Unique transcriptomic profile of peripheral blood monocytes in rheumatoid arthritis-associated interstitial lung disease.

OBJECTIVES: Though interstitial lung disease (ILD) contributes to excess morbidity and mortality in rheumatoid arthritis (RA), RA-ILD pathogenesis remains incompletely defined. As intermediate, non-classical and suppressed CD14+ monocytes are expanded in RA-ILD, this study sought to characterize gene expression profiles of circulating monocytes in RA-ILD. METHODS: Peripheral blood mononuclear cells were collected from patients with RA without lung disease (N = 5), RA-ILD (N = 5), idiopathic pulmonary fibrosis (IPF; N = 5), and controls without lung and autoimmune disease (N = 4). RNA was extracted from CD14+ isolated monocytes and subjected to transcriptional analysis of 1365 genes. Gene enrichment and pathway analyses were performed. RESULTS: Unsupervised clustering grouped patients with RA-ILD together with IPF for myeloid innate genes. For fibrosis genes, patients with RA-ILD clustered independent of comparator groups. There were 103, 66, and 64 upregulated and 66, 14, and 25 downregulated genes for RA-ILD, RA, and IPF, vs controls, respectively. For RA-ILD, there was increased expression of genes involved in regulating inflammation and fibrosis (SOCS3, CECAM1, LTB4R2, CLEC7A, IRF7, PHYKPL, GBP5, RAPGEF), epigenetic modification (KDM5D, KMT2D, OGT), and macrophage activation. Top canonical pathways included macrophage differentiation-activation, IL-12, neuroinflammatory, glucocorticoid receptor, and IL-27 signalling. CONCLUSIONS: Circulating monocytes in RA-ILD patients demonstrate unique gene expression profiles with innate immune gene features more aligned with IPF as opposed to RA in the absence of clinical lung disease with fibrosis gene expression that was distinct from RA and IPF. These studies are important for understanding disease pathogenesis and may provide information for future therapeutic targets in RA-ILD.

Critical Role of Zinc Transporter (ZIP8) in Myeloid Innate Immune Cell Function and the Host Response against Bacterial Pneumonia.

Zinc (Zn) is required for proper immune function and host defense. Zn homeostasis is tightly regulated by Zn transporters that coordinate biological processes through Zn mobilization. Zn deficiency is associated with increased susceptibility to bacterial infections, including Streptococcus pneumoniae, the most commonly identified cause of community-acquired pneumonia. Myeloid cells, including macrophages and dendritic cells (DCs), are at the front line of host defense against invading bacterial pathogens in the lung and play a critical role early on in shaping the immune response. Expression of the Zn transporter ZIP8 is rapidly induced following bacterial infection and regulates myeloid cell function in a Zn-dependent manner. To what extent ZIP8 is instrumental in myeloid cell function requires further study. Using a novel, myeloid-specific, Zip8 knockout model, we identified vital roles of ZIP8 in macrophage and DC function upon pneumococcal infection. Administration of S. pneumoniae into the lung resulted in increased inflammation, morbidity, and mortality in Zip8 knockout mice compared with wild-type counterparts. This was associated with increased numbers of myeloid cells, cytokine production, and cell death. In vitro analysis of macrophage and DC function revealed deficits in phagocytosis and increased cytokine production upon bacterial stimulation that was, in part, due to increased NF-κB signaling. Strikingly, alteration of myeloid cell function resulted in an imbalance of Th17/Th2 responses, which is potentially detrimental to host defense. These results (for the first time, to our knowledge) reveal a vital ZIP8- and Zn-mediated axis that alters the lung myeloid cell landscape and the host response against pneumococcus.

Malondialdehyde-Acetaldehyde Adduct Formation Decreases Immunoglobulin A Transport across Airway Epithelium in Smokers Who Abuse Alcohol.

Alcohol misuse and smoking are risk factors for pneumonia, yet the impact of combined cigarette smoke and alcohol on pneumonia remains understudied. Smokers who misuse alcohol form lung malondialdehyde-acetaldehyde (MAA) protein adducts and have decreased levels of anti-MAA secretory IgA (sIgA). Transforming growth factor-ß (TGF-ß) down-regulates polymeric Ig receptor (pIgR) on mucosal epithelium, resulting in decreased sIgA transcytosis to the mucosa. It is hypothesized that MAA-adducted lung protein increases TGF-ß, preventing expression of epithelial cell pIgR and decreasing sIgA. Cigarette smoke and alcohol co-exposure on sIgA and TGF-ß in human bronchoalveolar lavage fluid and in mice instilled with MAA-adducted surfactant protein D (SPD-MAA) were studied herein. Human bronchial epithelial cells (HBECs) and mouse tracheal epithelial cells were treated with SPD-MAA and sIgA and TGF-ß was measured. Decreased sIgA and increased TGF-ß were observed in bronchoalveolar lavage from combined alcohol and smoking groups in humans and mice. CD204 (MAA receptor) knockout mice showed no changes in sIgA. SPD-MAA decreased pIgR in HBECs. Conversely, SPD-MAA stimulated TGF-ß release in both HBECs and mouse tracheal epithelial cells, but not in CD204 knockout mice. SPD-MAA stimulated TGF-ß in alveolar macrophage cells. These data show that MAA-adducted surfactant protein stimulates lung epithelial cell TGF-ß, down-regulates pIgR, and decreases sIgA transcytosis. These data provide a mechanism for the decreased levels of sIgA observed in smokers who misuse alcohol.

COVID-19 patients with documented alcohol use disorder or alcohol-related complications are more likely to be hospitalized and have higher all-cause mortality.

BACKGROUND: Coronavirus Disease 2019 (COVID-19) has affected every country globally, with hundreds of millions of people infected with the SARS-CoV-2 virus and over 6 million deaths to date. It is unknown how alcohol use disorder (AUD) affects the severity and mortality of COVID-19. AUD is known to increase the severity and mortality of bacterial pneumonia and the risk of developing acute respiratory distress syndrome. Our objective is to determine whether individuals with AUD have increased severity and mortality from COVID-19. METHODS: We utilized a retrospective cohort study of inpatients and outpatients from 44 centers participating in the National COVID Cohort Collaborative. All were adult COVID-19 patients with and without documented AUDs. RESULTS: We identified 25,583 COVID-19 patients with an AUD and 1,309,445 without. In unadjusted comparisons, those with AUD had higher odds of hospitalization (odds ratio [OR] 2.00, 95% confidence interval [CI] 1.94 to 2.06, p < 0.001). After adjustment for age, sex, race/ethnicity, smoking, body mass index, and comorbidities, individuals with an AUD still had higher odds of requiring hospitalization (adjusted OR [aOR] 1.51, CI 1.46 to 1.56, p < 0.001). In unadjusted comparisons, individuals with AUD had higher odds of all-cause mortality (OR 2.18, CI 2.05 to 2.31, p < 0.001). After adjustment as above, individuals with an AUD still had higher odds of all-cause mortality (aOR 1.55, CI 1.46 to 1.65, p < 0.001). CONCLUSION: This work suggests that AUD can increase the severity and mortality of COVID-19 infection. This reinforces the need for clinicians to obtain an accurate alcohol history from patients hospitalized with COVID-19. For this study, our results are limited by an inability to quantify the daily drinking habits of the participants. Studies are needed to determine the mechanisms by which AUD increases the severity and mortality of COVID-19.

Amphiregulin modulates murine lung recovery and fibroblast function following exposure to agriculture organic dust.

Exposure to agricultural bioaerosols can lead to chronic inflammatory lung diseases. Amphiregulin (AREG) can promote the lung repair process but can also lead to fibrotic remodeling. The objective of this study was to determine the role of AREG in altering recovery from environmental dust exposure in a murine in vivo model and in vitro using cultured human and murine lung fibroblasts. C57BL/6 mice were intranasally exposed to swine confinement facility dust extract (DE) or saline daily for 1 wk or allowed to recover for 3-7 days while being treated with an AREG-neutralizing antibody or recombinant AREG. Treatment with the anti-AREG antibody prevented resolution of DE exposure-induced airway influx of total cells, neutrophils, and macrophages and increased levels of TNF-α, IL-6, and CXCL1. Neutrophils and activated macrophages (CD11c+CD11bhi) persisted after recovery in lung tissues of anti-AREG-treated mice. In murine and human lung fibroblasts, DE induced the release of AREG and inflammatory cytokines. Fibroblast recellularization of primary human lung mesenchymal matrix scaffolds and wound closure was inhibited by DE and enhanced with recombinant AREG alone. AREG treatment rescued the DE-induced inhibitory fibroblast effects. AREG intranasal treatment for 3 days during recovery phase reduced repetitive DE-induced airway inflammatory cell influx and cytokine release. Collectively, these studies demonstrate that inhibition of AREG reduced, whereas AREG supplementation promoted, the airway inflammatory recovery response following environmental bioaerosol exposure, and AREG enhanced fibroblast function, suggesting that AREG could be targeted in agricultural workers repetitively exposed to organic dust environments to potentially prevent and/or reduce disease.

Alcohol Use Disorders Are Associated With a Unique Impact on Airway Epithelial Cell Gene Expression.

BACKGROUND: Alcohol use disorders (AUDs) and cigarette smoking both increase risk for the development of community-acquired pneumonia (CAP), likely through adverse effects on proximal airway mucociliary clearance and pathogen recognition. Smoking-related alterations on airway gene expression are well described, but little is known about the impact of AUDs. We measured gene expression in human airway epithelial cells (AECs), hypothesizing that AUDs would be associated with novel differences in gene expression that could alter risk for CAP. METHODS: Bronchoscopy with airway brushings was performed in participants with AUDs and controls to obtain AECs. An AUD Identification Test was used to define AUD. RNA was extracted from AECs, and mRNA expression data were collected on an Agilent micro-array. Differential expression analyses were performed on the filtered and normalized data with correction for multiple testing. Enrichment analyses were performed using clusterProfiler. RESULTS: Expression data from 19 control and 18 AUD participants were evaluated. After adjustment for smoking, AUDs were associated with significant differential expression of 520 AEC genes, including genes for ribosomal proteins and genes involved in protein folding. Enrichment analyses indicated significant differential expression of 24 pathways in AUDs, including those implicated in protein targeting to membrane and viral gene expression. Smoking-associated AEC gene expression differences mirrored previous reports, but differed from those associated with AUDs. CONCLUSIONS: AUDs have a distinct impact on AEC gene expression that may influence proximal airway function independent of smoking. Alcohol-associated alterations may influence risk for CAP through modifying key mechanisms important in protecting proximal airway integrity.

MyD88 controls airway epithelial Muc5ac expression during TLR activation conditions from agricultural organic dust exposure.

Inflammation from airborne microbes can overwhelm compensatory mucociliary clearance mechanisms, leading to mucous cell metaplasia. Toll-like receptor (TLR) activation via myeloid differentiation factor 88 (MyD88) signaling is central to pathogen responses. We have previously shown that agricultural organic dust extract (ODE), with abundant microbial component diversity, activates TLR-induced airway inflammation. With the use of an established model, C57BL/6J wild-type (WT) and global MyD88 knockout (KO) mice were treated with intranasal inhalation of ODE or saline, daily for 1 wk. ODE primarily increased mucin (Muc)5ac levels relative to Muc5b. Compared with ODE-challenged WT mice, ODE-challenged, MyD88-deficient mice demonstrated significantly increased Muc5ac immunostaining, protein levels by immunoblot, and expression by quantitative PCR. The enhanced Muc5ac levels in MyD88-deficient mice were not explained by differences in the differentiation program of airway secretory cells in naïve mice. Increased Muc5ac levels in MyD88-deficient mice were also not explained by augmented inflammation, IL-17A, or neutrophil elastase levels. Furthermore, the enhanced airway mucins in the MyD88-deficient mice were not due to defective secretion, as the mucin secretory capacity of MyD88-KO mice remained intact. Finally, ODE-induced Muc5ac levels were enhanced in MyD88-deficient airway epithelial cells in vitro. In conclusion, MyD88 deficiency enhances airway mucous cell metaplasia under environments with high TLR activation.

Aging leads to dysfunctional innate immune responses to TLR2 and TLR4 agonists.

BACKGROUND: Sepsis is more common in the elderly. TNF⍺ is recognized as an important mediator in sepsis and Toll-like receptors (TLRs) play an important role in initiating signaling cascades to produce TNF⍺. Little is known about how innate immunity is altered in healthy human aging that predisposes to sepsis. AIMS AND METHODS: We tested the hypothesis that aging dysregulates the innate immune response to TLR 2 and 4 ligands. We performed whole blood assays on 554 healthy subjects aged 40-80 years. TNFα production was measured at baseline and after stimulation with the TLR2 agonists: peptidoglycan, lipoteichoic acid, Pam3CysK, Zymosan A and the TLR4 agonist lipopolysaccharide (LPS). In a subset of subjects (n = 250), we measured Toll-like receptor (TLR) 2, 4 and MyD88 expression using real-time PCR. RESULTS AND DISCUSSION: We measured a 2.5% increase per year in basal secretion of TNFα with aging (n = 554 p = 0.02). Likewise, TNFα secretion was increased with aging after stimulation with peptidoglycan (1.3% increase/year; p = 0.0005) and zymosan A (1.1% increase/year p = 0.03). We also examined the difference between baseline and stimulated TNFα for each individual. We found that the increase was driven by the elevated baseline levels. In fact, there was a diminished stimulated response to LPS (1.9% decrease/year; p = 0.05), lipoteichoic acid (2.1% decrease/year p = 0.03), and Pam3CysK (2.6% decrease/year p = 0.0007). There were no differences in TLR or MyD88 mRNA expression with aging, however, there was an inverse relationship between TLR expression and stimulated TNFα production. CONCLUSIONS: With aging, circulating leukocytes produce high levels of TNFα at baseline and have inadequate responses to TLR2 and TLR4 agonists. These defects likely contribute to the increased susceptibility to sepsis in older adults.

Oxidative stress associated with aging activates protein kinase Cε, leading to cilia slowing.

Older people are four times more likely to develop pneumonia than younger people. As we age, many components of pulmonary innate immunity are impaired, including slowing of mucociliary clearance. Ciliary beat frequency (CBF) is a major determinant of mucociliary clearance, and it slows as we age. We hypothesized that CBF is slowed in aging because of increased oxidative stress, which activates PKCε signaling. We pharmacologically inhibited PKCε in ex vivo mouse models of aging. We measured a slowing of CBF with aging that was reversed with inhibition using the novel PKC inhibitor, Ro-31-8220, as well as the PKCε inhibitor, PKCe141. Inhibition of PKCε using siRNA in mouse trachea also returned CBF to normal. In addition, antioxidants decrease PKCε activity and speed cilia. We also aged wild-type and PKCε KO mice and measured CBF. The PKCε KO mice were spared from the CBF slowing of aging. Using human airway epithelial cells from younger and older donors at air-liquid interface (ALI), we inhibited PKCε with siRNA. We measured a slowing of CBF with aging that was reversed with siRNA inhibition of PKCε. In addition, we measured bead clearance speeds in human ALI, which demonstrated a decrease in bead velocity with aging and a return to baseline after inhibition of PKCε. In summary, in human and mouse models, aging is associated with increased oxidant stress, which activates PKCε and slows CBF.

Docosahexaenoic acid enhances amphiregulin-mediated bronchial epithelial cell repair processes following organic dust exposure.

Injurious dust exposures in the agricultural workplace involve the release of inflammatory mediators and activation of epidermal growth factor receptor (EGFR) in the respiratory epithelium. Amphiregulin (AREG), an EGFR ligand, mediates tissue repair and wound healing in the lung epithelium. Omega-3 fatty acids such as docosahexaenoic acid (DHA) are also known modulators of repair and resolution of inflammatory injury. This study investigated how AREG, DHA, and EGFR modulate lung repair processes following dust-induced injury. Primary human bronchial epithelial (BEC) and BEAS-2B cells were treated with an aqueous extract of swine confinement facility dust (DE) in the presence of DHA and AREG or EGFR inhibitors. Mice were exposed to DE intranasally with or without EGFR inhibition and DHA. Using a decellularized lung scaffolding tissue repair model, BEC recolonization of human lung scaffolds was analyzed in the context of DE, DHA, and AREG treatments. Through these investigations, we identified an important role for AREG in mediating BEC repair processes. DE-induced AREG release from BEC, and DHA treatment following DE exposure, enhanced this release. Both DHA and AREG also enhanced BEC repair capacities and rescued DE-induced recellularization deficits. In vivo, DHA treatment enhanced AREG production following DE exposure, whereas EGFR inhibitor-treated mice exhibited reduced AREG in their lung homogenates. These data indicate a role for AREG in the process of tissue repair after inflammatory lung injury caused by environmental dust exposure and implicate a role for DHA in regulating AREG-mediated repair signaling in BEC.

Dimethylarginine dimethylaminohydrolase (DDAH) overexpression enhances wound repair in airway epithelial cells exposed to agricultural organic dust.

OBJECTIVE: Workers exposed to dusts from concentrated animal feeding operations have a high prevalence of pulmonary diseases. These exposures lead to chronic inflammation and aberrant airway remodeling. Previous work shows that activating cAMP-dependent protein kinase (PKA) enhances airway epithelial wound repair while activating protein kinase C (PKC) inhibits wound repair. Hog barn dust extracts slow cell migration and wound repair via a PKC-dependent mechanism. Further, blocking nitric oxide (NO) production in bronchial epithelial cells prevents PKA activation. We hypothesized that blocking an endogenous NO inhibitor, asymmetric dimethylarginine, by overexpressing dimethylarginine dimethylaminohydrolase mitigates the effects of hog dust extract on airway epithelial would repair. MATERIALS/METHODS: We cultured primary tracheal epithelial cells in monolayers from both wild-type (WT) and dimethylarginine dimethylaminohydrolase overexpressing C57Bl/6 (DDAH1 transgenic) mice and measured wound repair using the electric cell impedance sensing system. RESULTS: Wound closure in epithelial cells from WT mice occurred within 24 h in vitro. In contrast, treatment of the WT cell monolayers with 5% hog dust extract prevented significant NO-stimulated wound closure. In cells from DDAH1 transgenic mice, control wounds were repaired up to 8 h earlier than seen in WT mice. A significant enhancement of wound repair was observed in DDAH cells compared to WT cells treated with hog dust extract for 24 h. Likewise, cells from DDAH1 transgenic mice demonstrated increased NO and PKA activity and decreased hog dust extract-stimulated PKC. DISCUSSION/CONCLUSION: Preserving the NO signal through endogenous inhibition of asymmetric dimethylarginine enhances wound repair even in the presence of dust exposure.

Pulmonary health effects of agriculture.

PURPOSE OF REVIEW: Occupational exposures in the agricultural industry are associated with numerous lung diseases, including chronic obstructive pulmonary disease, asthma, hypersensitivity pneumonitis, lung cancer, and interstitial lung diseases. Efforts are ongoing to ascertain contributing factors to these negative respiratory outcomes and improve monitoring of environmental factors leading to disease. In this review, recently published studies investigating the deleterious effects of occupational exposures in the agricultural industry are discussed. RECENT FINDINGS: Occupational exposures to numerous agricultural environment aerosols, including pesticides, fungi, and bacteria are associated with impaired respiratory function and disease. Increases in certain farming practices, including mushroom and greenhouse farming, present new occupational exposure concerns. Improved detection methods may provide opportunities to better monitor safe exposure levels to known lung irritants. SUMMARY: In the agricultural industry, occupational exposures to organic and inorganic aerosols lead to increased risk for lung disease among workers. Increased awareness of respiratory risks and improved monitoring of agricultural environments are necessary to limit pulmonary health risks to exposed populations.

Lessons learned: critical care management of patients with Ebola in the United States.

OBJECTIVE: This report will describe the preparations for and the provision of care of two patients with Ebola virus disease in the biocontainment unit at the University of Nebraska Medical Center. DATA SOURCES: Patient medical records. STUDY SELECTION: Not applicable. DATA EXTRACTION: Not applicable. DATA SYNTHESIS: Not applicable. CONCLUSIONS: Safe and effective care of patients with Ebola virus disease requires significant communication and planning. Adherence to a predetermined isolation protocol is essential, including proper donning and doffing of personal protective equipment. Location of the patient care area and the logistics of laboratory testing, diagnostic imaging, and the removal of waste must be considered. Patients with Ebola virus disease are often dehydrated and need adequate vascular access for fluid resuscitation, nutrition, and phlebotomy for laboratory sampling. Advanced planning for acute life-threatening events and code status must be considered. Intensivist scheduling should account for the significant amount of time required for the care of patients with Ebola virus disease. With appropriate precautions and resources, designated hospitals in the United States can safely provide care for patients with Ebola virus disease.

TLR2 and TLR4 Expression and Inflammatory Cytokines are Altered in the Airway Epithelium of Those with Alcohol Use Disorders.

BACKGROUND: The lung has a highly regulated system of innate immunity to protect itself from inhaled microbes and toxins. The first line of defense is mucociliary clearance, but if invaders overcome this, inflammatory pathways are activated. Toll-like receptors (TLRs) are expressed on the airway epithelium. Their signaling initiates the inflammatory cascade and leads to production of inflammatory cytokines such as interleukin (IL)-6 and IL-8. We hypothesized that airway epithelial insults, including heavy alcohol intake or smoking, would alter the expression of TLRs on the airway epithelium. METHODS: Bronchoscopy with bronchoalveolar lavage and brushings of the airway epithelium was performed in otherwise healthy subjects who had normal chest radiographs and spirometry. A history of alcohol use disorders (AUDs) was ascertained using the Alcohol Use Disorders Identification Test (AUDIT), and a history of cigarette smoking was also obtained. Age, gender, and nutritional status in all groups were similar. We used real-time polymerase chain reaction (PCR) to quantitate TLR1 to 9 and enzyme-linked immune assay to measure tumor necrosis factor-α, IL-6, and IL-8. RESULTS: Airway brushings were obtained from 26 nonsmoking/non-AUD subjects, 28 smoking/non-AUD subjects, 36 smoking/AUD subjects, and 17 nonsmoking/AUD subjects. We found that TLR2 is up-regulated in AUD subjects, compared to nonsmoking/non-AUD subjects, and correlated with their AUDIT scores. We also measured a decrease in TLR4 expression in AUD subjects that correlated with AUDIT score. IL-6 and IL-8 were also increased in bronchial washings from AUD subjects. CONCLUSIONS: We have previously demonstrated in normal human bronchial epithelial cells that in vitro alcohol exposure up-regulates TLR2 through a NO/cGMP/PKG-dependent pathway, resulting in up-regulation of inflammatory cytokine production after Gram-positive bacterial product stimulation. Our current translational study confirms that TLR2 is also up-regulated in humans with AUDs.

Age Impacts Pulmonary Inflammation and Systemic Bone Response to Inhaled Organic Dust Exposure.

Agricultural workers have high rates of airway and skeletal health disease. Studies recently demonstrated that inhaled agricultural organic dust extract (ODE)-induced airway injury is associated with bone deterioration in an animal model. However, the effect of age in governing these responses to organic dusts is unclear, but might be important in future approaches. Young (7-9 wk) and older (12-14,o) male C57BL/6 mice received intranasal (i.n.) inhalation exposure to ODE from swine confinement facilities once or daily for 3 wk. Acute ODE-induced neutrophil influx and cytokine and chemokine (tumor necrosis factor [TNF]-α, interleukin [IL]-6, keratinocyte chemoattractant [CXCL1], macrophage inflammatory protein-2 [CXCL2]) airway production were reduced in older compared to young mice. Repetitive ODE treatment, however, increased lymphocyte recruitment and alveolar compartment histopathologic inflammatory changes in older mice. Whole lung cell infiltrate analysis revealed that young, but not older, mice repetitively treated with ODE demonstrated an elevated CD4:CD8 lymphocyte response. Acute inhalant ODE exposure resulted in a 4-fold and 1.5-fold rise in blood neutrophils in young and older mice, respectively. Serum IL-6 and CXCL1 levels were elevated in young and older mice i.n. exposed once to ODE, with increased CXCL1 levels in younger compared to older mice. Although older mice displayed reduced bone measurements compared to younger mice, younger rodents demonstrated ODE-induced decrease in bone mineral density, bone volume, and bone microarchitecture quality as determined by computed tomography (CT) analysis. Collectively, age impacts the airway injury and systemic inflammatory and bone loss response to inhalant ODE, suggesting an altered and enhanced immunologic response in younger as compared to older counterparts.

cAMP-dependent protein kinase activation decreases cytokine release in bronchial epithelial cells.

Lung injury caused by inhalation of dust from swine-concentrated animal-feeding operations (CAFO) involves the release of inflammatory cytokine interleukin 8 (IL-8), which is mediated by protein kinase C-ε (PKC-ε) in airway epithelial cells. Once activated by CAFO dust, PKC-ε is responsible for slowing cilia beating and reducing cell migration for wound repair. Conversely, the cAMP-dependent protein kinase (PKA) stimulates contrasting effects, such as increased cilia beating and an acceleration of cell migration for wound repair. We hypothesized that a bidirectional mechanism involving PKA and PKC regulates epithelial airway inflammatory responses. To test this hypothesis, primary human bronchial epithelial cells and BEAS-2B cells were treated with hog dust extract (HDE) in the presence or absence of cAMP. PKC-ε activity was significantly reduced in cells that were pretreated for 1 h with 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) before exposure to HDE (P < 0.05). HDE-induced IL-6, and IL-8 release was significantly lower in cells that were pretreated with 8-Br-cAMP (P < 0.05). To exclude exchange protein activated by cAMP (EPAC) involvement, cells were pretreated with either 8-Br-cAMP or 8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-CPT-2Me-cAMP) (EPAC agonist). 8-CPT-2Me-cAMP did not activate PKA and did not reduce HDE-stimulated IL-6 release. In contrast, 8-Br-cAMP decreased HDE-stimulated tumor necrosis factor (TNF)-α-converting enzyme (TACE; ADAM-17) activity and subsequent TNF-α release (P < 0.001). 8-Br-cAMP also blocked HDE-stimulated IL-6 and keratinocyte-derived chemokine release in precision-cut mouse lung slices (P < 0.05). These data show bidirectional regulation of PKC-ε via a PKA-mediated inhibition of TACE activity resulting in reduced PKC-ε-mediated release of IL-6 and IL-8.

Motile cilia harbor serum response factor as a mechanism of environment sensing and injury response in the airway.

Nonmotile primary cilia are recognized as important sensory organelles during development and normal biological functioning. For example, recent work demonstrates that transcriptional regulators of the sonic hedgehog signaling pathway localize to primary cilia and participate in sensing and transducing signals regarding the cellular environment. In contrast, motile cilia are traditionally viewed as mechanical machinery, vital for the movement of solutes and clearance of bacteria and debris, but not participants in cellular sensing and signaling mechanisms. Recently, motile cilia were found to harbor receptors responsible for sensing and responding to environmental stimuli. However, no transcription factors are known to be regulated by cilia localization as a sensing mechanism in vertebrates. Using a mouse model of organic dust-induced airway inflammation, we found that the transcription factor serum response factor (SRF) localizes to motile cilia of airway epithelial cells and alters its localization in response to inflammatory stimuli. Furthermore, inhibition of SRF signaling using the small molecule CCG-1423 reduces organic dust-induced IL-8 release from bronchial epithelial cells and stimulates cilia beat frequency in ciliated mouse tracheal epithelial cells. Immunohistochemical analyses reveal that SRF localizes to the cilia of mouse brain ependymal and ovarian epithelial cells as well. These data reveal a novel mechanism by which a transcription factor localizes to motile cilia and modulates cell activities including cilia motility and inflammation response. These data challenge current dogma regarding motile cilia functioning and may lead to significant contributions in understanding motile ciliary signaling dynamics, as well as mechanisms involving SRF-mediated responses to inflammation and injury.

mTOR signaling regulates aberrant epithelial cell proliferative and migratory behaviors characteristic of airway mucous metaplasia in asthma.

In asthma, the airway epithelium is hyperplastic, hypertrophied, and lined with numerous large MUC5AC-containing goblet cells (GC). Furthermore, the normal epithelial architecture is disorganized with numerous, what we here describe as, ectopic goblet cells (eGC) deep within the thickened epithelial layer disconnected from the lumenal surface. mTOR is a highly conserved pathway that regulates cell size and proliferation. We hypothesized that the balance between mTOR and autophagy signaling regulates key features of the asthma epithelial layer. Airway histological sections from subjects with asthma had increased frequency of eGC and increased levels of mTOR phosphorylation target-Ribosomal S6. Using human airway epithelial cells (hAECs) with IL-13 stimulation and timed withdrawal to stimulate resolution, we found that multiple key downstream phosphorylation targets downstream from the mTOR complex were increased during early IL-13-mediated mucous metaplasia, and then significantly declined during resolution. The IL-13-mediated changes in mTOR signaling were paralleled by morphologic changes with airway epithelial hypertrophy, hyperplasia, and frequency of eGC. We then examined the relationship between mTOR and autophagy using mice deficient in autophagy protein Atg16L1. Despite having increased cytoplasmic mucins, mouse AECs from Atg16L1 deficient mice had no significant difference in mTOR downstream signaling. mTOR inhibition with rapamycin led to a loss of IL-13-mediated epithelial hypertrophy, hyperplasia, ectopic GC distribution, and reduction in cytoplasmic MUC5AC levels. mTOR inhibition was also associated with a reduction in aberrant IL-13-mediated hAEC proliferation and migration. Our findings demonstrate that mTOR signaling is associated with mucous metaplasia and is crucial to the disorganized airway epithelial structure and function characteristic of muco-obstructive airway diseases such as asthma. Graphical Abstract Key Concepts: The airway epithelium in asthma is disorganized and characterized by cellular proliferation, aberrant migration, and goblet cell mucous metaplasia.mTOR signaling is a dynamic process during IL-13-mediated mucous metaplasia, increasing with IL-13 stimulation and declining during resolution.mTOR signaling is strongly increased in the asthmatic airway epithelium.mTOR signaling is associated with the development of key features of the metaplastic airway epithelium including cell proliferation and ectopic distribution of goblet cells and aberrant cellular migration.Inhibition of mTOR leads to decreased epithelial hypertrophy, reduced ectopic goblet cells, and cellular migration.

Aconitate decarboxylase 1 mediates the acute airway inflammatory response to environmental exposures.

Background: Environmental lipopolysaccharide (LPS) and microbial component-enriched organic dusts cause significant lung disease. These environmental exposures induce the recruitment and activation of distinct lung monocyte/macrophage subpopulations involved in disease pathogenesis. Aconitate decarboxylase 1 (Acod1) was one of the most upregulated genes following LPS (vs. saline) exposure of murine whole lungs with transcriptomic profiling of sorted lung monocyte/macrophage subpopulations also highlighting its significance. Given monocyte/macrophage activation can be tightly linked to metabolism, the objective of these studies was to determine the role of the immunometabolic regulator ACOD1 in environmental exposure-induced lung inflammation. Methods: Wild-type (WT) mice were intratracheally (i.t.) instilled with 10 µg of LPS or saline. Whole lungs were profiled using bulk RNA sequencing or sorted to isolate monocyte/macrophage subpopulations. Sorted subpopulations were then characterized transcriptomically using a NanoString innate immunity multiplex array 48 h post-exposure. Next, WT and Acod1-/- mice were instilled with LPS, 25% organic dust extract (ODE), or saline, whereupon serum, bronchoalveolar lavage fluid (BALF), and lung tissues were collected. BALF metabolites of the tricarboxylic acid (TCA) cycle were quantified by mass spectrometry. Cytokines/chemokines and tissue remodeling mediators were quantitated by ELISA. Lung immune cells were characterized by flow cytometry. Invasive lung function testing was performed 3 h post-LPS with WT and Acod1-/- mice. Results: Acod1-/- mice treated with LPS demonstrated decreased BALF levels of itaconate, TCA cycle reprogramming, decreased BALF neutrophils, increased lung CD4+ T cells, decreased BALF and lung levels of TNF-α, and decreased BALF CXCL1 compared to WT animals. In comparison, Acod1-/- mice treated with ODE demonstrated decreased serum pentraxin-2, BALF levels of itaconate, lung total cell, neutrophil, monocyte, and B-cell infiltrates with decreased BALF levels of TNF-α and IL-6 and decreased lung CXCL1 vs. WT animals. Mediators of tissue remodeling (TIMP1, MMP-8, MMP-9) were also decreased in the LPS-exposed Acod1-/- mice, with MMP-9 also reduced in ODE-exposed Acod1-/- mice. Lung function assessments demonstrated a blunted response to LPS-induced airway hyperresponsiveness in Acod1-/- animals. Conclusion: Acod1 is robustly upregulated in the lungs following LPS exposure and encodes a key immunometabolic regulator. ACOD1 mediates the proinflammatory response to acute inhaled environmental LPS and organic dust exposure-induced lung inflammation.

Expansion of distinct peripheral blood myeloid cell subpopulations in patients with rheumatoid arthritis-associated interstitial lung disease.

OBJECTIVES: Interstitial lung disease (ILD) is associated with significant mortality in rheumatoid arthritis (RA) patients with key cellular players remaining largely unknown. This study aimed to characterize inflammatory and myeloid derived suppressor cell (MDSC) subpopulations in RA-ILD as compared to RA, idiopathic pulmonary fibrosis (IPF) without autoimmunity, and controls. METHODS: Peripheral blood was collected from patients with RA, RA-ILD, IPF, and controls (N = 60, 15/cohort). Myeloid cell subpopulations were identified phenotypically by flow cytometry using the following markers:CD45,CD3,CD19,CD56,CD11b,HLA-DR,CD14,CD16,CD15,CD125,CD33. Functionality of subsets were identified with intracellular arginase-1 (Arg-1) and inducible nitric oxide synthase (iNOS) expression. RESULTS: There was increased intermediate (CD14++CD16+) and nonclassical (CD14+/-CD16++) and decreased classical (CD14++CD16-) monocytes in RA, RA-ILD, and IPF vs. control. Intermediate monocytes were higher and classical monocytes were lower in RA-ILD vs. RA but not IPF. Monocytic (m)MDSCs were higher in RA-ILD vs. control and RA but not IPF. Granulocytic (g)MDSCs did not significantly differ. In contrast, neutrophils were increased in IPF and RA-ILD patients with elevated expression of Arg-1 sharing similar dimensional clustering pattern. Eosinophils were increased in RA-ILD vs. controls, RA and IPF. Across cohorts, iNOS was decreased in intermediate/nonclassical monocytes but increased in mMDSCs vs. classical monocytes. In RA-ILD, iNOS positive mMDSCs were increased versus classic monocytes. CONCLUSIONS: Myeloid cell subpopulations are significantly modulated in RA-ILD patients with expansion of CD16+ monocytes, mMDSCs, and neutrophils, a phenotypic profile more aligned with IPF than other RA patients. Eosinophil expansion was unique to RA-ILD, potentially facilitating disease pathogenesis and providing a future therapeutic target.