Proteomic Profiling of Hypoplastic Lungs Suggests an Underlying Inflammatory Response in the Pathogenesis of Abnormal Lung Development in Congenital Diaphragmatic Hernia.

The pathogenesis of lung hypoplasia in congenital diaphragmatic hernia (CDH), a common birth defect, is poorly understood. The diaphragmatic defect can be repaired surgically, but the abnormal lung development contributes to a high mortality in these patients. To understand the underlying pathobiology, we compared the proteomic profiles of fetal rat lungs at the alveolar stage (E21) that were either exposed to nitrofen in utero (CDH lungs, n=5) or exposed to vehicle only (non-CDH control lungs, n=5). Pathway analysis of proteomic datasets showed significant enrichment in inflammatory response proteins associated with cytokine signaling and Epstein Barr Virus in nitrofen CDH lungs. Among the 218 significantly altered proteins between CDH and non-CDH control lungs were Tenascin C, CREBBP, LYN, and STAT3. We showed that Tenascin C was decreased around the distal airway branches in nitrofen rat lungs and human CDH lungs, obtained from stillborn fetuses that did not receive pre- or postnatal treatment. In contrast, STAT3 was significantly increased in the airway epithelium of nitrofen lungs at E21. STAT3 inhibition after direct nitrofen exposure to fetal rat lung explants (E14.5) partially rescued the hypoplastic lung phenotype ex vivo by increasing peripheral lung budding. Moreover, we demonstrated that several STAT3-associated cytokines (IL-15, IL-9, andIL-2) are increased in fetal tracheal aspirates of CDH survivors compared with nonsurvivors after fetoscopic endoluminal tracheal occlusion. With our unbiased proteomics approach, we showed for the first time that downstream inflammatory processes are likely involved in the pathogenesis of abnormal lung development in CDH.

IFNγ-mediated IL-33 production is dependent on the aryl hydrocarbon receptor in human bronchial epithelial cells.

IL-33 is an alarmin produced by stromal cells and is known to promote airway inflammation. IL-33 is a critical mediator of steroid-unresponsiveness in severe asthma. We have previously shown that IFNγ, a cytokine known to be elevated in airway inflammation and severe asthma, enhances the abundance of IL-33 in bronchial epithelial cells. Previous studies have shown that environmental insults such as particulate matter results in activation of the aryl hydrocarbon receptor (AhR) and IL-33 production. However, the role of AhR in cytokine-mediated IL-33 production is unknown. In this study, we demonstrate that the knockdown of AhR results in significant decrease in IFNγ-mediated IL-33 production and phosphorylation of STAT1 (Y701), in human bronchial epithelial cells. The findings of this report suggest that AhR may be an essential component in IFNγ-mediated IL-33 production in the lungs.

Defining the effects of traffic-related air pollution on the human plasma proteome using an aptamer proteomic array: A dose-dependent increase in atherosclerosis-related proteins.

BACKGROUND: Traffic-related air pollution (TRAP) is a critical risk factor and major contributor to respiratory and cardiovascular disease (CVD). The effects of TRAP beyond the lungs can be related to changes in circulatory proteins. However, such TRAP-mediated changes have not been defined in an unbiased manner using a controlled human model. OBJECTIVE: To detail global protein changes (the proteome) in plasma following exposure to inhaled diesel exhaust (DE), a paradigm of TRAP, using controlled human exposures. METHODS: In one protocol, ex-smokers and never-smokers were exposed to filtered air (FA) and DE (300 µg PM2.5/m3), on order-randomized days, for 2 h. In a second protocol, independent never-smoking participants were exposed to lower concentrations of DE (20, 50 or 150 µg PM2.5/m3) and FA, for 4 h, on order-randomized days. Each exposure was separated by 4 weeks of washout. Plasma samples obtained 24 h post-exposure from ex-smokers (n = 6) were first probed using Slow off-rate modified aptamer proteomic array. Plasma from never-smokers (n = 11) was used for independent assessment of proteins selected from the proteomics study by immunoblotting. RESULTS: Proteomics analyses revealed that DE significantly altered 342 proteins in plasma of ex-smokers (n = 6). The top 20 proteins therein were primarily associated with inflammation and CVD. Plasma from never-smokers (n = 11) was used for independent assessment of 6 proteins, amongst the top 10 proteins increased by DE in the proteomics study, for immunoblotting. The abundance of all six proteins (fractalkine, apolipoproteins (APOB and APOM), IL18R1, MIP-3 and MMP-12) was significantly increased by DE in plasma of these never-smokers. DE-mediated increase was shown to be concentration-dependent for fractalkine, APOB and MMP-12, all biomarkers of atherosclerosis, which correlated with plasma levels of IL-6, a subclinical marker of CVD, in independent participants. CONCLUSION: This investigation details changes in the human plasma proteome due to TRAP. We identify specific atherosclerosis-related proteins that increase concentration-dependently across a range of TRAP levels applicable worldwide.

The Small RNAs PA2952.1 and PrrH as Regulators of Virulence, Motility, and Iron Metabolism in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that undergoes swarming motility in response to semisolid conditions with amino acids as a nitrogen source. With a genome encoding hundreds of potential intergenic small RNAs (sRNAs), P. aeruginosa can easily adapt to different conditions and stresses. We previously identified 20 sRNAs that were differentially expressed (DE) under swarming conditions. Here, these sRNAs were overexpressed in strain PAO1 and were subjected to an array of phenotypic screens. Overexpression of the PrrH sRNA resulted in decreased swimming motility, whereas a ΔprrH mutant had decreased cytotoxicity and increased pyoverdine production. Overexpression of the previously uncharacterized PA2952.1 sRNA resulted in decreased swarming and swimming motilities, increased gentamicin and tobramycin resistance under swarming conditions, and increased trimethoprim susceptibility. Transcriptome sequencing (RNA-Seq) and proteomic analysis were performed on the wild type (WT) overexpressing PA2952.1 compared to the empty vector control under swarming conditions, and these revealed the differential expression (absolute fold change [FC] ≥ 1.5) of 784 genes and the differential abundance (absolute FC ≥ 1.25) of 59 proteins. Among these were found 73 transcriptional regulators, two-component systems, and sigma and anti-sigma factors. Downstream effectors included downregulated pilus and flagellar genes, the upregulated efflux pump MexGHI-OpmD, and the upregulated arn operon. Genes involved in iron and zinc uptake were generally upregulated, and certain pyoverdine genes were upregulated. Overall, the sRNAs PA2952.1 and PrrH appeared to be involved in regulating virulence-related programs in P. aeruginosa, including iron acquisition and motility.IMPORTANCE Due to the rising incidence of multidrug-resistant (MDR) strains and the difficulty of eliminating P. aeruginosa infections, it is important to understand the regulatory mechanisms that allow this bacterium to adapt to and thrive under a variety of conditions. Small RNAs (sRNAs) are one regulatory mechanism that allows bacteria to change the amount of protein synthesized. In this study, we overexpressed 20 different sRNAs in order to investigate how this might affect different bacterial behaviors. We found that one of the sRNAs, PrrH, played a role in swimming motility and virulence phenotypes, indicating a potentially important role in clinical infections. Another sRNA, PA2952.1, affected other clinically relevant phenotypes, including motility and antibiotic resistance. RNA-Seq and proteomics of the strain overexpressing PA2952.1 revealed the differential expression of 784 genes and 59 proteins, with a total of 73 regulatory factors. This substantial dysregulation indicates an important role for the sRNA PA2952.1.

The importance of reporting house dust mite endotoxin abundance: impact on the lung transcriptome.

The abundance of lipopolysaccharide (LPS) in house dust mite (HDM) preparations is broad and mirrors the variability seen in the homes of people with asthma. LPS in commercially available stocks ranges from 31 to 5,2000 endotoxin units. The influence of vastly different LPS loads on the mechanisms that define the immune and inflammatory phenotype of HDM-challenged mice has not been defined. This aim of the study was to understand the lung phenotype of mice challenged with HDM extract containing high or low levels of LPS. Female BALB/c mice were sensitized for 2 wk with commercial HDM extract containing either high (36,000 endotoxin units; HHDM) or low (615 endotoxin units; LHDM) levels of LPS. Lung phenotype was characterized by measuring lung function, total and differential cell counts, cytokine abundance, and the lung transcriptome by RNA-sequencing. LPS levels in HDM stocks used for preclinical asthma research in mice remain poorly reported. In 2019, only 14% of papers specified LPS concentration in HDM lots. Specific differences existed in airway responsiveness between mice challenged with HHDM or LHDM. HHDM- and LHDM-induced cytokine profiles of bronchial lavage were significantly different and the lung transcriptome was differentially enriched for genes involved in DNA damage repair or cilium movement, following HHDM or LHDM challenge, respectively. The abundance of LPS in commercially available HDM influences the phenotype of allergic airways inflammation in mice. Failure to report the level of LPS in HDM extracts used in animal models of airway disease will lead to inconsistency in reproducibility and reliability of published data.

Antimicrobial Host Defence Peptides: Immunomodulatory Functions and Translational Prospects.

Cationic host defence peptides (CHDPs), also known as antimicrobial peptides, exhibit a wide range of activities contributing to immune responses and resolution of infections. CHDPs are expressed across diverse species, are generally amphipathic with less than 50 amino acids in length, and differ significantly in sequence and structure. This chapter focuses on the role of these peptides in immunity. CHDPs are known to function in both innate and adaptive immune responses. These peptides exert both pro- and anti-inflammatory properties, which are likely context dependent based on cell and tissue type, concentration of the peptides, and its interaction with other factors in the microenvironment. Furthermore, the crosstalk between CHDPs and the microbiome and how this may influence mucosal immunity is a rapidly emerging field of research. Overall, the immunomodulatory functions of CHDPs play an important role in the control of infections, regulation of inflammation, and maintaining immune homeostasis. It is thus not surprising that dysregulation of expression of CHDPs is implicated in the susceptibility, pathology, and progression of various diseases. In this chapter, we summarize the immunomodulatory functions of CHDPs, its clinical relevance, and the translational opportunities that these peptides provide for the development of new therapies.

Immunomodulatory innate defence regulator (IDR) peptide alleviates airway inflammation and hyper-responsiveness.

BACKGROUND: Exacerbation in asthma is associated with decreased expression of specific host defence peptides (HDPs) in the lungs. We examined the effects of a synthetic derivative of HDP, innate defence regulator (IDR) peptide IDR-1002, in house dust mite (HDM)-challenged murine model of asthma, in interleukin (IL)-33-challenged mice and in human primary bronchial epithelial cells (PBECs). METHODS: IDR-1002 (6 mg/kg per mouse) was administered (subcutaneously) in HDM-challenged and/or IL-33-challenged BALB/c mice. Lung function analysis was performed with increasing dose of methacholine by flexiVent small animal ventilator, cell differentials in bronchoalveolar lavage performed by modified Wright-Giemsa staining, and cytokines monitored by MesoScale Discovery assay and ELISA. PBECs stimulated with tumour necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ), with or without IDR-1002, were analysed by western blots. RESULTS: IDR-1002 blunted HDM challenge-induced airway hyper-responsiveness (AHR), and lung leucocyte accumulation including that of eosinophils and neutrophils, in HDM-challenged mice. Concomitantly, IDR-1002 suppressed HDM-induced IL-33 in the lungs. IFN-γ/TNF-α-induced IL-33 production was abrogated by IDR-1002 in PBECs. Administration of IL-33 in HDM-challenged mice, or challenge with IL-33 alone, mitigated the ability of IDR-1002 to control leucocyte accumulation in the lungs, suggesting that the suppression of IL-33 is essential for the anti-inflammatory activity of IDR-1002. In contrast, the peptide significantly reduced either HDM, IL-33 or HDM+IL-33 co-challenge-induced AHR in vivo. CONCLUSION: This study demonstrates that an immunomodulatory IDR peptide controls the pathophysiology of asthma in a murine model. As IL-33 is implicated in steroid-refractory severe asthma, our findings on the effects of IDR-1002 may contribute to the development of novel therapies for steroid-refractory severe asthma.

Inhaled diesel exhaust alters the allergen-induced bronchial secretome in humans.

Diesel exhaust (DE) is a paradigm for traffic-related air pollution. Human adaptation to DE is poorly understood and currently based on oversimplified models. DE promotes allergic responses, but protein expression changes mediated by this interaction have not been systematically investigated. The aim of this study was to define the effect of inhaled DE on allergen-induced proteins in the lung.We performed a randomised and blinded controlled human crossover exposure study. Participants inhaled filtered air or DE; thereafter, contralateral lung segments were challenged with allergen or saline. Using label-free quantitative proteomics, we comprehensively defined DE-mediated alteration of allergen-driven secreted proteins (secretome) in bronchoalveolar lavage. We further examined expression of proteins selected from the secretome data in independent validation experiments using Western blots, ELISA and immunohistochemistry.We identified protein changes unique to co-exposure (DE+allergen), undetected with mono-exposures (DE or allergen alone). Validation studies confirmed that specific proteins (e.g. the antimicrobial peptide cystatin-SA) were significantly enhanced with DE+allergen compared to either mono-exposure.This study demonstrates that common environmental co-exposures can uniquely alter protein responses in the lungs, illuminating biology that mono-exposures cannot. This study highlights the value of complex human in vivo models in detailing airway responses to inhaled pollution.

Buprenorphine Alters Inflammatory and Oxidative Stress Molecular Markers in Arthritis.

Buprenorphine is recommended for use as an analgesic in animal models including in murine models of collagen-induced arthritis (CIA). However, the effect of buprenorphine on the expression of disease-associated biomarkers is not well defined. We examined the effect of buprenorphine administration on disease progression and the expression of inflammatory and oxidative stress markers, in a murine model of CIA. Buprenorphine administration altered the expression of cytokines, IFN-γ, IL-6, and MMP-3, and oxidative markers, for example, iNOS, superoxide dismutase (SOD1), and catalase (CAT), in the CIA mice. As buprenorphine is an analgesic, we further monitored the association of expression of these biomarkers with pain scores in a human cohort of early rheumatoid arthritis (RA). Serum MMP-3 levels and blood mRNA expression of antioxidants sod1 and cat correlated with pain scores in the RA cohort. We have demonstrated that administration of buprenorphine alters the expression of inflammatory and oxidative stress-related molecular markers in a murine model of CIA. This caveat needs to be considered in animal experiments using buprenorphine as an analgesic, as it can be a confounding factor in murine studies used for prediction of response to therapy. Furthermore, the antioxidant enzymes that showed an association with pain scores in the human cohort may be explored as biomarkers for pain in future studies.

Amphiphilic tobramycins with immunomodulatory properties.

Amphiphilic aminoglycosides (AAGs) are an emerging source of antibacterials to combat infections caused by antibiotic-resistant bacteria. Mode-of-action studies indicate that AAGs predominately target bacterial membranes, thereby leading to depolarization and increased permeability. To assess whether AAGs also induce host-directed immunomodulatory responses, we determined the AAG-dependent induction of cytokines in macrophages in the absence or presence of lipopolysaccharide (LPS). Our results show for the first time that AAGs can boost the innate immune response, specifically the recruitment of immune cells such as neutrophils required for the resolution of infections. Moreover, AAGs can selectively control inflammatory responses induced in the presence of endotoxins to prevent septic shock. In conclusion, our study demonstrates that AAGs possess multifunctional properties that combine direct antibacterial activity with host-directed clearance effects reminiscent of those of host-defense peptides.

Human cathelicidin LL-37 and its derivative IG-19 regulate interleukin-32-induced inflammation.

Human cathelicidin LL-37 protects against infections and endotoxin-induced inflammation. In a recent study we have shown that IG-19, an LL-37-derived peptide, protects in a murine model of arthritis. Cytokine interleukin-32 (IL-32) is elevated and directly associated with the disease severity of inflammatory arthritis. Therefore, in this study we examined the effects of LL-37 and IG-19 on IL-32-induced responses in human peripheral blood-derived mononuclear cells (PBMC) and macrophages. We showed that CD14(+) monocytes are the primary cells that produce pro-inflammatory tumour necrosis factor-α (TNF-α) following stimulation of PBMC with IL-32. We demonstrated that LL-37 and IG-19 significantly suppress IL-32-induced production of pro-inflammatory cytokines, e.g. TNF-α and IL-1ß, without altering chemokine production. In contrast, LL-37 and IG-19 enhance the production of the anti-inflammatory cytokine IL-1RA. Further mechanistic studies revealed that LL-37 and IG-19 suppress IL-32-mediated phosphorylation of Fyn (Y420) Src kinase. In contrast, IL-32-mediated phosphorylation of AKT-1 (T308) and MKP-1 (S359) is not suppressed by the peptides. LL-37 and IG-19 alone induce the phosphorylation of MKP-1 (S359), which is a known negative regulator of inflammation. Furthermore, the peptides induce the activity of p44/42 mitogen-activated protein kinase, which is known to phosphorylate MKP-1 (S359). This is the first study to demonstrate the regulation of IL-32-induced inflammation by LL-37 and its derivative peptide IG-19. The mechanistic results from this study suggest that regulation of immune-mediated inflammation by these peptides may be controlled by the dual phosphatase MKP-1. We speculate that LL-37 and its derivatives may contribute to the control of immune-mediated inflammatory diseases.

Structure-activity relationships in ultrashort cationic lipopeptides: the effects of amino acid ring constraint on antibacterial activity.

Taking a minimalistic approach in efforts to lower the cost for the development of new synthetic antimicrobial peptides, ultrashort cationic lipopeptides were designed to mimic the amphiphilic nature crucial for their activity but with only a very short peptide sequence ligated to a lipidic acid. Nine ultrashort cationic lipopeptides were prepared to study the effects of ring constraint in the amino acid side chain of the peptide component. USCL-PCat1, consisting of only four L-4R-aminoproline residues and acylated with palmitic acid at the N-terminus, was found to populate a polyproline II helical secondary conformation that is stable to different pHs and temperatures using circular dichroism. The synthesized lipopeptides were found to have a micellar structure in water using negative staining transmission electron microscopy. We found that constraining the side chain of the amino acid component is not beneficial to the antimicrobial activity. USCL-Dab1, USCL-Dab3 and USCL-K1 showed promising activity against a panel of laboratory reference and clinically isolated Gram-positive and Gram-negative bacterial strains, some of which are multidrug resistant. No appreciable cytotoxicity against human monocytic THP-1 cells was observed up to concentrations of 20-40 µM for all synthesized compounds. Moreover, all USCLs did not induce the production of either pro-inflammatory cytokines or chemokines up to 40 µM.

Concentration-dependent alterations in the human plasma proteome following controlled exposure to diesel exhaust.

Traffic-related air pollution (TRAP) exposure is associated with systemic health effects, which can be studied using blood-based markers. Although we have previously shown that high TRAP concentrations alter the plasma proteome, the concentration-response relationship between blood proteins and TRAP is unexplored in controlled human exposure studies. We aimed to identify concentration-dependent plasma markers of diesel exhaust (DE), a model of TRAP. Fifteen healthy non-smokers were enrolled into a double-blinded, crossover study where they were exposed to filtered air (FA) and DE at 20, 50 and 150 µg/m3 PM2.5 for 4h, separated by ≥ 4-week washouts. We collected blood at 24h post-exposure and used label-free mass spectrometry to quantify proteins in plasma. Proteins exhibiting a concentration-response, as determined by linear mixed effects models (LMEMs), were assessed for pathway enrichment using WebGestalt. Top candidates, identified by sparse partial least squares discriminant analysis and LMEMs, were confirmed using enzyme-linked immunoassays. Thereafter, we assessed correlations between proteins that showed a DE concentration-response and acute inflammatory endpoints, forced expiratory volume in 1 s (FEV1) and methacholine provocation concentration causing a 20% drop in FEV1 (PC20). DE exposure was associated with concentration-dependent alterations in 45 proteins, which were enriched in complement pathways. Of the 9 proteins selected for confirmatory immunoassays, based on complementary bioinformatic approaches to narrow targets and availability of high-quality assays, complement factor I (CFI) exhibited a significant concentration-dependent decrease (-0.02 µg/mL per µg/m3 of PM2.5, p = 0.04). Comparing to FA at discrete concentrations, CFI trended downward at 50 (-2.14 ± 1.18, p = 0.08) and significantly decreased at 150 µg/m3 PM2.5 (-2.93 ± 1.18, p = 0.02). CFI levels were correlated with FEV1, PC20 and nasal interleukin (IL)-6 and IL-1ß. This study details concentration-dependent alterations in the plasma proteome following DE exposure at concentrations relevant to occupational and community settings. CFI shows a robust concentration-response and association with established measures of airway function and inflammation.

Human Host Defense Peptide LL-37 Suppresses TNFα-Mediated Matrix Metalloproteinases MMP9 and MMP13 in Human Bronchial Epithelial Cells.

INTRODUCTION: TNFα-inducible matrix metalloproteinases play a critical role in the process of airway remodeling in respiratory inflammatory disease including asthma. The cationic host defense peptide LL-37 is elevated in the lungs during airway inflammation. However, the impact of LL-37 on TNFα-driven processes is not well understood. Here, we examined the effect of LL-37 on TNFα-mediated responses in human bronchial epithelial cells (HBECs). METHODS: We used a slow off-rate modified aptamer-based proteomics approach to define the HBEC proteome altered in response to TNFα. Abundance of selected protein candidates and signaling intermediates was examined using immunoassays, ELISA and Western blots, and mRNA abundance was examined by qRT-PCR. RESULTS: Proteomics analysis revealed that 124 proteins were significantly altered, 12 proteins were enhanced by ≥2-fold compared to unstimulated cells, in response to TNFα. MMP9 was the topmost increased protein in response to TNFα, enhanced by ∼10-fold, and MMP13 was increased by ∼3-fold, compared to unstimulated cells. Furthermore, we demonstrated that LL-37 significantly suppressed TNFα-mediated MMP9 and MMP13 in HBEC. Mechanistic data revealed that TNFα-mediated MMP9 and MMP13 production is controlled by SRC kinase and that LL-37 enhances related upstream negative regulators, namely, phospho-AKT (T308) and TNFα-mediated TNFAIP3 or A20. CONCLUSIONS: The findings of this study suggest that LL-37 may play a role in intervening in the process of airway remodeling in chronic inflammatory respiratory disease such as asthma.

Inflammatory cytokines IL-32 and IL-17 have common signaling intermediates despite differential dependence on TNF-receptor 1.

Cytokines IL-32 and IL-17 are emerging as critical players in the pathophysiology of immune-mediated chronic inflammatory diseases. It has been speculated that the molecular mechanisms governing IL-32- and IL-17-mediated cellular responses are differentially dependent on the TNF pathway. In this study, kinome analysis demonstrated that following stimulation with cytokine IL-32, but not IL-17, there was increased phosphorylation of a peptide target corresponding to TNF-R1. Consistent with this observation, blocking TNF-R1 resulted in a suppression of IL-32-induced downstream responses, indicating that IL-32-mediated activity may be dependent on TNF-R1. In contrast, blocking TNF-R1 did not affect IL-17-induced downstream responses. Kinome analysis also implicated p300 (transcriptional coactivator) and death-associated protein kinase-1 (DAPK-1) as signaling intermediates for both IL-32 and IL-17. Phosphorylation of p300 and DAPK-1 upon stimulation with either IL-32 or IL-17 was confirmed by immunoblots. The presence of common targets was supported by results demonstrating similar downstream responses induced in the presence of IL-32 and IL-17, such as transcriptional responses and the direct activation of NF-κB. Furthermore, knockdown of p300 and DAPK-1 altered downstream responses induced by IL-32 and IL-17, and impacted certain cellular responses induced by TNF-α and IL-1ß. We hypothesize that p300 and DAPK-1 represent nodes where the inflammatory networks of IL-32 and IL-17 overlap, and that these proteins would affect both TNF-R1-dependent and -independent pathways. Therefore, p300 and DAPK-1 are viable potential therapeutic targets for chronic inflammatory diseases.

A bioavailable form of curcumin suppresses cationic host defence peptides cathelicidin and calprotectin in a murine model of collagen-induced arthritis.

Curcumin, a component of the South-Asian spice turmeric, elicits anti-inflammatory functions. We have previously demonstrated that a highly bioavailable formulation of cucurmin, Cureit/Acumin™ (CUR), can suppress disease onset and severity, in a collagen-induced arthritis (CIA) mouse model. In a previous study, we have also shown that the abundance of antimicrobial host defence peptides, specifically cathelicidin (CRAMP) and calprotectin (S100A8 and S100A9), is significantly increased in the joint tissues of CIA mice. Elevated levels of cathelicidin and calprotectin have been associated with the pathogenesis of rheumatoid arthritis. Therefore, in this study, we examined the effect CUR administration on the abundance of cathelicidin and calprotectin in the joints, in a CIA mouse model. Here, we demonstrate that daily oral administration of CUR significantly reduces the elevated levels of CRAMP and calprotectin to baseline in the joints of CIA mice. We also show a linear correlation between the abundance of these peptides in the joints with serum inflammatory cytokines TNFα, IFNγ, and MCP-1. Overall, our results suggest that oral administration of a bioavailable CUR can suppress cathelicidin and calprotectin in the joints and regulate both local (joints) and systemic (serum) inflammation, in inflammatory arthritis.

Controlled Diesel Exhaust Exposure Induces a Concentration-dependent Increase in Airway Inflammation: A Clinical Trial.

Rationale: Air pollution exposure is harmful to human airways, and its impacts are best studied using concentration-response relationships. However, most concentration-response research on airway health has investigated chronic exposures, with less being known about acute effects, which can be robustly studied using controlled human exposures. Objectives: To investigate the concentration relationship between airway health measures and diesel exhaust (DE). Methods: We conducted a double-blind crossover study with 17 healthy nonsmokers exposed to filtered air and DE standardized to 20, 50, and 150 µg/m3 of particulate matter ⩽2.5 µm in aerodynamic diameter for 4 hours. Before, during, and up to 24 hours from the exposure start, we measured lung function, airway responsiveness, and airway inflammation using spirometry, methacholine challenge, and fractional exhaled nitric oxide (FeNO), respectively. In addition, we measured nasal airway inflammation using differential cell counts and cytokines in nasal lavage and epithelial lining fluid at 24 hours. We assessed DE concentration responses and associations between outcomes using linear mixed effects models and repeated measures correlations, respectively, thereafter adjusting for multiple comparisons. Results: DE exposure increased percentage ΔFeNO at 4 hours (ß = 0.16 ± 0.06). Compared with filtered air, percentage ΔFeNO trended toward an increase at concentrations of 20 µg/m3 (ß = 18.66 ± 8.76) and 50 µg/m3 (ß = 19.33 ± 8.92) and increased significantly at 150 µg/m3 (ß = 34.43 ± 8.92). In addition, DE exposure induced a trend toward increased nasal IL-6 at 24 hours (percentage difference, 0.88; 95% confidence interval, 0.08, 1.70). There were no effects of DE exposure on FeNO at 24 hours, lung function, airway responsiveness, or nasal cell counts. Conclusions: DE induces a concentration-dependent increase in FeNO, indicating that it may be a sensitive marker of an acute inflammatory response in the airways. We report responses at concentrations below those in previous controlled DE exposure studies, and we document particulate matter ⩽2.5 µm in aerodynamic diameter concentration-response estimates at exposure levels routinely experienced in the community and occupational settings. Clinical trial registered with www.clinicaltrials.gov (NCT03234790).

Rodent Lung Tissue Sample Preparation and Processing for Shotgun Proteomics.

Mass spectrometry (MS) is a routinely used approach to characterize global protein profile in various biological samples. Here we describe rodent lung tissue homogenization, sample preparation, and liquid chromatography with tandem mass spectrometry (LC-MS/MS) method for shotgun proteomics.

Sex Dimorphism of Allergen-Induced Secreted Proteins in Murine and Human Lungs.

Biological sex influences disease severity, prevalence and response to therapy in allergic asthma. However, allergen-mediated sex-specific changes in lung protein biomarkers remain undefined. Here, we report sex-related differences in specific proteins secreted in the lungs of both mice and humans, in response to inhaled allergens. Female and male BALB/c mice (7-8 weeks) were intranasally challenged with the allergen house dust mite (HDM) for 2 weeks. Bronchoalveolar lavage fluid (BALF) was collected 24 hour after the last HDM challenge from allergen-naïve and HDM-challenged mice (N=10 per group, each sex). In a human study, adult participants were exposed to nebulized (2 min) allergens (based on individual sensitivity), BALF was obtained after 24 hour (N=5 each female and male). The BALF samples were examined in immunoblots for the abundance of 10 proteins shown to increase in response to allergen in both murine and human BALF, selected from proteomics studies. We showed significant sex-bias in allergen-driven increase in five out of the 10 selected proteins. Of these, increase in eosinophil peroxidase (EPX) was significantly higher in females compared to males, in both mice and human BALF. We also showed specific sex-related differences between murine and human samples. For example, allergen-driven increase in S100A8 and S100A9 was significantly higher in BALF of females compared to males in mice, but significantly higher in males compared to females in humans. Overall, this study provides sex-specific protein biomarkers that are enhanced in response to allergen in murine and human lungs, informing and motivating translational research in allergic asthma.