LOX-1 expression in the lungs, whereby macrophages are capable of further induction during pneumonia and neutrophils exhibit a rapid, but heterogenous, elevation of LOX-1 in the infected lung. Blockade of LOX-1 led to dysregulated immune signaling in alveolar macrophages, marked by alterations in activation markers and a concomitant elevation of inflammatory gene networks. However, bone marrow chimeras also suggested a prominent role for neutrophils in LOX-1-mediated lung protection, further supported by LOX-1+ neutrophils exhibiting transcriptional changes consistent with reparative processes. Taken together, this work establishes LOX-1 as a tissue-protective factor in the lungs during pneumonia, possibly mediated by its influence on immune signaling in alveolar macrophages and LOX-1+ airspace neutrophils.

IL-27 is a heterodimeric IL-12 family cytokine formed by noncovalent association of the promiscuous EBI3 subunit and selective p28 subunit. IL-27 is produced by mononuclear phagocytes and unfolds pleiotropic immune-modulatory functions through ligation to IL-27 receptor α (IL-27RA). Although IL-27 is known to contribute to immunity and to limit inflammation after various infections, its relevance for host defense against multicellular parasites is still poorly defined. Here, we investigated the role of IL-27 during infection with the soil-transmitted hookworm, Nippostrongylus brasiliensis, in its early host intrapulmonary life cycle. IL-27(p28) was detectable in bronchoalveolar lavage fluid of C57BL/6J wild-type mice on day 1 after s.c. inoculation. IL-27RA expression was most abundant on lung-invading γδ T cells. Il27ra-/- mice showed increased lung parasite burden together with aggravated pulmonary hemorrhage and higher alveolar total protein leakage as a surrogate for epithelial-vascular barrier disruption. Conversely, injections of recombinant mouse (rm)IL-27 into wild-type mice reduced lung injury and parasite burden. In multiplex screens, higher airway accumulations of IL-6, TNF-α, and MCP-3 (CCL7) were observed in Il27ra-/- mice, whereas rmIL-27 treatment showed a reciprocal effect. Importantly, γδ T cell numbers in airways were enhanced by endogenous or administered IL-27. Further analysis revealed a direct antihelminthic function of IL-27 on γδ T cells as adoptive intratracheal transfer of rmIL-27-treated γδ T cells during primary N. brasiliensis lung infection conferred protection in mice. In summary, this report demonstrates protective functions of IL-27 to control the early lung larval stage of hookworm infection.

During bacterial pneumonia, alveolar epithelial cells are critical for maintaining gas exchange and providing antimicrobial as well as pro-immune properties. We previously demonstrated that leukemia inhibitory factor (LIF), an IL-6 family cytokine, is produced by type II alveolar epithelial cells (ATII) and is critical for tissue protection during bacterial pneumonia. However, the target cells and mechanisms of LIF-mediated protection remain unknown. Here, we demonstrate that antibody-induced LIF blockade remodels the lung epithelial transcriptome in association with increased apoptosis. Based on these data, we performed pneumonia studies using a novel mouse model in which LIFR (the unique receptor for LIF) is absent in lung epithelium. Although LIFR is expressed on the surface of epithelial cells, its absence only minimally contributed to tissue protection during pneumonia. Single-cell RNA-sequencing (scRNAseq) was conducted to identify adult murine lung cell types most prominently expressing Lifr, revealing endothelial cells, mesenchymal cells, and ATIIs as major sources of Lifr. Sequencing data indicated that ATII cells were significantly impacted by pneumonia, with additional differences observed in response to LIF neutralization, including but not limited to gene programs related to cell death, injury, and inflammation. Overall, our data suggest that LIF signaling on epithelial cells alters responses in this cell type during pneumonia. However, our results also suggest separate and perhaps more prominent roles of LIFR in other cell types, such as endothelial cells or mesenchymal cells, which provide grounds for future investigation.