Mechanism of TLR4-Mediated Anti-Inflammatory Response Induced by Exopolysaccharide from the Probiotic Bacillus subtilis.

Intestinal inflammatory diseases affect millions of people worldwide, and one class of drugs showing promise toward treatment of several inflammatory diseases is probiotics. Numerous studies have been performed using probiotics to prevent and treat intestinal inflammatory diseases. Most of these studies used intact bacteria, and neither the active molecule nor the molecular mechanisms by which they affect immune responses are known. We have shown that the probiotic Bacillus subtilis is anti-inflammatory and can protect mice from acute colitis induced by the enteric pathogen Citrobacter rodentium. We identified and purified the active molecule, exopolysaccharide (EPS), and showed that it protects mice from C. rodentium-induced colitis by inducing anti-inflammatory M2 macrophages or inhibitory dendritic cells (DCs), both of which inhibit excessive T cell responses. We showed previously that EPS affects macrophages and DCs in a TLR4-dependent manner, and in the current study we asked how EPS induces these anti-inflammatory cells and how they function to inhibit T cells. By investigating the signaling downstream of TLR4 that leads to acquisition of inhibitory properties of macrophages and DCs, we found that EPS induces expression of the inhibitory molecule IDO in bone marrow-derived DCs, and that inhibition of T cell proliferation by IDO-expressing bone marrow-derived DCs utilizes the kynurenine/aryl hydrocarbon receptor circuit. Furthermore, unlike LPS, EPS does not induce inflammatory cytokines upon injection in vivo, directly demonstrating different outcomes induced by two different TLR4 agonists.

Single Cell Analysis Reveals Novel Immune Perturbations in Fibrotic Hypersensitivity Pneumonitis.

Rationale: Fibrotic hypersensitivity pneumonitis is a debilitating interstitial lung disease driven by incompletely understood immune mechanisms. Objectives: To elucidate immune aberrations in fibrotic hypersensitivity pneumonitis in single-cell resolution. Methods: Single-cell 5' RNA sequencing was conducted on peripheral blood mononuclear cells and bronchoalveolar lavage cells obtained from 45 patients with fibrotic hypersensitivity pneumonitis, 63 idiopathic pulmonary fibrosis, 4 non-fibrotic hypersensitivity pneumonitis, and 36 healthy controls in the United States and Mexico. Analyses included differential gene expression (Seurat), transcription factor activity imputation (DoRothEA-VIPER), and trajectory analyses (Monocle3/Velocyto-scVelo-CellRank). Measurements and Main Results: Overall, 501,534 peripheral blood mononuclear cells from 110 patients and controls and 88,336 bronchoalveolar lavage cells from 19 patients were profiled. Compared to controls, fibrotic hypersensitivity pneumonitis has elevated classical monocytes (adjusted-p=2.5e-3) and are enriched in CCL3hi/CCL4hi and S100Ahi classical monocytes (adjusted-p<2.2e-16). Trajectory analyses demonstrate that S100Ahi classical monocytes differentiate into SPP1hi lung macrophages associated with fibrosis. Compared to both controls and idiopathic pulmonary fibrosis, fibrotic hypersensitivity pneumonitis patient cells are significantly enriched in GZMhi cytotoxic T cells. These cells exhibit transcription factor activities indicative of TGFß and TNFα/NFκB pathways. These results are publicly available at https://ildimmunecellatlas.org. Conclusions: Single-cell transcriptomics of fibrotic hypersensitivity pneumonitis patients uncovered novel immune perturbations, including previously undescribed increases in GZMhi cytotoxic CD4+ and CD8+ T cells - reflecting this disease's unique inflammatory T-cell driven nature - as well as increased S100Ahi and CCL3hi/CCL4hi classical monocytes also observed in idiopathic pulmonary fibrosis. Both cell populations may guide the development of new biomarkers and therapeutic interventions.

Local, Quantitative Morphometry of Fibroproliferative Lung Injury Using Laminin.

Investigations into the mechanisms of injury and repair in fibroproliferative disease require consideration of the spatial heterogeneity inherent in the disease. Most scoring of fibrotic remodeling in preclinical animal models relies on the modified Ashcroft score, which is an ordinal rubric of macroscopic resolution. The obvious limitations of manual histopathologic scoring have generated an unmet need for unbiased, repeatable scoring of fibroproliferative burden in tissue. Using computer vision approaches on immunofluorescence imaging of the extracellular matrix component laminin, we generated a robust and repeatable quantitative remodeling scorer. In the bleomycin lung injury model, the quantitative remodeling scorer shows significant agreement with the modified Ashcroft scale. This antibody-based approach is easily integrated into larger multiplex immunofluorescence experiments, which we demonstrate by testing the spatial apposition of tertiary lymphoid structures to fibroproliferative tissue, a poorly characterized phenomenon observed in both human interstitial lung diseases and preclinical models of lung fibrosis. The tool reported in this article is available as a stand-alone application that is usable without programming knowledge.

A Gata3 enhancer necessary for ILC2 development and function.

The type 2 helper effector program is driven by the master transcription factor GATA3 and can be expressed by subsets of both innate lymphoid cells (ILCs) and adaptive CD4+ T helper (Th) cells. While ILC2s and Th2 cells acquire their type 2 differentiation program under very different contexts, the distinct regulatory mechanisms governing this common program are only partially understood. Here we show that the differentiation of ILC2s, and their concomitant high level of GATA3 expression, are controlled by a Gata3 enhancer, Gata3 +674/762, that plays only a minimal role in Th2 cell differentiation. Mice lacking this enhancer exhibited defects in several but not all type 2 inflammatory responses, depending on the respective degree of ILC2 and Th2 cell involvement. Our study provides molecular insights into the different gene regulatory pathways leading to the acquisition of the GATA3-driven type 2 helper effector program in innate and adaptive lymphocytes.

Identification of Clinically Significant Cytokine Signature Clusters in Patients With Septic Shock.

OBJECTIVES: To identify cytokine signature clusters in patients with septic shock. DESIGN: Prospective observational cohort study. SETTING: Single academic center in the United States. PATIENTS: Adult (≥ 18 yr old) patients admitted to the medical ICU with septic shock requiring vasoactive medication support. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: One hundred fourteen patients with septic shock completed cytokine measurement at time of enrollment (t 1 ) and 24 hours later (t 2 ). Unsupervised random forest analysis of the change in cytokines over time, defined as delta (t 2 -t 1 ), identified three clusters with distinct cytokine profiles. Patients in cluster 1 had the lowest initial levels of circulating cytokines that decreased over time. Patients in cluster 2 and cluster 3 had higher initial levels that decreased over time in cluster 2 and increased in cluster 3. Patients in clusters 2 and 3 had higher mortality compared with cluster 1 (clusters 1-3: 11% vs 31%; odds ratio [OR], 3.56 [1.10-14.23] vs 54% OR, 9.23 [2.89-37.22]). Cluster 3 was independently associated with in-hospital mortality (hazard ratio, 5.24; p = 0.005) in multivariable analysis. There were no significant differences in initial clinical severity scoring or steroid use between the clusters. Analysis of either t 1 or t 2 cytokine measurements alone or in combination did not reveal clusters with clear clinical significance. CONCLUSIONS: Longitudinal measurement of cytokine profiles at initiation of vasoactive medications and 24 hours later revealed three distinct cytokine signature clusters that correlated with clinical outcomes.

Advancing Lung Immunology Research: An Official American Thoracic Society Workshop Report.

The mammalian airways and lungs are exposed to a myriad of inhaled particulate matter, allergens, and pathogens. The immune system plays an essential role in protecting the host from respiratory pathogens, but a dysregulated immune response during respiratory infection can impair pathogen clearance and lead to immunopathology. Furthermore, inappropriate immunity to inhaled antigens can lead to pulmonary diseases. A complex network of epithelial, neural, stromal, and immune cells has evolved to sense and respond to inhaled antigens, including the decision to promote tolerance versus a rapid, robust, and targeted immune response. Although there has been great progress in understanding the mechanisms governing immunity to respiratory pathogens and aeroantigens, we are only beginning to develop an integrated understanding of the cellular networks governing tissue immunity within the lungs and how it changes after inflammation and over the human life course. An integrated model of airway and lung immunity will be necessary to improve mucosal vaccine design as well as prevent and treat acute and chronic inflammatory pulmonary diseases. Given the importance of immunology in pulmonary research, the American Thoracic Society convened a working group to highlight central areas of investigation to advance the science of lung immunology and improve human health.

Gut microbiota modulates bleomycin-induced acute lung injury response in mice.

BACKGROUND: Airway instillation of bleomycin (BLM) in mice is a widely used, yet challenging, model for acute lung injury (ALI) with high variability in treatment scheme and animal outcomes among investigators. Whether the gut microbiota plays any role in the outcome of BLM-induced lung injury is currently unknown. METHODS: Intratracheal instillation of BLM into C57BL/6 mice was performed. Fecal microbiomes were analyzed by 16s rRNA amplicon and metagenomic sequencing. Germ-free mice conventionalization and fecal microbiota transfer between SPF mice were performed to determine dominant commensal species that are associated with more severe BLM response. Further, lungs and gut draining lymph nodes of the mice were analyzed by flow cytometry to define immunophenotypes associated with the BLM-sensitive microbiome. RESULTS: Mice from two SPF barrier facilities at the University of Chicago exhibited significantly different mortality and weight loss during BLM-induced lung injury. Conventionalizing germ-free mice with SPF microbiota from two different housing facilities recapitulated the respective donors' response to BLM. Fecal microbiota transfer from the facility where the mice had worse mortality into the mice in the facility with more survival rendered recipient mice more susceptible to BLM-induced weight loss in a dominant negative manner. BLM-sensitive phenotype was associated with the presence of Helicobacter and Desulfovibrio in the gut, decreased Th17-neutrophil axis during steady state, and augmented lung neutrophil accumulation during the acute phase of the injury response. CONCLUSION: The composition of gut microbiota has significant impact on BLM-induced wasting and death suggesting a role of the lung-gut axis in lung injury.

Hypersensitivity pneumonitis: Current concepts in pathogenesis, diagnosis, and treatment.

Hypersensitivity pneumonitis is an immune-mediated interstitial lung disease caused by an aberrant response to an inhaled exposure, which results in mostly T cell-mediated inflammation, granuloma formation, and fibrosis in some cases. HP is diagnosed by exposure identification, HRCT findings of ground-glass opacities, centrilobular nodules, and mosaic attenuation, with traction bronchiectasis and honeycombing in fibrotic cases. Additional testing including serum IgG testing for the presence of antigen exposure, bronchoalveolar lavage lymphocytosis, and lung biopsy demonstrating granulomas, inflammation, and fibrosis, increases the diagnostic confidence. Treatment for HP includes avoidance of the implicated exposure, immunosuppression, and anti-fibrotic therapy in select cases. This narrative review presents the recent literature in the understanding of the immunopathological mechanisms, diagnosis, and treatment of HP.

IL-33-mediated Eosinophilia Protects against Acute Lung Injury.

Pneumonia-induced lung injury and acute respiratory distress syndrome can develop because of an inappropriate inflammatory response to acute infections, leading to a compromised alveolar barrier. Recent work suggests that hospitalized patients with allergies/asthma are less likely to die of pulmonary infections and that there is a correlation between survival from acute respiratory distress syndrome and higher eosinophil counts; thus, we hypothesized that eosinophils associated with a type 2 immune response may protect against pneumonia-induced acute lung injury. To test this hypothesis, mice were treated with the type 2-initiating cytokine IL-33 intratracheally 3 days before induction of pneumonia with airway administration of a lethal dose of Staphylococcus aureus. Interestingly, IL-33 pretreatment promoted survival by inhibiting acute lung injury: amount of BAL fluid proinflammatory cytokines and pulmonary edema were both reduced, with an associated increase in oxygen saturation. Pulmonary neutrophilia was also reduced, whereas eosinophilia was strongly increased. This eosinophilia was key to protection; eosinophil reduction eliminated both IL-33-mediated protection against mortality and inhibition of neutrophilia and pulmonary edema. Together, these data reveal a novel role for eosinophils in protection against lung injury and suggest that modulation of pulmonary type 2 immunity may represent a novel therapeutic strategy.

Temperature Trajectory Subphenotypes Correlate With Immune Responses in Patients With Sepsis.

OBJECTIVES: We recently found that distinct body temperature trajectories of infected patients correlated with survival. Understanding the relationship between the temperature trajectories and the host immune response to infection could allow us to immunophenotype patients at the bedside using temperature. The objective was to identify whether temperature trajectories have consistent associations with specific cytokine responses in two distinct cohorts of infected patients. DESIGN: Prospective observational study. SETTING: Large academic medical center between 2013 and 2019. SUBJECTS: Two cohorts of infected patients: 1) patients in the ICU with septic shock and 2) hospitalized patients with Staphylococcus aureus bacteremia. INTERVENTIONS: Clinical data (including body temperature) and plasma cytokine concentrations were measured. Patients were classified into four temperature trajectory subphenotypes using their temperature measurements in the first 72 hours from the onset of infection. Log-transformed cytokine levels were standardized to the mean and compared with the subphenotypes in both cohorts. MEASUREMENTS AND MAIN RESULTS: The cohorts consisted of 120 patients with septic shock (cohort 1) and 88 patients with S. aureus bacteremia (cohort 2). Patients from both cohorts were classified into one of four previously validated temperature subphenotypes: "hyperthermic, slow resolvers" (n = 19 cohort 1; n = 13 cohort 2), "hyperthermic, fast resolvers" (n = 18 C1; n = 24 C2), "normothermic" (n = 54 C1; n = 31 C2), and "hypothermic" (n = 29 C1; n = 20 C2). Both "hyperthermic, slow resolvers" and "hyperthermic, fast resolvers" had high levels of G-CSF, CCL2, and interleukin-10 compared with the "hypothermic" group when controlling for cohort and timing of cytokine measurement (p < 0.05). In contrast to the "hyperthermic, slow resolvers," the "hyperthermic, fast resolvers" showed significant decreases in the levels of several cytokines over a 24-hour period, including interleukin-1RA, interleukin-6, interleukin-8, G-CSF, and M-CSF (p < 0.001). CONCLUSIONS: Temperature trajectory subphenotypes are associated with consistent cytokine profiles in two distinct cohorts of infected patients. These subphenotypes could play a role in the bedside identification of cytokine profiles in patients with sepsis.

Prognosticating Outcomes in Interstitial Lung Disease by Mediastinal Lymph Node Assessment. An Observational Cohort Study with Independent Validation.

RATIONALE: Mediastinal lymph node (MLN) enlargement on chest computed tomography (CT) is prevalent in patients with interstitial lung disease (ILD) and may reflect immunologic activation and subsequent cytokine-mediated immune cell trafficking. OBJECTIVES: We aimed to determine whether MLN enlargement on chest CT predicts clinical outcomes and circulating cytokine levels in ILD. METHODS: MLN measurements were obtained from chest CT scans of patients with ILD at baseline evaluation over a 10-year period. Patients with sarcoidosis and drug toxicity-related ILD were excluded. MLN diameter and location were assessed. Plasma cytokine levels were analyzed in a subset of patients. The primary outcome was transplant-free survival (TFS). Secondary outcomes included all-cause and respiratory hospitalizations, lung function, and plasma cytokine concentrations. Cox regression was used to assess mortality risk. Outcomes were assessed in three independent ILD cohorts. MEASUREMENTS AND MAIN RESULTS: Chest CT scans were assessed in 1,094 patients (mean age, 64 yr; 52% male). MLN enlargement (≥10 mm) was present in 66% (n = 726) and strongly predicted TFS (hazard ratio [HR], 1.53; 95% confidence interval [CI], 1.12-2.10; P = 0.008) and risk of all-cause and respiratory hospitalizations (internal rate of return [IRR], 1.52; 95% CI, 1.17-1.98; P = 0.002; and IRR, 1.71; 95% CI, 1.15-2.53; P = 0.008, respectively) when compared with subjects with MLN <10 mm. Patients with MLN enlargement had lower lung function and decreased plasma concentrations of soluble CD40L (376 pg/ml vs. 505 pg/ml, P = 0.001) compared with those without MLN enlargement. Plasma IL-10 concentration >45 pg/ml predicted mortality (HR, 4.21; 95% CI, 1.21-14.68; P = 0.024). Independent analysis of external datasets confirmed these findings. CONCLUSIONS: MLN enlargement predicts TFS and hospitalization risk in ILD and is associated with decreased levels of a key circulating cytokine, soluble CD40L. Incorporating MLN and cytokine findings into current prediction models might improve ILD prognostication.

Single-Cell Transcriptomic Analysis of Human Lung Provides Insights into the Pathobiology of Pulmonary Fibrosis.

Rationale: The contributions of diverse cell populations in the human lung to pulmonary fibrosis pathogenesis are poorly understood. Single-cell RNA sequencing can reveal changes within individual cell populations during pulmonary fibrosis that are important for disease pathogenesis. Objectives: To determine whether single-cell RNA sequencing can reveal disease-related heterogeneity within alveolar macrophages, epithelial cells, or other cell types in lung tissue from subjects with pulmonary fibrosis compared with control subjects. Methods: We performed single-cell RNA sequencing on lung tissue obtained from eight transplant donors and eight recipients with pulmonary fibrosis and on one bronchoscopic cryobiospy sample from a patient with idiopathic pulmonary fibrosis. We validated these data using in situ RNA hybridization, immunohistochemistry, and bulk RNA-sequencing on flow-sorted cells from 22 additional subjects. Measurements and Main Results: We identified a distinct, novel population of profibrotic alveolar macrophages exclusively in patients with fibrosis. Within epithelial cells, the expression of genes involved in Wnt secretion and response was restricted to nonoverlapping cells. We identified rare cell populations including airway stem cells and senescent cells emerging during pulmonary fibrosis. We developed a web-based tool to explore these data. Conclusions: We generated a single-cell atlas of pulmonary fibrosis. Using this atlas, we demonstrated heterogeneity within alveolar macrophages and epithelial cells from subjects with pulmonary fibrosis. These results support the feasibility of discovery-based approaches using next-generation sequencing technologies to identify signaling pathways for targeting in the development of personalized therapies for patients with pulmonary fibrosis.

T-cell phenotypes are associated with serum IgE levels in Amish and Hutterite children.

OBJECTIVES: Amish children raised on traditional farms have lower atopy and asthma risk than Hutterite children raised on modern farms. In our previous study we established that the Amish environment affects the innate immune response to decrease asthma and atopy risk. Here we investigated T-cell phenotypes in the same Amish and Hutterite children as in our earlier study to elucidate how this altered innate immunity affects adaptive T cells. METHODS: Blood was collected from 30 Amish and 30 Hutterite age- and sex-matched children; cells were cryopreserved until analysis. Flow cytometry was used to analyze cell subsets. Atopy was determined based on allergen-specific and total IgE levels. RESULTS: Children exposed to Amish farms had increased activated regulatory CD4+ T-cell phenotypes, whereas conventional CD4 T cells expressed lower levels of costimulation molecules and other activation markers. The increase in numbers of circulating activated regulatory CD4+ T cells was associated with an increase in inhibitory receptors on monocytes in Amish, but not Hutterite, children. Strikingly, the Amish children had a higher proportion of CD28null CD8 T cells than the Hutterite children (P < .0001, nonparametric t test), a difference that remained even after accounting for the effects of age and sex (conditional log regression exponential ß = 1.08, P = .0053). The proportion of these cells correlated with high T-cell IFN-γ production (rs = 0.573, P = .005) and low serum IgE levels (rs = -0.417, P = .025). Furthermore, CD28null CD8 T-cell numbers were increased in Amish children, with high expression of the innate genes TNF and TNF-α-induced protein 3 (TNFAIP3) in peripheral blood leukocytes. CONCLUSION: Amish children's blood leukocytes are not only altered in their innate immune status but also have distinct T-cell phenotypes that are often associated with increased antigen exposure.

Associations between fungal and bacterial microbiota of airways and asthma endotypes.

BACKGROUND: The relationship between asthma, atopy, and underlying type 2 (T2) airway inflammation is complex. Although the bacterial airway microbiota is known to differ in asthmatic patients, the fungal and bacterial markers that discriminate T2-high (eosinophilic) and T2-low (neutrophilic/mixed-inflammation) asthma and atopy are still incompletely identified. OBJECTIVES: The aim of this study was to demonstrate the fungal microbiota structure of airways in asthmatic patients associated with T2 inflammation, atopy, and key clinical parameters. METHODS: We collected endobronchial brush (EB) and bronchoalveolar lavage (BAL) samples from 39 asthmatic patients and 19 healthy subjects followed by 16S gene and internal transcribed spacer-based microbiota sequencing. The microbial sequences were classified into exact sequence variants. The T2 phenotype was defined by using a blood eosinophil count with a threshold of 300 cells/µL. RESULTS: Fungal diversity was significantly lower in EB samples from patients with T2-high compared with T2-low inflammation; key fungal genera enriched in patients with T2-high inflammation included Trichoderma species, whereas Penicillium species was enriched in patients with atopy. In BAL fluid samples the dominant genera were Cladosporium, Fusarium, Aspergillus, and Alternaria. Using generalized linear models, we identified significant associations between specific fungal exact sequence variants and FEV1, fraction of exhaled nitric oxide values, BAL fluid cell counts, and corticosteroid use. Investigation of interkingdom (bacterial-fungal) co-occurrence patterns revealed different topologies between asthmatic patients and healthy control subjects. Random forest models with fungal classifiers predicted asthma status with 75% accuracy for BAL fluid samples and 80% accuracy for EB samples. CONCLUSIONS: We demonstrate clear differences in bacterial and fungal microbiota in asthma-associated phenotypes. Our study provides additional support for considering microbial signatures in delineating asthma phenotypes.

Effects of an FcγRIIA polymorphism on leukocyte gene expression and cytokine responses to anti-CD3 and anti-CD28 antibodies.

The low affinity Fcγ receptor, FcγRIIA, harbors a common missense mutation, rs1801274 (G>A, Arg131His) that modifies binding affinity to human IgG2 and mouse IgG1 antibodies and is associated with increased risk of autoimmune disease. Despite the important role of the Arg131His variant, little is understood about heterozygous genotype effects on global gene expression and cytokine production during an FcγR-dependent response. To address this gap in knowledge, we treated human whole-blood samples from 130 individuals with mouse IgG1 anti-CD3 and anti-CD28 antibodies and characterized the genome-wide gene expression profiles and cytokine production among individuals stratified by rs1801274 genotype. Our analysis revealed that the levels of four cytokines (IFNγ, IL-12, IL-2, TNFα) and global gene expression patterns differed between all three genotype classes. Surprisingly, the heterozygotes showed suboptimal T cell activation compared to cells from individuals homozygous for the higher-affinity FcγRIIA allele (GG; Arg/Arg). The results of this study demonstrate that IgG response varies among all rs1801274 genotype classes and results in profound differences in both cytokine responses and gene expression patterns in blood leukocytes. Because even heterozygotes showed dampened global responses, our data may provide insight into the heterogeneity of outcomes in cytokine release assays and immunotherapy efficacy.

Improving the Quality and Reproducibility of Flow Cytometry in the Lung. An Official American Thoracic Society Workshop Report.

Defining responses of the structural and immune cells in biologic systems is critically important to understanding disease states and responses to injury. This requires accurate and sensitive methods to define cell types in organ systems. The principal method to delineate the cell populations involved in these processes is flow cytometry. Although researchers increasingly use flow cytometry, technical challenges can affect its accuracy and reproducibility, thus significantly limiting scientific advancements. This challenge is particularly critical to lung immunology, as the lung is readily accessible and therefore used in preclinical and clinical studies to define potential therapeutics. Given the importance of flow cytometry in pulmonary research, the American Thoracic Society convened a working group to highlight issues and technical challenges to the performance of high-quality pulmonary flow cytometry, with a goal of improving its quality and reproducibility.

Innate Immunity and Asthma Risk in Amish and Hutterite Farm Children.

BACKGROUND: The Amish and Hutterites are U.S. agricultural populations whose lifestyles are remarkably similar in many respects but whose farming practices, in particular, are distinct; the former follow traditional farming practices whereas the latter use industrialized farming practices. The populations also show striking disparities in the prevalence of asthma, and little is known about the immune responses underlying these disparities. METHODS: We studied environmental exposures, genetic ancestry, and immune profiles among 60 Amish and Hutterite children, measuring levels of allergens and endotoxins and assessing the microbiome composition of indoor dust samples. Whole blood was collected to measure serum IgE levels, cytokine responses, and gene expression, and peripheral-blood leukocytes were phenotyped with flow cytometry. The effects of dust extracts obtained from Amish and Hutterite homes on immune and airway responses were assessed in a murine model of experimental allergic asthma. RESULTS: Despite the similar genetic ancestries and lifestyles of Amish and Hutterite children, the prevalence of asthma and allergic sensitization was 4 and 6 times as low in the Amish, whereas median endotoxin levels in Amish house dust was 6.8 times as high. Differences in microbial composition were also observed in dust samples from Amish and Hutterite homes. Profound differences in the proportions, phenotypes, and functions of innate immune cells were also found between the two groups of children. In a mouse model of experimental allergic asthma, the intranasal instillation of dust extracts from Amish but not Hutterite homes significantly inhibited airway hyperreactivity and eosinophilia. These protective effects were abrogated in mice that were deficient in MyD88 and Trif, molecules that are critical in innate immune signaling. CONCLUSIONS: The results of our studies in humans and mice indicate that the Amish environment provides protection against asthma by engaging and shaping the innate immune response. (Funded by the National Institutes of Health and others.).

Distinct T-helper cell responses to Staphylococcus aureus bacteremia reflect immunologic comorbidities and correlate with mortality.

BACKGROUND: The dysregulated host immune response that defines sepsis varies as a function of both the immune status of the host and the distinct nature of the pathogen. The degree to which immunocompromising comorbidities or immunosuppressive medications affect the immune response to infection is poorly understood because these patients are often excluded from studies about septic immunity. The objectives of this study were to determine the immune response to a single pathogen (Staphylococcus aureus) among a diverse case mix of patients and to determine whether comorbidities affect immune and clinical outcomes. METHODS: Blood samples were drawn from 95 adult inpatients at multiple time points after the first positive S. aureus blood culture. Cox proportional hazards modeling was used to determine the associations between admission neutrophil counts, admission lymphocyte counts, cytokine levels, and 90-day mortality. A nested case-control flow cytometric analysis was conducted to determine T-helper type 1 (Th1), Th2, Th17, and regulatory T-cell (Treg) subsets among a subgroup of 28 patients. In a secondary analysis, we categorized patients as either having immunocompromising disorders (human immunodeficiency virus and hematologic malignancies), receiving immunosuppressive medications, or being not immunocompromised. RESULTS: Higher neutrophil-to-lymphocyte count ratios and higher Th17 cytokine responses relative to Th1 cytokine responses early after infection were independently associated with mortality and did not depend on the immune state of the patient (HR 1.93, 95% CI 1.17-3.17, p = 0.01; and HR 1.13, 95% CI 1.01-1.27, p = 0.03, respectively). On the basis of flow cytometric analysis of CD4 T-helper subsets, an increasing Th17/Treg response over the course of the infection was most strongly associated with increased mortality (HR 4.41, 95% CI 1.69-11.5, p < 0.01). This type of immune response was most common among patients who were not immunocompromised. In contrast, among immunocompromised patients who died, a decreasing Th1/Treg response was most common. CONCLUSIONS: The association of both increased Th17 responses and increased neutrophil counts relative to lymphocyte counts with mortality suggests that an overwhelming inflammatory response is detrimental. However, the differential responses of patients according to immune state suggest that immune status is an important clinical indicator that should be accounted for in the management of septic patients, as well as in the development of novel immunomodulatory therapies.

Genome-Wide Methylation Study Identifies an IL-13-induced Epigenetic Signature in Asthmatic Airways.

RATIONALE: Epigenetic changes to airway cells have been proposed as important modulators of the effects of environmental exposures on airway diseases, yet no study to date has shown epigenetic responses to exposures in the airway that correlate with disease state. The type 2 cytokine IL-13 is a key mediator of allergic airway diseases, such as asthma, and is up-regulated in response to many asthma-promoting exposures. OBJECTIVES: To directly study the epigenetic response of airway epithelial cells (AECs) to IL-13 and test whether IL-13-induced epigenetic changes differ between individuals with and without asthma. METHODS: Genome-wide DNA methylation and gene expression patterns were studied in 58 IL-13-treated and untreated primary AEC cultures and validated in freshly isolated cells of subjects with and without asthma using the Illumina Human Methylation 450K and HumanHT-12 BeadChips. IL-13-mediated comethylation modules were identified and correlated with clinical phenotypes using weighted gene coexpression network analysis. MEASUREMENTS AND MAIN RESULTS: IL-13 altered global DNA methylation patterns in cultured AECs and were significantly enriched near genes associated with asthma. Importantly, a significant proportion of this IL-13 epigenetic signature was validated in freshly isolated AECs from subjects with asthma and clustered into two distinct modules, with module 1 correlated with asthma severity and lung function and module 2 with eosinophilia. CONCLUSIONS: These results suggest that a single exposure of IL-13 may selectively induce long-lasting DNA methylation changes in asthmatic airways that alter specific AEC pathways and contribute to asthma phenotypes.

Intrinsic functional defects of type 2 innate lymphoid cells impair innate allergic inflammation in promyelocytic leukemia zinc finger (PLZF)-deficient mice.

BACKGROUND: The transcription factor promyelocytic leukemia zinc finger (PLZF) is transiently expressed during development of type 2 innate lymphoid cells (ILC2s) but is not present at the mature stage. We hypothesized that PLZF-deficient ILC2s have functional defects in the innate allergic response and represent a tool for studying innate immunity in a mouse with a functional adaptive immune response. OBJECTIVE: We determined the consequences of PLZF deficiency on ILC2 function in response to innate and adaptive immune stimuli by using PLZF(-/-) mice and mixed wild-type:PLZF(-/-) bone marrow chimeras. METHODS: PLZF(-/-) mice, wild-type littermates, or mixed bone marrow chimeras were treated with the protease allergen papain or the cytokines IL-25 and IL-33 or infected with the helminth Nippostrongylus brasiliensis to induce innate type 2 allergic responses. Mice were sensitized with intraperitoneal ovalbumin-alum, followed by intranasal challenge with ovalbumin alone, to induce adaptive TH2 responses. Lungs were analyzed for immune cell subsets, and alveolar lavage fluid was analyzed for ILC2-derived cytokines. In addition, ILC2s were stimulated ex vivo for their capacity to release type 2 cytokines. RESULTS: PLZF-deficient lung ILC2s exhibit a cell-intrinsic defect in the secretion of IL-5 and IL-13 in response to innate stimuli, resulting in defective recruitment of eosinophils and goblet cell hyperplasia. In contrast, the adaptive allergic inflammatory response to ovalbumin and alum was unimpaired. CONCLUSIONS: PLZF expression at the innate lymphoid cell precursor stage has a long-range effect on the functional properties of mature ILC2s and highlights the importance of these cells for innate allergic responses in otherwise immunocompetent mice.