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.

Nrf2 through Aryl Hydrocarbon Receptor Regulates IL-22 Response in CD4+ T Cells.

IL-17A and IL-22 derived from Th17 cells play a significant role in mucosal immunity and inflammation. TGF-ß and IL-6 promote Th17 differentiation; however, these cytokines have multiple targets. The identification and screening of additional molecules that regulate IL-17A and IL-22 responses in certain inflammatory conditions is of great clinical significance. In this study, we show that CDDO-Im, a specific Nrf2 activator, promotes IL-17A and IL-22 responses in murine Th17 cells. In contrast, CDDO-Im inhibits IL-17A response in multiple sclerosis patient-derived PBMCs. However, Nrf2 specifically regulates IL-22 response in vivo. Nrf2 acts through the regulation of antioxidant response element (ARE) binding motifs in target genes to induce or repress transcription. Promoter analysis revealed that Il17a, Rorc, and Ahr genes have several ARE motifs. We showed that Nrf2 bound to ARE repressor (ARE-R2) of Rorc and inhibited Rorc-dependent IL-17A transactivation. The luciferase reporter assay data showed that CDDO-Im regulated Ahr promoter activity. Chromatin immunoprecipitation quantitative PCR data showed that Nrf2 bound to ARE of AhR. Finally, we confirmed that the CDDO-Im-mediated induction of IL-22 production in CD4+ T cells was abrogated in CD4-specific Ahr knockout mice (AhrCD4 ). CH-223191, a specific AhR antagonist, inhibits CDDO-Im-induced IL-22 production in CD4+ T cells, which further confirmed the AhR-dependent regulation. Collectively, our data showed that Nrf2 via AhR pathways regulated IL-22 response in CD4+ T cells.

A Comparison of Growth Factors and Cytokines in Fresh Frozen Plasma and Never Frozen Plasma.

BACKGROUND: Fresh frozen plasma (FFP) contains proinflammatory mediators released from cellular debris during frozen storage. In addition, recent studies have shown that transfusion of never-frozen plasma (NFP), instead of FFP, may be superior in trauma patients. We hypothesized that FFP would have higher levels of inflammatory mediators when compared to NFP. MATERIALS AND METHODS: FFP (n = 8) and NFP (n = 8) samples were obtained from an urban, level 1 trauma center blood bank. The cytokines in these samples were compared using a Milliplex (Milliplex Sigma) human cytokine magnetic bead panel multiplex assay for 41 different biomarkers. RESULTS: Growth factors that were higher in NFP included platelet-derived growth factor-AA (PDGF-AA; 8.09 versus 108.00 pg/mL, P < 0.001) and PDGF-AB (0.00 versus 215.20, P= 0.004). Soluble CD40-ligand (sCD40L), a platelet activator and pro-coagulant, was higher in NFP (31.81 versus 80.45 pg/mL, P< 0.001). RANTES, a leukocyte chemotactic cytokine was higher in NFP (26.19 versus 1418.00 pg/mL, P< 0.001). Interleukin-4 (5.70 versus 0.00 pg/mL, P= 0.03) and IL-8 (2.20 versus 0.52 pg/ml, P= 0.03) levels were higher in were higher in FFP. CONCLUSIONS: Frozen storage of plasma may result in decrease of several growth factors and/or pro-coagulants found in NFP. In addition, the freezing and thawing process may induce release of pro-inflammatory chemokines. Further studies are needed to determine if these cytokines result in improved outcomes with NFP over FFP in transfusion of trauma patients.

The role of the innate immune system on pulmonary infections.

Inhalation is required for respiration and life in all vertebrates. This process is not without risk, as it potentially exposes the host to environmental pathogens with every breath. This makes the upper respiratory tract one of the most common routes of infection and one of the leading causes of morbidity and mortality in the world. To combat this, the lung relies on the innate immune defenses. In contrast to the adaptive immune system, the innate immune system does not require sensitization, previous exposure or priming to attack foreign particles. In the lung, the innate immune response starts with the epithelial barrier and mucus production and is reinforced by phagocytic cells and T cells. These cells are vital for the production of cytokines, chemokines and anti-microbial peptides that are critical for clearance of infectious agents. In this review, we discuss all aspects of the innate immune response, with a special emphasis on ways to target aspects of the immune response to combat antibiotic resistant bacteria.

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.

Interleukin-17 is required for T helper 1 cell immunity and host resistance to the intracellular pathogen Francisella tularensis.

The importance of T helper type 1 (Th1) cell immunity in host resistance to the intracellular bacterium Francisella tularensis is well established. However, the relative roles of interleukin (IL)-12-Th1 and IL-23-Th17 cell responses in immunity to F. tularensis have not been studied. The IL-23-Th17 cell pathway is critical for protective immunity against extracellular bacterial infections. In contrast, the IL-23-Th17 cell pathway is dispensable for protection against intracellular pathogens such as Mycobacteria. Here we show that the IL-23-Th17 pathway regulates the IL-12-Th1 cell pathway and was required for protective immunity against F.tularensis live vaccine strain. We show that IL-17A, but not IL-17F or IL-22, induced IL-12 production in dendritic cells and mediated Th1 responses. Furthermore, we show that IL-17A also induced IL-12 and interferon-gamma production in macrophages and mediated bacterial killing. Together, these findings illustrate a biological function for IL-17A in regulating IL-12-Th1 cell immunity and host responses to an intracellular pathogen.

Pulmonary Th17 Antifungal Immunity Is Regulated by the Gut Microbiome.

Commensal microbiota are critical for the development of local immune responses. In this article, we show that gut microbiota can regulate CD4 T cell polarization during pulmonary fungal infections. Vancomycin drinking water significantly decreased lung Th17 cell numbers during acute infection, demonstrating that Gram-positive commensals contribute to systemic inflammation. We next tested a role for RegIIIγ, an IL-22-inducible antimicrobial protein with specificity for Gram-positive bacteria. Following infection, increased accumulation of Th17 cells in the lungs of RegIIIγ(-/-) and Il22(-/-) mice was associated with intestinal segmented filamentous bacteria (SFB) colonization. Although gastrointestinal delivery of rRegIIIγ decreased lung inflammatory gene expression and protected Il22(-/-) mice from weight loss during infection, it had no direct effect on SFB colonization, fungal clearance, or lung Th17 immunity. We further show that vancomycin only decreased lung IL-17 production in mice colonized with SFB. To determine the link between gut microbiota and lung immunity, serum-transfer experiments revealed that IL-1R ligands increase the accumulation of lung Th17 cells. These data suggest that intestinal microbiota, including SFB, can regulate pulmonary adaptive immune responses.

A Novel CD4+ T Cell-Dependent Murine Model of Pneumocystis-driven Asthma-like Pathology.

RATIONALE: Infection with Pneumocystis, an opportunistic fungal pathogen, can result in fulminant pneumonia in the clinical setting of patients with immunosuppression. In murine models, Pneumocystis has previously been shown to induce a CD4+ T cell-dependent eosinophilic response in the lung capable of providing protection. OBJECTIVES: We sought to explore the role of Pneumocystis in generating asthma-like lung pathology, given the natural eosinophilic response to infection. METHODS: Pneumocystis infection or antigen treatment was used to induce asthma-like pathology in wild-type mice. The roles of CD4+ T cells and eosinophils were examined using antibody depletion and knockout mice, respectively. The presence of anti-Pneumocystis antibodies in human serum samples was detected by ELISA and Western blotting. MEASUREMENTS AND MAIN RESULTS: Pneumocystis infection generates a strong type II response in the lung that requires CD4+ T cells. Pneumocystis infection was capable of priming a Th2 response similar to that of a commonly studied airway allergen, the house dust mite. Pneumocystis antigen treatment was also capable of inducing allergic inflammation in the lung, resulting in anti-Pneumocystis IgE production, goblet cell hyperplasia, and increased airway resistance. In the human population, patients with severe asthma had increased levels of anti-Pneumocystis IgG and IgE compared with healthy control subjects. Patients with severe asthma with elevated anti-Pneumocystis IgG levels had worsened symptom scores and lung parameters such as decreased forced expiratory volume and increased residual volume compared with patients with severe asthma who had low anti-Pneumocystis IgG. CONCLUSIONS: The present study demonstrates for the first time, to our knowledge, that Pneumocystis is an airway allergen capable of inducing asthma-like lung pathology.

Therapeutic Role of Interleukin 22 in Experimental Intra-abdominal Klebsiella pneumoniae Infection in Mice.

Interleukin 22 (IL-22) is an IL-10-related cytokine produced by T helper 17 (Th17) cells and other immune cells that signals via IL-22 receptor alpha 1 (IL-22Ra1), which is expressed on epithelial tissues, as well as hepatocytes. IL-22 has been shown to have hepatoprotective effects that are mediated by signal transducer and activator of transcription 3 (STAT3) signaling. However, it is unclear whether IL-22 can directly regulate antimicrobial programs in the liver. To test this hypothesis, hepatocyte-specific IL-22Ra1 knockout (Il22Ra1(Hep-/-)) and Stat3 knockout (Stat3(Hep-/-)) mice were generated and subjected to intra-abdominal infection with Klebsiella pneumoniae, which results in liver injury and necrosis. We found that overexpression of IL-22 or therapeutic administration of recombinant IL-22 (rIL-22), given 2 h postinfection, significantly reduced the bacterial burden in both the liver and spleen. The antimicrobial activity of rIL-22 required hepatic Il22Ra1 and Stat3. Serum from rIL-22-treated mice showed potent bacteriostatic activity against K. pneumoniae, which was dependent on lipocalin 2 (LCN2). However, in vivo, rIL-22-induced antimicrobial activity was only partially reduced in LCN2-deficient mice. We found that rIL-22 also induced serum amyloid A2 (SAA2) and that SAA2 had anti-K. pneumoniae bactericidal activity in vitro. These results demonstrate that IL-22, through IL-22Ra1 and STAT3 singling, can induce intrinsic antimicrobial activity in the liver, which is due in part to LCN2 and SAA2. Therefore, IL-22 may be a useful adjunct in treating hepatic and intra-abdominal infections.

Mucosal pre-exposure to Th17-inducing adjuvants exacerbates pathology after influenza infection.

Mucosal vaccines are thought to confer superior protection against mucosal infectious diseases. In addition, mucosal routes of vaccine delivery preferentially induce the generation of T helper 17 (Th17) cells, which produce the cytokine IL-17. Th17 cells are critical in mediating vaccine-induced immunity against several mucosal infectious diseases. However, IL-17 is also a potent proinflammatory cytokine, and we recently showed that IL-17 mediates immunopathology and lung injury after influenza infection in mice. In the present study, we tested the hypothesis that mucosal pre-exposure to Th17-inducing adjuvants can promote disease exacerbation upon subsequent infection with influenza virus. Mice mucosally pre-exposed to Th17-inducing adjuvants, such as type II heat-labile enterotoxin or cholera toxin, resulted in increased morbidity and exacerbated lung inflammation upon subsequent infection with influenza virus. Furthermore, the increased morbidity was accompanied by increased expression of inflammatory chemokines and increased accumulation of neutrophils. Importantly, blockade of the IL-17 pathway in mice pre-exposed to Th17-inducing adjuvants resulted in attenuation of the inflammatory phenotype seen in influenza-infected mice. Our findings indicate that, before mucosal Th17-inducing adjuvants can be used in vaccine strategies, the short- and long-term detrimental effects of such adjuvants on disease exacerbation and lung injury in response to infections, such as influenza, should be carefully studied.

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.

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.

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.

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.

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.

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.

Dimethyl sulfoxide as a novel therapy in a murine model of acute lung injury.

INTRODUCTION: The endothelial glycocalyx 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 endothelial glycocalyx. We hypothesized that DMSO would protect against protein leak by preserving the endothelial glycocalyx in a murine model of acute respiratory distress syndrome (ARDS). METHODS: C57BL/6 mice were given ARDS via intratracheally administered lipopolysaccharide (LPS). Dimethyl sulfoxide (220 mg/kg) was administered intravenously for 4 days. Animals were sacrificed postinjury day 4 after bronchoalveolar lavage (BAL). Bronchoalveolar lavage cell counts and protein content were quantified. Lung sections were stained with fluorescein isothiocyanate-labeled wheat germ agglutinin to quantify the endothelial glycocalyx. Human umbilical vein endothelial cells (HUVECs) were exposed to LPS. Endothelial glycocalyx was measured using fluorescein isothiocyanate-labeled wheat germ agglutinin, and co-immunoprecipitation was performed to measure interaction between sheddases and syndecan-1. RESULTS: Dimethyl sulfoxide treatment resulted in greater endothelial glycocalyx staining intensity in the lung when compared with sham (9,641 vs. 36,659 arbitrary units, p < 0.001). Total BAL cell counts were less for animals receiving DMSO (6.93 × 10 6 vs. 2.49 × 10 6 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 µg/µL, p = 0.02). Dimethyl sulfoxide prevented LPS-induced endothelial glycocalyx loss in HUVECs and reduced the interaction between matrix metalloproteinase 16 and syndecan-1. CONCLUSION: Systemically administered DMSO protects the endothelial glycocalyx in the pulmonary vasculature, mitigating pulmonary capillary leak after acute lung injury. Dimethyl sulfoxide also results in decreased inflammatory response. Dimethyl sulfoxide reduced the interaction between matrix metalloproteinase 16 and syndecan-1 and prevented LPS-induced glycocalyx damage in HUVECs. Dimethyl sulfoxide may be a novel therapeutic for ARDS.

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.

COVID-19 and influenza infections mediate distinct pulmonary cellular and transcriptomic changes.

SARS-CoV-2 infection can cause persistent respiratory sequelae. However, the underlying mechanisms remain unclear. Here we report that sub-lethally infected K18-human ACE2 mice show patchy pneumonia associated with histiocytic inflammation and collagen deposition at 21 and 45 days post infection (DPI). Transcriptomic analyses revealed that compared to influenza-infected mice, SARS-CoV-2-infected mice had reduced interferon-gamma/alpha responses at 4 DPI and failed to induce keratin 5 (Krt5) at 6 DPI in lung, a marker of nascent pulmonary progenitor cells. Histologically, influenza- but not SARS-CoV-2-infected mice showed extensive Krt5+ "pods" structure co-stained with stem cell markers Trp63/NGFR proliferated in the pulmonary consolidation area at both 7 and 14 DPI, with regression at 21 DPI. These Krt5+ "pods" structures were not observed in the lungs of SARS-CoV-2-infected humans or nonhuman primates. These results suggest that SARS-CoV-2 infection fails to induce nascent Krt5+ cell proliferation in consolidated regions, leading to incomplete repair of the injured lung.