Endothelial cell expression of a STING gain-of-function mutation initiates pulmonary lymphocytic infiltration.

Patients afflicted with Stimulator of interferon gene (STING) gain-of-function mutations frequently present with debilitating interstitial lung disease (ILD) that is recapitulated in mice expressing the STINGV154M mutation (VM). Prior radiation chimera studies revealed an unexpected and critical role for non-hematopoietic cells in initiating ILD. To identify STING-expressing non-hematopoietic cell types required for the development of ILD, we use a conditional knockin (CKI) model and direct expression of the VM allele to hematopoietic cells, fibroblasts, epithelial cells, or endothelial cells. Only endothelial cell-targeted VM expression results in enhanced recruitment of immune cells to the lung associated with elevated chemokine expression and the formation of bronchus-associated lymphoid tissue, as seen in the parental VM strain. These findings reveal the importance of endothelial cells as instigators of STING-driven lung disease and suggest that therapeutic targeting of STING inhibitors to endothelial cells could potentially mitigate inflammation in the lungs of STING-associated vasculopathy with onset in infancy (SAVI) patients or patients afflicted with other ILD-related disorders.

GL7 ligand expression defines a novel subset of CD4+ TRM cells in lungs recovered from pneumococcus.

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) RORγT+, GL7+ TRM cells exhibited higher levels of (T-box expressed in T cells) T-bet and Gata-3, corresponding with increased synthesis of interferon-γ, 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.

Expression of a STING Gain-of-function Mutation in Endothelial Cells Initiates Lymphocytic Infiltration of the Lungs.

Patients afflicted with STING gain-of-function mutations frequently present with debilitating interstitial lung disease ( ILD ) that is recapitulated in mice expressing the STING V154M mutation ( VM ). Prior radiation chimera studies revealed an unexpected and critical role for non-hematopoietic cells in the initiation of ILD. To identify STING-expressing non-hematopoietic cell types relevant to ILD, we generated a conditional knock-in ( CKI ) model in which expression of the VM allele was directed to hematopoietic cells, fibroblasts, epithelial cells, or endothelial cells. Only endothelial cell-targeted expression of the mutant allele resulted in the recruitment of immune cells to the lung and the formation of bronchus-associated lymphoid tissue, as seen in the parental VM strain. These findings reveal the importance of endothelial cells as instigators of STING-driven lung disease and suggest that therapeutic targeting of STING inhibitors to endothelial cells could potentially mitigate inflammation in the lungs of SAVI patients or patients afflicted with other ILD-related disorders. Summary: Patients with STING gain-of-function (GOF) mutations develop life-threatening lung autoinflammation. In this study, Gao et al. utilize a mouse model of conditional STING GOF to demonstrate a role for endothelial STING GOF in initiating immune cell recruitment into lung tissues of SAVI mice.

Innate immune responses in pneumonia.

The lungs are an immunologically unique environment; they are exposed to innumerable pathogens and particulate matter daily. Appropriate clearance of pathogens and response to pollutants is required to prevent overwhelming infection, while preventing tissue damage and maintaining efficient gas exchange. Broadly, the innate immune system is the collection of immediate, intrinsic immune responses to pathogen or tissue injury. In this review, we will examine the innate immune responses of the lung, with a particular focus on their role in pneumonia. We will discuss the anatomic barriers and antimicrobial proteins of the lung, pathogen and injury recognition, and the role of leukocytes (macrophages, neutrophils, and innate lymphocytes) and lung stromal cells in innate immunity. Throughout the review, we will focus on new findings in innate immunity as well as features that are unique to the lung.

Lectin-like oxidized low-density lipoprotein receptor 1 attenuates pneumonia-induced lung injury.

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.

Recruitment and training of alveolar macrophages after pneumococcal pneumonia.

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.

Epithelial LIF signaling limits apoptosis and lung injury during bacterial pneumonia.

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.

Antigen presentation by lung epithelial cells directs CD4+ TRM cell function and regulates barrier immunity.

Barrier tissues are populated by functionally plastic CD4+ resident memory T (TRM) cells. Whether the barrier epithelium regulates CD4+ TRM cell locations, plasticity and activities remains unclear. Here we report that lung epithelial cells, including distinct surfactant protein C (SPC)lowMHChigh epithelial cells, function as anatomically-segregated and temporally-dynamic antigen presenting cells. In vivo ablation of lung epithelial MHC-II results in altered localization of CD4+ TRM cells. Recurrent encounters with cognate antigen in the absence of epithelial MHC-II leads CD4+ TRM cells to co-express several classically antagonistic lineage-defining transcription factors, changes their cytokine profiles, and results in dysregulated barrier immunity. In addition, lung epithelial MHC-II is needed for surface expression of PD-L1, which engages its ligand PD-1 to constrain lung CD4+ TRM cell phenotypes. Thus, we establish epithelial antigen presentation as a critical regulator of CD4+ TRM cell function and identify epithelial-CD4+ TRM cell immune interactions as core elements of barrier immunity.

Liver-Dependent Lung Remodeling during Systemic Inflammation Shapes Responses to Secondary Infection.

Systemic duress, such as that elicited by sepsis, burns, or trauma, predisposes patients to secondary pneumonia, demanding better understanding of host pathways influencing this deleterious connection. These pre-existing circumstances are capable of triggering the hepatic acute-phase response (APR), which we previously demonstrated is essential for limiting susceptibility to secondary lung infections. To identify potential mechanisms underlying protection afforded by the lung-liver axis, our studies aimed to evaluate liver-dependent lung reprogramming when a systemic inflammatory challenge precedes pneumonia. Wild-type mice and APR-deficient littermate mice with hepatocyte-specific deletion of STAT3 (hepSTAT3-/-), a transcription factor necessary for full APR initiation, were challenged i.p. with LPS to induce endotoxemia. After 18 h, pneumonia was induced by intratracheal Escherichia coli instillation. Endotoxemia elicited significant transcriptional alterations in the lungs of wild-type and hepSTAT3-/- mice, with nearly 2000 differentially expressed genes between genotypes. The gene signatures revealed exaggerated immune activity in the lungs of hepSTAT3-/- mice, which were compromised in their capacity to launch additional cytokine responses to secondary infection. Proteomics revealed substantial liver-dependent modifications in the airspaces of pneumonic mice, implicating a network of dispatched liver-derived mediators influencing lung homeostasis. These results indicate that after systemic inflammation, liver acute-phase changes dramatically remodel the lungs, resulting in a modified landscape for any stimuli encountered thereafter. Based on the established vulnerability of hepSTAT3-/- mice to secondary lung infections, we believe that intact liver function is critical for maintaining the immunological responsiveness of the lungs.

Roles of interleukin-11 during acute bacterial pneumonia.

Interleukin-11 (IL-11) is an interleukin-6 (IL-6) family cytokine shown to play a protective role in acute inflammatory settings including systemic infection. In this study we addressed the role of IL-11 in acute bacterial pneumonia using a mouse model of E. coli pneumonia. Compared with other related cytokines, IL-11 protein was maintained at high levels in the lung at baseline, with only mild alterations in whole lung and BALF levels during acute infection. The primary source of IL-11 in the lung was the epithelium, but steady state production was not dependent on the inflammatory transcription factor nuclear factor kappa B in cells of either myeloid or epithelial lineage. Blockade of IL-11 with neutralizing antibodies resulted in a mild but significant decrease in neutrophil recruitment and increase in pulmonary edema during pneumonia, without detectable alterations in bacterial clearance. Exogenous IL-11 administration, however, had no effect at baseline or during infection. Overall, we conclude that maintenance of lung IL-11 concentrations may influence acute pulmonary inflammation during infection, albeit modestly.

NF-κB RelA Is Required for Hepatoprotection during Pneumonia and Sepsis.

Pneumonia and sepsis are distinct but integrally linked public health concerns. The hepatic acute-phase response (APR), which is largely dependent on transcription factors NF-κB RelA and STAT3, is a hallmark of these pathologies and other injurious conditions. Inactivation of the APR can promote liver injury, a frequently observed organ dysfunction during sepsis. However, whether or how the acute-phase changes promote liver tissue resilience during infections is unclear. To determine the hepatoprotective role of the hepatic APR, we utilized mice bearing hepatocyte-specific deletions of either RelA or STAT3. Mice were challenged intratracheally (i.t.), intravenously (i.v.), or intraperitoneally (i.p.) with Escherichia coli, Klebsiella pneumoniae, Streptococcus pneumoniae, lipopolysaccharide (LPS), or alpha-galactosylceramide (αGalCer) to induce pneumonia, sepsis, or NKT cell activation. Liver injury was observed in RelA-null (hepRelAΔ/Δ) mice but not STAT3-null (hepSTAT3Δ/Δ) mice during pneumonia. The absence of RelA resulted in hepatotoxicity across several models of pneumonia, sepsis, and NKT cell activation. Injury was associated with increased levels of activated caspase-3 and -8 and substantial alteration of the hepatic transcriptome. Hepatotoxicity in the absence of RelA could be reversed by neutralization of tumor necrosis factor alpha (TNF-α). These results indicate the requirement of RelA-dependent inducible hepatoprotection during pneumonia and sepsis. Further, the results demonstrate that RelA-dependent gene programs are critical for maintaining liver homeostasis against TNF-α-driven immunotoxicity.

Genome-wide methylation analysis reveals differentially methylated loci that are associated with an age-dependent increase in bovine fibroblast response to LPS.

BACKGROUND: Differences in DNA methylation are known to contribute to the development of immune-related disorders in humans but relatively little is known about how methylation regulates immune function in cattle. Utilizing whole-transcriptome analyses of bovine dermal fibroblasts, we have previously identified an age and breed-dependent up-regulation of genes within the toll-like receptor 4 (TLR4) pathway that correlates with enhanced fibroblast production of IL-8 in response to lipopolysaccharide (LPS). Age-dependent differences in IL-8 production are abolished by treatment with 5-aza-2-deoxycytidine and Trichostatin A (AZA-TSA), suggesting epigenetic regulation of the innate response to LPS. In the current study, we performed reduced representation bisulfite sequencing (RRBS) on fibroblast cultures isolated from the same animals at 5- and 16-months of age to identify genes that exhibit variable methylation with age. To validate the role of methylation in gene expression, six innate response genes that were hyper-methylated in young animals were assessed by RT-qPCR in fibroblasts from animals at different ages and from different breeds. RESULTS: We identified 14,094 differentially methylated CpGs (DMCs) that differed between fibroblast cultures at 5- versus 16-months of age. Of the 5065 DMCs that fell within gene regions, 1117 were located within promoters, 1057 were within gene exons and 2891 were within gene introns and 67% were more methylated in young cultures. Transcription factor enrichment of the promoter regions hyper-methylated in young cultures revealed significant regulation by the key pro-inflammatory regulator, NF-κB. Additionally, five out of six chosen genes (PIK3R1, FES, NFATC1, TNFSF13 and RORA) that were more methylated in young cultures showed a significant reduction in expression post-LPS treatment in comparison with older cultures. Two of these genes, FES and NFATC1, were similarly down-regulated in Angus cultures that also exhibit a low LPS response phenotype. CONCLUSIONS: Our study has identified immune-related loci regulated by DNA methylation in cattle that may contribute to differential cellular response to LPS, two of which exhibit an identical expression profile in both low-responding age and breed phenotypes. Methylation biomarkers of differential immunity may prove useful in developing selection strategies for replacement cows that are less susceptible to severe infections, such as coliform mastitis.

In utero exposure to LPS alters the postnatal acute-phase response in beef heifers.

The potential effect of prenatal LPS exposure on the postnatal acute phase response (APR) to an LPS challenge in heifers was determined. Pregnant crossbred cows were separated into prenatal immune stimulation (PIS) and saline groups (Control). From these treatments, heifer calves were identified at weaning to subsequently receive an exogenous LPS challenge. Sickness behavior scores (SBS) were recorded and blood samples were collected at 30-min intervals from -2 to 8 h and again at 24 h relative to the LPS challenge. There was a treatment × time interaction for the change in vaginal temperature (VT) such that the change in VT was greater in Control than PIS from 150 to 250 min, yet it was greater in PIS than Control from 355 to 440 min and from 570 to 1145 min. There was also a treatment × time interaction for SBS such that scores were greater in Control than PIS at 0.5 h, yet were greater in PIS than Control from 2.5 to 4 h post-LPS. There was a tendency for a treatment × time interaction for serum concentrations of IL-6, which were greater in PIS than Control heifers from 5.5 to 6 h and from 7 to 8 h post-challenge. Thus, a single exposure to LPS during gestation can alter the postnatal APR to LPS in heifer calves.