Dimethyl Sulfoxide as a Novel Therapy in a Murine Model of Acute Lung Injury.

INTRODUCTION: The endothelial glycocalyx (EGX) on the luminal surface of endothelial cells contributes to the permeability barrier of the pulmonary vasculature. Dimethyl sulfoxide (DMSO) has a disordering effect on plasma membranes, which prevents the formation of ordered membrane domains important in the shedding of the EGX. We hypothesized that DMSO would protect against protein leak by preserving the EGX in a murine model of acute respiratory distress syndrome (ARDS). METHODS: C57BL/6 mice were given ARDS via intra-tracheally administered lipopolysaccharide (LPS). DMSO (220 mg/kg) was administered intravenously for 4 days. Animals were sacrificed post-injury day 4 after bronchoalveolar lavage (BAL). BAL cell counts and protein content was quantified. Lung sections were stained with FITC-labelled wheat germ agglutinin (FITC-WGA) to quantify the EGX. Cultured endothelial cells (HUVECs) were exposed to LPS. EGX was measured using FITC-WGA, and co-immunoprecipitation was performed to measure interaction between sheddases and syndecan-1. RESULTS: DMSO treatment resulted in greater EGX staining intensity in the lung when compared to sham (9,641 vs. 36,659 A.U. p < 0.001). Total BAL cell counts were less for animals receiving DMSO (6.93 x 106 vs. 2.49 x 106 cells, p = 0.04). The treated group had less BAL macrophages (189.2 vs. 76.9 cells, p = 0.02) and lymphocytes (527.7 vs. 200.0 cells, p = 0.02). Interleukin-6 levels were lower in DMSO treated. Animals that received DMSO had less protein leak in BAL (1.48 vs. 1.08 ug/ul, p = 0.02). DMSO prevented LPS-induced EGX loss in HUVECs, and reduced the interaction between Matrix Metalloproteinase (MMP) 16 and syndecan-1. CONCLUSIONS: Systemically administered DMSO protects the EGX in the pulmonary vasculature, mitigating pulmonary capillary leak after acute lung injury. DMSO also results in decreased inflammatory response. DMSO reduced the interaction between MMP16 and Syndecan-1 and prevented LPS-induced glycocalyx damage in cultured endothelial cells. DMSO may be a novel therapeutic for ARDS. LEVEL OF EVIDENCE: Not applicable (animal studies).

Dimethyl malonate protects the lung in a murine model of acute respiratory distress syndrome.

BACKGROUND: Succinate is a proinflammatory citric acid cycle metabolite that accumulates in tissues during pathophysiological states. Oxidation of succinate after ischemia-reperfusion leads to reversal of the electron transport chain and generation of reactive oxygen species. Dimethyl malonate (DMM) is a competitive inhibitor of succinate dehydrogenase, which has been shown to reduce succinate accumulation. We hypothesized that DMM would protect against inflammation in a murine model of ARDS. METHODS: C57BL/6 mice were given ARDS via 67.7 µg of intratracheally administered lipopolysaccharide. Dimethyl malonate (50 mg/kg) was administered via tail vein injection 30 minutes after injury, then daily for 3 days. The animals were sacrificed on day 4 after bronchoalveolar lavage (BAL). Bronchoalveolar lavage cell counts were performed to examine cellular influx. Supernatant protein was quantified via Bradford protein assay. Animals receiving DMM (n = 8) were compared with those receiving sham injection (n = 8). Cells were fixed and stained with FITC-labeled wheat germ agglutinin to quantify the endothelial glycocalyx (EGX). RESULTS: Total cell counts in BAL was less for animals receiving DMM (6.93 × 10 6 vs. 2.46 × 10 6 , p = 0.04). The DMM group had less BAL macrophages (168.6 vs. 85.1, p = 0.04) and lymphocytes (527.7 vs. 248.3; p = 0.04). Dimethyl malonate-treated animals had less protein leak in BAL than sham treated (1.48 vs. 1.15 µg/µl, p = 0.03). Treatment with DMM resulted in greater staining intensity of the EGX in the lung when compared with sham (12,016 vs. 15,186 arbitrary units, p = 0.03). Untreated animals had a greater degree of weight loss than treated animals (3.7% vs. 1.1%, p = 0.04). Dimethyl malonate prevented the upregulation of monocyte chemoattractant protein-1 (1.66 vs. 0.92 RE, p = 0.02) and ICAM-1 (1.40 vs. 1.01 RE, p = 0.05). CONCLUSION: Dimethyl malonate reduces lung inflammation and capillary leak in ARDS. This may be mediated by protection of the EGX and inhibition of monocyte chemoattractant protein-1 and ICAM-1. Dimethyl malonate may be a novel therapeutic for ARDS.

IL-22 Binding Protein Controls IL-22-Driven Bleomycin-Induced Lung Injury.

The high mortality rates of acute lung injury and acute respiratory distress syndrome challenge the field to identify biomarkers and factors that can be exploited for therapeutic approaches. IL-22 is a cytokine that has antibacterial and reparative properties in the lung. However, it also can exacerbate inflammation and requires tight control by the extracellular inhibitory protein known as IL-22 binding protein (IL-22BP) (Il22ra2). This study showed the necessity of IL-22BP in controlling and preventing acute lung injury using IL-22BP knockout mice (Il22ra2-/-) in the bleomycin model of acute lung injury/acute respiratory distress syndrome. Il22ra2-/- mice had greater sensitivity (weight loss and death) and pulmonary inflammation in the acute phase (first 7 days) of the injury compared with wild-type C57Bl/6 controls. The inflammation was driven by excess IL-22 production, inducing the influx of pathogenic IL-17A+ γδ T cells to the lung. Interestingly, this inflammation was initiated in part by the noncanonical IL-22 signaling to macrophages, which express the IL-22 receptor (Il22ra1) in vivo after bleomycin challenge. This study further showed that IL-22 receptor alpha-1+ macrophages can be stimulated by IL-22 to produce a number of IL-17-inducing cytokines such as IL-1ß, IL-6, and transforming growth factor-ß1. Together, the results suggest that IL-22BP prevents IL-22 signaling to macrophages and reduces bleomycin-mediated lung injury.

EXOSOMES AND MICROVESICLES FROM ADIPOSE-DERIVED MESENCHYMAL STEM CELLS PROTECTS THE ENDOTHELIAL GLYCOCALYX FROM LPS INJURY.

ABSTRACT: Introduction: Endothelial glycocalyx damage occurs in numerous pathological conditions and results in endotheliopathy. Extracellular vesicles, including exosomes and microvesicles, isolated from adipose-derived mesenchymal stem cells (ASCs) have therapeutic potential in multiple disease states; however, their role in preventing glycocalyx shedding has not been defined. We hypothesized that ASC-derived exosomes and microvesicles would protect the endothelial glycocalyx from damage by LPS injury in cultured endothelial cells. Methods : Exosomes and microvesicles were collected from ASC conditioned media by centrifugation (10,000 g for microvesicles, 100,000 g for exosomes). Human umbilical vein endothelial cells (HUVECs) were exposed to 1 µg/mL lipopolysaccharide (LPS). LPS-injured cells (n = 578) were compared with HUVECS with concomitant LPS injury plus 1.0 µg/mL of exosomes (n = 540) or microvesicles (n = 510) for 24 hours. These two cohorts were compared with control HUVECs that received phosphate-buffered saline only (n = 786) and HUVECs exposed to exosomes (n = 505) or microvesicles (n = 500) alone. Cells were fixed and stained with FITC-labeled wheat germ agglutinin to quantify EGX. Real-time quantitative reverse-transcription polymerase chain reaction was used on HUVECs cell lystate to quantify hyaluron synthase-1 (HAS1) expression. Results: Exosomes alone decreased endothelial glycocalyx staining intensity when compared with control (4.94 vs. 6.41 AU, P < 0.001), while microvesicles did not cause a change glycocalyx staining intensity (6.39 vs. 6.41, P = 0.99). LPS injury resulted in decreased glycocalyx intensity as compared with control (5.60 vs. 6.41, P < 0.001). Exosomes (6.85 vs. 5.60, P < 0.001) and microvesicles (6.35 vs. 5.60, P < 0.001) preserved endothelial glycocalyx staining intensity after LPS injury. HAS1 levels were found to be higher in the exosome (1.14 vs. 3.67 RE, P = 0.02) and microvesicle groups (1.14 vs. 3.59 RE, P = 0.02) when compared with LPS injury. Hyaluron synthase-2 and synthase-3 expressions were not different in the various experimental groups. Conclusions: Exosomes alone can damage the endothelial glycocalyx. However, in the presence of LPS injury, both exosomes and microvesicles protect the glycocalyx layer. This effect seems to be mediated by HAS1. Level of Evidence : Basic science study.

Interleukin-22 mitigates acute respiratory distress syndrome (ARDS).

BACKGROUND: The goal of this study was to determine if IL-22:Fc would Acute Respiratory Distress Syndrome (ARDS). SUMMARY BACKGROUND DATA: No therapies exist for ARDS and treatment is purely supportive. Interleukin-22 (IL-22) plays an integral component in recovery of the lung from infection. IL-22:Fc is a recombinant protein with a human FC immunoglobulin that increases the half-life of IL-22. STUDY DESIGN: ARDS was induced in C57BL/6 mice with intra-tracheal lipopolysaccharide (LPS) at a dose of 33.3 or 100 ug. In the low-dose LPS group (LDG), IL-22:FC was administered via tail vein injection at 30 minutes (n = 9) and compared to sham (n = 9). In the high-dose LPS group (HDG), IL-22:FC was administered (n = 11) then compared to sham (n = 8). Euthanasia occurred after bronchioalveolar lavage (BAL) on post-injury day 4. RESULTS: In the LDG, IL-22:FC resulted in decreased protein leak (0.15 vs. 0.25 ug/uL, p = 0.02). BAL protein in animals receiving IL-22:Fc in the HDG was not different. For the HDG, animals receiving IL-22:Fc had lower BAL cell counts (539,636 vs 3,147,556 cells/uL, p = 0.02). For the HDG, IL-6 (110.6 vs. 527.1 pg/mL, p = 0.04), TNF-α (5.87 vs. 25.41 pg/mL, p = 0.04), and G-CSF (95.14 vs. 659.6, p = 0.01) levels were lower in the BAL fluid of IL-22:Fc treated animals compared to sham. CONCLUSIONS: IL-22:Fc decreases lung inflammation and lung capillary leak in ARDS. IL-22:Fc may be a novel therapy for ARDS.

SARS-CoV-2 infection of primary human lung epithelium for COVID-19 modeling and drug discovery.

Coronavirus disease 2019 (COVID-19) is the latest respiratory pandemic caused by severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). Although infection initiates in the proximal airways, severe and sometimes fatal symptoms of the disease are caused by infection of the alveolar type 2 (AT2) cells of the distal lung and associated inflammation. In this study, we develop primary human lung epithelial infection models to understand initial responses of proximal and distal lung epithelium to SARS-CoV-2 infection. Differentiated air-liquid interface (ALI) cultures of proximal airway epithelium and alveosphere cultures of distal lung AT2 cells are readily infected by SARS-CoV-2, leading to an epithelial cell-autonomous proinflammatory response with increased expression of interferon signaling genes. Studies to validate the efficacy of selected candidate COVID-19 drugs confirm that remdesivir strongly suppresses viral infection/replication. We provide a relevant platform for study of COVID-19 pathobiology and for rapid drug screening against SARS-CoV-2 and emergent respiratory pathogens.

Interleukin 22 mitigates endothelial glycocalyx shedding after lipopolysaccharide injury.

BACKGROUND: The endothelial glycocalyx (EG) on the luminal surface of endothelial cells contributes to the permeability barrier of vessels and prevents activation of the coagulation cascade. Endothelial glycocalyx damage, which occurs in the shock state, results in endotheliopathy. Interleukin (IL)-22 is a cytokine with both proinflammatory and anti-inflammatory properties, and how IL-22 affects the EG has not been studied. We hypothesized that IL-22:Fc, a recombinant fusion protein with human IL-22 and the Fc portion of human immunoglobulin G1 (which extends the protein half-life), would not affect EG shedding in endothelium after injury. METHODS: Human umbilical vein endothelial cells (HUVECs) were exposed to 1 µg/mL lipopolysaccharide (LPS). Lipopolysaccharide-injured cells (n = 284) were compared with HUVECs with LPS injury plus 0.375 µg/mL of IL-22:Fc treatment (n = 293) for 12 hours. These two cohorts were compared with control HUVECs (n = 286) and HUVECs exposed to IL-22:Fc alone (n = 269). Cells were fixed and stained with fluorescein isothiocyanate-labeled wheat germ agglutinin to quantify EG. Total RNA was collected, and select messenger RNAs were quantified by real time - quantitative polymerase chain reaction (RT-qPCR) using SYBR green fluorescence. RESULTS: Exposure of HUVECs to LPS resulted in degradation of the EG compared with control (5.86 vs. 6.09 arbitrary unit [AU], p = 0.01). Interleukin-22:Fc alone also resulted in degradation of EG (5.08 vs. 6.09 AU, p = 0.01). Treatment with IL-22:Fc after LPS injury resulted in less degradation of EG compared with LPS injury alone (5.86 vs. 5.08 AU, p = 0.002). Expression of the IL-22Ra1 receptor was not different for IL-22:Fc treated compared with LPS injury only (0.69 vs. 0.86 relative expression, p = 0.10). Treatment with IL-22:Fc after LPS injury resulted in less matrix metalloproteinase 2 (0.79 vs. 1.70 relative expression, p = 0.005) and matrix metalloproteinase 14 (0.94 vs. 2.04 relative expression, p = 0.02). CONCLUSIONS: Interleukin-22:Fc alone induces EG degradation. However, IL-22:Fc treatment after LPS injury appears to mitigate EG degradation. This protective effect appears to be mediated via reduced expression of metalloproteinases.

Murine models of Pneumocystis infection recapitulate human primary immune disorders.

Despite the discovery of key pattern recognition receptors and CD4+ T cell subsets in laboratory mice, there is ongoing discussion of the value of murine models to reflect human disease. Pneumocystis is an AIDS-defining illness, in which risk of infection is inversely correlated with peripheral CD4+ T cell counts. Due to medical advances in the control of HIV, the current epidemiology of Pneumocystis infection is predominantly due to primary human immunodeficiencies and immunosuppressive therapies. To this end, we found that every human genetic immunodeficiency associated with Pneumocystis infection that has been tested in mice recapitulated susceptibility. For example, humans with a loss-of-function IL21R mutation are severely immunocompromised. We found that IL-21R, in addition to CD4+ T cell intrinsic STAT3 signaling, were required for generating protective antifungal class-switched antibody responses, as well as effector T cell-mediated protection. Furthermore, CD4+ T cell intrinsic IL-21R/STAT3 signaling was required for CD4+ T cell effector responses, including IL-22 production. Recombinant IL-22 administration to Il21r-/- mice induced the expression of a fungicidal peptide, cathelicidin antimicrobial peptide, which showed in vitro fungicidal activity. In conclusion, SPF laboratory mice faithfully replicate many aspects of human primary immunodeficiency and provide useful tools to understand the generation and nature of effector CD4+ T cell immunity.

Epigenetic and Transcriptomic Regulation of Lung Repair during Recovery from Influenza Infection.

Seasonal and pandemic influenza is a cause of morbidity and mortality worldwide. Most people infected with influenza virus display mild-to-moderate disease phenotypes and recover within a few weeks. Influenza is known to cause persistent alveolitis in animal models; however, little is known about the molecular pathways involved in this phenotype. We challenged C57BL/6 mice with influenza A/PR/8/34 and examined lung pathologic processes and inflammation, as well as transcriptomic and epigenetic changes at 21 to 60 days after infection. Influenza induced persistent parenchymal lung inflammation, alveolar epithelial metaplasia, and epithelial endoplasmic reticulum stress that were evident after the clearance of virus and resolution of morbidity. Influenza infection induced robust changes in the lung transcriptome, including a significant impact on inflammatory and extracellular matrix protein expression. Despite the robust changes in lung gene expression, preceding influenza (21 days) did not exacerbate secondary Staphylococcus aureus infection. Finally, we examined the impact of influenza on miRNA expression in the lung and found an increase in miR-155. miR-155 knockout mice recovered from influenza infection faster than controls and had decreased lung inflammation and endoplasmic reticulum stress. These data illuminate the dynamic molecular changes in the lung in the weeks after influenza infection and characterize the repair process, identifying a novel role for miR-155.

Intraductal infusion of taurocholate followed by distal common bile duct ligation leads to a severe necrotic model of pancreatitis in mice.

OBJECTIVE: The most common etiology of acute pancreatitis results from the impaction of gallstones or sludge in the distal common bile duct (CBD). The result is pancreatic duct obstruction, diversion of bile into the pancreas, or cholestasis. In the current study, we examined whether combining both aspects, that is, infusion of the bile acid taurocholate (TC) followed by bile duct ligation (BDL), could yield a more severe form of pancreatitis that mimics biliary pancreatitis. METHODS: In mice, after laparotomy, the CBD was infused with either normal saline (NS) or TC. Subsequently, the CBD was ligated at the ampulla. RESULTS: Mice receiving TC infusion followed by BDL (TC + BDL) had higher mortality compared with animals receiving intraductal NS with BDL (NS + BDL). The TC + BDL arm developed more severe and diffuse pancreatic necrosis. In addition, serum amylase, IL-6, and bilirubin were significantly higher. However, pancreatic edema as well as lung and liver injury were unchanged between TC + BDL and NS + BDL. CONCLUSIONS: In summary, the combination of bile infusion into the pancreas followed by BDL causes a more severe, necrotizing pancreatitis. We believe that this novel model of pancreatitis is useful because it can be used in transgenic mice and recapitulates several aspects of biliary pancreatitis.

Promotion of lung tumor growth by interleukin-17.

Recent findings demonstrate that inhaled cigarette smoke, the predominant lung carcinogen, elicits a T helper 17 (Th17) inflammatory phenotype. Interleukin-17A (IL-17), the hallmark cytokine of Th17 inflammation, displays pro- and antitumorigenic properties in a manner that varies according to tumor type and assay system. To investigate the role of IL-17 in lung tumor growth, we used an autochthonous tumor model (K-Ras(LA1) mice) with lung delivery of a recombinant adenovirus that expresses IL-17A. Virus-mediated expression of IL-17A in K-Ras(LA1) mice at 8-10 wk of age doubled lung tumor growth in 3 wk relative to littermates that received a green fluorescent protein-expressing control adenovirus. IL-17 induced matrix metalloproteinase-9 (MMP-9) expression in vivo and in vitro. In accord with this finding, selective and specific inhibitors of MMP-9 repressed the increased motility and invasiveness of IL-17-treated lung tumor cells in culture. Knockdown or mutation of p53 promoted the motility of murine lung tumor cells and abrogated the promigratory role of IL-17. Coexpression of siRNA-resistant wild-type, but not mutant, human p53 rescued both IL-17-mediated migration and MMP-9 mRNA induction in p53 knockdown lung tumor cells. IL-17 increased MMP-9 mRNA stability by reducing interaction with the mRNA destabilizing serine/arginine-rich splicing factor 1 (SRSF1). Taken together, our results indicate that IL-17 stimulates lung tumor growth and regulates MMP-9 mRNA levels in a p53- and SRSF1-dependent manner.

Acute alcohol intoxication impairs methicillin-resistant Staphylococcus aureus clearance in the lung by impeding epithelial production of Reg3γ.

The incidence of community-associated methicillin-resistant Staphylococcus aureus (MRSA) pneumonia in previously healthy individuals has increased in the past 5 years. Such infections are associated with bronchiectasis and high mortality rates, making them a significant public health concern. The mechanisms of host defense against this pathogen are not well characterized. However, patients diagnosed with MRSA, as opposed to methicillin-susceptible S. aureus (MSSA), are more likely to have abused alcohol in the past, and these patients are more likely to die from sepsis. In the United States, USA300 is the predominant strain that causes necrotizing pneumonia. To investigate whether acute ethanol exacerbates MRSA pneumonia, mice were intraperitoneally (i.p.) administered 2 or 4 g/kg of ethanol 30 min prior to oropharyngeal inoculation of 2 × 10(7) CFU of USA300. An increased pulmonary bacterial burden was observed in alcohol-intoxicated mice at 16 and 24 h and was associated with decreased levels of interleukin 6 (IL-6). IL-6 activates signal transducer and activator of transcription 3 (STAT3) as part of an acute-phase response of infection. Reg3γ is an antimicrobial C-type lectin that is induced by STAT3 signaling in response to Gram-positive bacteria. Previously, in situ hybridization studies showed that Reg3g is highly expressed in lung epithelium. In the present study, we found that acute ethanol exacerbated USA300 in a murine model of USA300 pneumonia. This was associated with reduced IL-6 expression in vivo as well as inhibition of IL-6 induction of STAT3 signaling and Reg3g expression in mouse lung epithelial (MLE12) cells in vitro. Furthermore, recombinant Reg3γ administration 4 h after MRSA infection in alcohol-intoxicated mice rescued USA300 clearance in vivo. Therefore, acute alcohol intoxication leads to decreased MRSA clearance in part by inhibiting IL-6/STAT3 induction of the antimicrobial protein Reg3γ in the pulmonary epithelium.

IL-22 is essential for lung epithelial repair following influenza infection.

Influenza infection is widespread in the United States and the world. Despite low mortality rates due to infection, morbidity is common and little is known about the molecular events involved in recovery. Influenza infection results in persistent distal lung remodeling, and the mechanism(s) involved are poorly understood. Recently IL-22 has been found to mediate epithelial repair. We propose that IL-22 is critical for recovery of normal lung function and architecture after influenza infection. Wild-type and IL-22(-/-) mice were infected with influenza A PR8/34 H1N1 and were followed up for up to 21 days post infection. IL-22 receptor was localized to the airway epithelium in naive mice but was expressed at the sites of parenchymal lung remodeling induced by influenza infection. IL-22(-/-) mice displayed exacerbated lung injury compared with wild-type mice, which correlated with decreased lung function 21 days post infection. Epithelial metaplasia was observed in wild-type mice but was not evident in IL-22(-/-) animals that were characterized with an increased fibrotic phenotype. Gene expression analysis revealed aberrant expression of epithelial genes involved in repair processes, among changes in several other biological processes. These data indicate that IL-22 is required for normal lung repair after influenza infection. IL-22 represents a novel pathway involved in interstitial lung disease.

Innate Stat3-mediated induction of the antimicrobial protein Reg3γ is required for host defense against MRSA pneumonia.

Pulmonary Staphylococcus aureus (SA) infections are a public health concern and a major complication of hyper-IgE syndrome, caused by mutations in STAT3. In contrast to previous findings of skin infection, we observed that clearance of SA from the lung did not require T, B, or NK cells but did require Stat3 activation. Immunohistochemistry showed robust Stat3 phosphorylation in the lung epithelium. We identified that a critical Stat3 target gene in lung epithelium is Reg3g (regenerating islet-derived 3 γ), a gene which is highly expressed in gastrointestinal epithelium but whose role in pulmonary host defense is uncharacterized. Stat3 regulated Reg3g transcription through direct binding at the Reg3g promoter region. Recombinant Reg3γ bound to SA and had both bacteriostatic and bactericidal activity in a dose-dependent fashion. Stat3 inhibition in vivo reduced Reg3g transcripts in the lung, and more importantly, recombinant Reg3γ rescued mice from defective SA clearance. These findings reveal an antibacterial function for lung epithelium through Stat3-mediated induction of Reg3γ.

Patients with cystic fibrosis have inducible IL-17+IL-22+ memory cells in lung draining lymph nodes.

BACKGROUND: IL-17 is an important cytokine signature of the TH differentiation pathway TH17. This T-cell subset is crucial in mediating autoimmune disease or antimicrobial immunity in animal models, but its presence and role in human disease remain to be completely characterized. OBJECTIVE: We set out to determine the frequency of TH17 cells in patients with cystic fibrosis (CF), a disease in which there is recurrent infection with known pathogens. METHODS: Explanted lungs from patients undergoing transplantation or organ donors (CF samples=18; non-CF, nonbronchiectatic samples=10) were collected. Hilar nodes and parenchymal lung tissue were processed and examined for TH17 signature by using immunofluorescence and quantitative real-time PCR. T cells were isolated and stimulated with antigens from Pseudomonas aeruginosa and Aspergillus species. Cytokine profiles and staining with flow cytometry were used to assess the reactivity of these cells to antigen stimulation. RESULTS: We found a strong IL-17 phenotype in patients with CF compared with that seen in control subjects without CF. Within this tissue, we found pathogenic antigen-responsive CD4+IL-17+ cells. There were double-positive IL-17+IL-22+ cells [TH17(22)], and the IL-22+ population had a higher proportion of memory characteristics. Antigen-specific TH17 responses were stronger in the draining lymph nodes compared with those seen in matched parenchymal lungs. CONCLUSION: Inducible proliferation of TH17(22) with memory cell characteristics is seen in the lungs of patients with CF. The function of these individual subpopulations will require further study regarding their development. T cells are likely not the exclusive producers of IL-17 and IL-22, and this will require further characterization.

Cutting edge: IL-6 is a marker of inflammation with no direct role in inflammasome-mediated mouse models.

IL-6 is a known downstream target of IL-1ß and is consistently increased in serum from patients with NLRP3 inflammasome-mediated conditions. Therefore, IL-6 could be a therapeutic target in the treatment of IL-1ß-provoked inflammation. IL-6 was increased in serum with accompanying neutrophilia in tissues of an inducible mouse model of Muckle-Wells syndrome. However, an IL-6-null background failed to provide phenotypic rescue and did not significantly impact inflammatory cytokine levels. In a second model of IL-1ß-driven inflammation, NLRP3 activation by monosodium urate crystals similarly increased IL-6. Consistent with our Muckle-Wells syndrome model, ablation of IL-6 did not impact an acute neutrophilic response in this in vivo evaluation of gouty arthritis. Taken together, our results indicate that IL-6 is a reliable marker of inflammation, with no direct role in inflammasome-mediated disease.

IL-17RA is required for CCL2 expression, macrophage recruitment, and emphysema in response to cigarette smoke.

Chronic Obstructive Pulmonary Disease (COPD) is characterized by airspace enlargement and peribronchial lymphoid follicles; however, the immunological mechanisms leading to these pathologic changes remain undefined. Here we show that cigarette smoke is a selective adjuvant that augments in vitro and in vivo Th17, but not Th1, cell differentiation via the aryl hydrocarbon receptor. Smoke exposed IL-17RA(-/-) mice failed to induce CCL2 and MMP12 compared to WT mice. Remarkably, in contrast to WT mice, IL-17RA(-/-) mice failed to develop emphysema after 6 months of cigarette smoke exposure. Taken together, these data demonstrate that cigarette smoke is a potent Th17 adjuvant and that IL-17RA signaling is required for chemokine expression necessary for MMP12 induction and tissue emphysema.

γδ T cells attenuate bleomycin-induced fibrosis through the production of CXCL10.

γδ T cells are a subset of T cells associated with epithelial mucosal tissues and play a prominent role in both promoting and dampening inflammatory responses to pathogens; in addition, they strongly mediate epithelial repair. By using a bleomycin model of pulmonary fibrosis, we found that γδ T-cell populations dramatically increased after bleomycin administration. To determine the importance of these cells, we exposed mice lacking the δ chain of the γδ T-cell receptor (γδ knockout [KO]) to bleomycin. Pulmonary fibrosis was more severe in γδ KO mice, as measured by collagen deposition (hydroxyproline) and histopathological features. Furthermore, there was no evidence of resolution of the fibrotic response up to 45 days after bleomycin therapy. In contrast to control mice, γδ KO mice had decreased concentrations of IL-6, granulocyte colony stimulating factor, chemokine CXC ligand (CXCL) 1, and interferon inducible protein 10/CXCL10. In vitro culture of γδ T cells purified from lungs 17 days after bleomycin exposure (a time of peak influx of these cells) demonstrated that γδ T cells produced substantial quantities of all four of these cytokines, suggesting that γδ T cells are a predominant source of these proteins. To demonstrate that γδ T cells are effector cells in the fibrotic response, we performed adoptive transfer experiments with γδ T cells sorted from bleomycin-treated lungs; these cells were sufficient to resolve fibrosis in γδ KO mice and restore CXCL10 levels comparable to wild-type mice. Furthermore, overexpression of CXCL10 in the lung decreased the severity of fibrosis seen in the γδ KO mice. Finally, adoptive transfer of γδ T cells from CXCL10(-/-) mice failed to reverse the severe fibrosis in γδ KO mice. These results indicate that γδ T cells promote the resolution of fibrosis through the production of CXCL10.

Small interfering RNAs (siRNAs) targeting TGF-beta1 mRNA suppress asbestos-induced expression of TGF-beta1 and CTGF in fibroblasts.

Interstitial lung disease (ILD) afflicts millions of people worldwide. ILD can be caused by a number of agents, including inhaled asbestos, and may ultimately result in respiratory failure and death. Currently, there are no effective treatments for ILD. Transforming growth factor-beta1 (TGF-beta1) is thought to play an important role in the development of pulmonary fibrosis, and asbestos has been shown to induce TGF-beta1 expression in a murine model of ILD. To better define the role of TGF-beta1 in ILD, we developed several small interfering RNAs (siRNAs) that target TGF-beta1 mRNA for degradation. To assess the efficacy of each siRNA in reducing asbestos-induced TGF-beta1 expression, Swiss 3T3 fibroblasts were transfected with TGF-beta1 siRNAs and then treated with chrysotile asbestos for 48 h. Two independent siRNAs targeting TGF-beta1 mRNA knocked-down asbestos-induced expression of TGF-beta1 mRNA by 72-89% and protein by 70-84%. Interestingly, siRNA knockdown of TGF-beta1 also reduced asbestos-induced expression of connective tissue growth factor (CTGF). CTGF can be upregulated by TGF-beta1 and appears to play an important role in the development of pulmonary fibrosis. These results suggest that siRNAs could be effective in preventing or possibly arresting the progression of pulmonary fibrosis. Studies are underway in vivo to test this postulate.

Critical role of IL-17RA in immunopathology of influenza infection.

Acute lung injury due to influenza infection is associated with high mortality, an increase in neutrophils in the airspace, and increases in tissue myeloperoxidase (MPO). Because IL-17A and IL-17F, ligands for IL-17 receptor antagonist (IL-17RA), have been shown to mediate neutrophil migration into the lung in response to LPS or Gram-negative bacterial pneumonia, we hypothesized that IL-17RA signaling was critical for acute lung injury in response to pulmonary influenza infection. IL-17RA was critical for weight loss and both neutrophil migration and increases in tissue myeloperoxidase (MPO) after influenza infection. However, IL-17RA was dispensable for the recruitment of CD8(+) T cells specific for influenza hemagglutinin or nucleocapsid protein. Consistent with this, IL-17RA was not required for viral clearance. However, in the setting of influenza infection, IL-17RA(-/-) mice showed significantly reduced levels of oxidized phospholipids, which have previously been shown to be an important mediator in several models of acute lung injury, including influenza infection and gastric acid aspiration. Taken together, these data support targeting IL-17 or IL-17RA in acute lung injury due to acute viral infection.