Inhibition of ABCC1 Decreases cAMP Egress and Promotes Human Airway Smooth Muscle Cell Relaxation.

In most living cells, the second-messenger roles for adenosine 3',5'-cyclic monophosphate (cAMP) are short-lived, confined to the intracellular space, and tightly controlled by the binary switch-like actions of Gαs (stimulatory G protein)-activated adenylyl cyclase (cAMP production) and cAMP-specific PDE (cAMP breakdown). Here, by using human airway smooth muscle (HASM) cells in culture as a model, we report that activation of the cell-surface ß2AR (ß2-adrenoceptor), a Gs-coupled GPCR (G protein-coupled receptor), evokes cAMP egress to the extracellular space. Increased extracellular cAMP levels ([cAMP]e) are long-lived in culture and are induced by receptor-dependent and receptor-independent mechanisms in such a way as to define a universal response class of increased intracellular cAMP levels ([cAMP]i). We find that HASM cells express multiple ATP-binding cassette (ABC) membrane transporters, with ABCC1 (ABC subfamily member C 1) being the most highly enriched transcript mapped to MRPs (multidrug resistance-associated proteins). We show that pharmacological inhibition or downregulation of ABCC1 with siRNA markedly reduces ß2AR-evoked cAMP release from HASM cells. Furthermore, inhibition of ABCC1 activity or expression decreases basal tone and increases ß-agonist-induced HASM cellular relaxation. These findings identify a previously unrecognized role for ABCC1 in the homeostatic regulation of [cAMP]i in HASM that may be conserved traits of the Gs-GPCRs (Gs-coupled family of GPCRs). Hence, the general features of this activation mechanism may uncover new disease-modifying targets in the treatment of airflow obstruction in asthma. Surprisingly, we find that serum cAMP levels are elevated in a small cohort of patients with asthma as compared with control subjects, which warrants further investigation.

CEBPD modulates the airway smooth muscle transcriptomic response to glucocorticoids.

BACKGROUND: CCAAT/Enhancer Binding Protein D (CEBPD), a pleiotropic glucocorticoid-responsive transcription factor, modulates inflammatory responses. Of relevance to asthma, expression of CEBPD in airway smooth muscle (ASM) increases with glucocorticoid exposure. We sought to characterize CEBPD-mediated transcriptomic responses to glucocorticoid exposure in ASM by measuring changes observed after knockdown of CEBPD and its impact on asthma-related ASM function. METHODS: Primary ASM cells derived from four donors were transfected with CEBPD or non-targeting (NT) siRNA and exposed to vehicle control, budesonide (100 nM, 18 h), TNFα (10 ng/ml, 18 h), or both budesonide and TNFα. Subsequently, RNA-Seq was used to measure gene expression levels, and pairwise differential expression results were obtained for exposures versus vehicle and knockdown versus control conditions. Weighted gene co-expression analysis was performed to identify groups of genes with similar expression patterns across the various experimental conditions (i.e., CEBPD knockdown status, exposures). RESULTS: CEBPD knockdown altered expression of 3037 genes under at least one exposure (q-value < 0.05). Co-expression analysis identified sets of 197, 152 and 290 genes that were correlated with CEBPD knockdown status, TNFα exposure status, and both, respectively. JAK-STAT signaling pathway genes, including IL6R and SOCS3, were among those influenced by both TNFα and CEBPD knockdown. Immunoblot assays revealed that budesonide-induced IL-6R protein expression and augmented IL-6-induced STAT3 phosphorylation levels were attenuated by CEBPD knockdown in ASM. CONCLUSIONS: CEBPD modulates glucocorticoid responses in ASM, in part via modulation of IL-6 receptor signaling.

RGS4 promotes allergen- and aspirin-associated airway hyperresponsiveness by inhibiting PGE2 biosynthesis.

BACKGROUND: Allergens elicit host production of mediators acting on G-protein-coupled receptors to regulate airway tone. Among these is prostaglandin E2 (PGE2), which, in addition to its role as a bronchodilator, has anti-inflammatory actions. Some patients with asthma develop bronchospasm after the ingestion of aspirin and other nonsteroidal anti-inflammatory drugs, a disorder termed aspirin-exacerbated respiratory disease. This condition may result in part from abnormal dependence on the bronchoprotective actions of PGE2. OBJECTIVE: We sought to understand the functions of regulator of G protein signaling 4 (RGS4), a cytoplasmic protein expressed in airway smooth muscle and bronchial epithelium that regulates the activity of G-protein-coupled receptors, in asthma. METHODS: We examined RGS4 expression in human lung biopsies by immunohistochemistry. We assessed airways hyperresponsiveness (AHR) and lung inflammation in germline and airway smooth muscle-specific Rgs4-/- mice and in mice treated with an RGS4 antagonist after challenge with Aspergillus fumigatus. We examined the role of RGS4 in nonsteroidal anti-inflammatory drug-associated bronchoconstriction by challenging aspirin-exacerbated respiratory disease-like (ptges1-/-) mice with aspirin. RESULTS: RGS4 expression in respiratory epithelium is increased in subjects with severe asthma. Allergen-induced AHR was unexpectedly diminished in Rgs4-/- mice, a finding associated with increased airway PGE2 levels. RGS4 modulated allergen-induced PGE2 secretion in human bronchial epithelial cells and prostanoid-dependent bronchodilation. The RGS4 antagonist CCG203769 attenuated AHR induced by allergen or aspirin challenge of wild-type or ptges1-/- mice, respectively, in association with increased airway PGE2 levels. CONCLUSIONS: RGS4 may contribute to the development of AHR by reducing airway PGE2 biosynthesis in allergen- and aspirin-induced asthma.

Airway Smooth Muscle-Specific Transcriptomic Signatures of Glucocorticoid Exposure.

Glucocorticoids, commonly used asthma controller medications, decrease symptoms in most patients, but some remain symptomatic despite high-dose treatment. The physiological basis underlying the glucocorticoid response, especially in asthma patients with severe, refractory disease, is not fully understood. We sought to identify differences between the transcriptomic response of airway smooth muscle (ASM) cells derived from donors with fatal asthma and donors without asthma to glucocorticoid exposure and to compare ASM-specific changes with those observed in other cell types. In cells derived from nine donors with fatal asthma and eight donors without asthma, RNA sequencing was used to measure ASM transcriptome changes after exposure to budesonide (100 nM 24 h) or control vehicle (DMSO). Differential expression results were obtained for this dataset, as well as 13 publicly available glucocorticoid-response transcriptomic datasets corresponding to seven cell types. Specific genes were differentially expressed in response to glucocorticoid exposure (7,835 and 6,957 in ASM cells derived from donors with fatal asthma and donors without asthma, respectively; adjusted P value < 0.05). Transcriptomic changes in response to glucocorticoid exposure were similar in ASM derived from donors with fatal asthma and donors without asthma, with enriched ontological pathways that included cytokine- and chemokine-related categories. A comparison of glucocorticoid-induced changes in the nonasthma ASM transcriptome with those observed in six other cell types showed that ASM has a distinct glucocorticoid-response signature that is also present in ASM cells from donors with fatal asthma.

Salicylic acid amplifies Carbachol-induced bronchoconstriction in human precision-cut lung slices.

BACKGROUND: Asthma exacerbations evoke emergency room visits, progressive loss of lung function and increased mortality. Environmental and industrial toxicants exacerbate asthma, although the underlying mechanisms are unknown. We assessed whether 3 distinct toxicants, salicylic acid (SA), toluene diisocyanate (TDI), and 1-chloro-2,4-dinitrobenzene (DNCB) induced airway hyperresponsiveness (AHR) through modulating excitation-contraction coupling in human airway smooth muscle (HASM) cells. The toxicants include a non-sensitizing irritant (SA), respiratory sensitizer (TDI) and dermal sensitizer (DNCB), respectively. We hypothesized that these toxicants induce AHR by modulating excitation-contraction (EC) coupling in airway smooth muscle (ASM) cells. METHODS: Carbachol-induced bronchoconstriction was measured in precision-cut human lung slices (hPCLS) following exposure to SA, TDI, DNCB or vehicle. Culture supernatants of hPCLS were screened for mediator release. In HASM cells treated with the toxicants, surrogate readouts of EC coupling were measured by phosphorylated myosin light chain (pMLC) and agonist-induced Ca2+ mobilization ([Ca2+]i). In addition, Nrf-2-dependent antioxidant response was determined by NAD(P) H quinone oxidoreductase 1 (NQO1) expression in HASM cells. RESULTS: In hPCLS, SA, but not TDI or DNCB, potentiated carbachol-induced bronchoconstriction. The toxicants had little effect on release of inflammatory mediators, including IL-6, IL-8 and eotaxin from hPCLS. In HASM cells, TDI amplified carbachol-induced MLC phosphorylation. The toxicants also had little effect on agonist-induced [Ca2+]i. CONCLUSION: SA, a non-sensitizing irritant, amplifies agonist-induced bronchoconstriction in hPCLS via mechanisms independent of inflammation and Ca2+ homeostasis in HASM cells. The sensitizers TDI and DNCB, had little effect on bronchoconstriction or inflammatory mediator release in hPCLS. IMPLICATIONS: Our findings suggest that non-sensitizing irritant salicylic acid may evoke AHR and exacerbate symptoms in susceptible individuals or in those with underlying lung disease.

Serum amyloid A: an ozone-induced circulating factor with potentially important functions in the lung-brain axis.

Accumulating evidence suggests that O3 exposure may contribute to CNS dysfunction. Here, we posit that inflammatory and acute-phase proteins in the circulation increase after O3 exposure and systemically convey signals of O3 exposure to the CNS. To model acute O3 exposure, female Balb/c mice were exposed to 3 ppm O3 or forced air for 2 h and were studied after 6 or 24 h. Of 23 cytokines and chemokines, only KC/CXCL1 was increased in blood 6 h after O3 exposure. The acute-phase protein serum amyloid A (A-SAA) was significantly increased by 24 h, whereas C-reactive protein was unchanged. A-SAA in blood correlated with total leukocytes, macrophages, and neutrophils in bronchoalveolar lavage from O3-exposed mice. A-SAA mRNA and protein were increased in the liver. We found that both isoforms of A-SAA completely crossed the intact blood-brain barrier, although the rate of SAA2.1 influx was approximately 5 times faster than that of SAA1.1. Finally, A-SAA protein, but not mRNA, was increased in the CNS 24 h post-O3 exposure. Our findings suggest that A-SAA is functionally linked to pulmonary inflammation in our O3 exposure model and that A-SAA could be an important systemic signal of O3 exposure to the CNS.-Erickson, M. A., Jude, J., Zhao, H., Rhea, E. M., Salameh, T. S., Jester, W., Pu, S., Harrowitz, J., Nguyen, N., Banks, W. A., Panettieri, R. A., Jr., Jordan-Sciutto, K. L. Serum amyloid A: an ozone-induced circulating factor with potentially important functions in the lung-brain axis.

Formaldehyde Induces Rho-Associated Kinase Activity to Evoke Airway Hyperresponsiveness.

Formaldehyde, a common indoor air pollutant, exacerbates asthma and synergizes with allergen to induce airway hyperresponsiveness (AHR) in animal models. The mechanisms mediating formaldehyde-induced AHR remain poorly understood. We posit that formaldehyde modulates agonist-induced contractile response of human airway smooth muscle (HASM) cells to elicit AHR. HASM cells were exposed to formaldehyde or vehicle and agonist-induced intracellular Ca2+ ([Ca2+]i) and myosin light-chain phosphatase (MYPT1) phosphorylation were determined. Air-liquid interface-differentiated human bronchial epithelial (HBE) cells were exposed to formaldehyde or vehicle and cocultured with HASM cells. Agonist-induced [Ca2+]i and MYPT1 phosphorylation were determined in the cocultured HASM cells. Precision-cut human lung slices were exposed to PBS or varying concentrations of formaldehyde, and then carbachol-induced airway narrowing was determined 24 hours after exposure. HASM cells were transfected with nontargeting or nuclear factor erythroid-derived 2, like 2 (Nrf-2)-targeting small interfering RNA and exposed to formaldehyde or vehicle, followed by determination of antioxidant response (quinone oxido-reductase 1 and thioredoxin 1) and basal and agonist-induced MYPT1 phosphorylation. Formaldehyde enhanced the basal Rho-kinase activity and MYPT1 phosphorylation with little effect on agonist-induced [Ca2+]i in HASM cells. Formaldehyde induced Nrf-2-dependent antioxidant response in HASM cells, although the MYPT1 phosphorylation was independent of Nrf-2 induction. Although HBE cells exposed to formaldehyde had little effect on agonist-induced [Ca2+]i or MYPT1 phosphorylation in cocultured HASM cells, formaldehyde enhanced carbachol-induced airway responsiveness in precision-cut human lung slices. In conclusion, formaldehyde induces phosphorylation of the regulatory subunit of MYPT1, independent of formaldehyde-induced Nrf-2 activation in HASM cells. The findings suggest that the Rho kinase-dependent Ca2+ sensitization pathway plays a role in formaldehyde-induced AHR.

MicroRNA-10a controls airway smooth muscle cell proliferation via direct targeting of the PI3 kinase pathway.

Airway smooth muscle (ASM) cells play important physiological roles in the lung, and abnormal proliferation of ASM directly contributes to the airway remodeling during development of lung diseases such as asthma. MicroRNAs are small yet versatile gene tuners that regulate a variety of cellular processes, including cell growth and proliferation; however, little is known about the precise role of microRNAs in the proliferation of the ASM. Here we report that a specific microRNA (miR-10a) controls ASM proliferation through directly inhibiting the phosphoinositide 3-kinase (PI3K) pathway. Next-generation sequencing identified miR-10a as the most abundant microRNA expressed in primary human airway smooth muscle (HASM) cells, accounting for > 20% of all small RNA reads. Overexpression of miR-10a reduced mitogen-induced HASM proliferation by ∼50%, whereas inhibition of miR-10a increased HASM proliferation by ∼40%. Microarray profiling of HASM cells expressing miR-10a mimics identified 52 significantly down-regulated genes as potential targets of miR-10a, including the catalytic subunit α of PI3K (PIK3CA), the central component of the PI3K pathway. MiR-10a directly suppresses PIK3CA expression by targeting the 3'-untranslated region (3'-UTR) of the gene. Inhibition of PIK3CA by miR-10a reduced V-akt murine thymoma viral oncogene homolog 1 (AKT) phosphorylation and blunted the expression of cyclins and cyclin-dependent kinases that are required for HASM proliferation. Together, our study identifies a novel microRNA-mediated regulatory mechanism for PI3K signaling and ASM proliferation and further suggests miR-10a as a potential therapeutic target for lung diseases whose etiology resides in abnormal ASM proliferation.

Loss of regulator of G protein signaling 5 promotes airway hyperresponsiveness in the absence of allergic inflammation.

BACKGROUND: Although eosinophilic inflammation typifies allergic asthma, it is not a prerequisite for airway hyperresponsiveness (AHR), suggesting that underlying abnormalities in structural cells, such as airway smooth muscle (ASM), contribute to the asthmatic diathesis. Dysregulation of procontractile G protein-coupled receptor (GPCR) signaling in ASM could mediate enhanced contractility. OBJECTIVE: We explored the role of a regulator of procontractile GPCR signaling, regulator of G protein signaling 5 (RGS5), in unprovoked and allergen-induced AHR. METHODS: We evaluated GPCR-evoked Ca(2+) signaling, precision-cut lung slice (PCLS) contraction, and lung inflammation in naive and Aspergillus fumigatus-challenged wild-type and Rgs5(-/-) mice. We analyzed lung resistance and dynamic compliance in live anesthetized mice using invasive plethysmography. RESULTS: Loss of RGS5 promoted constitutive AHR because of enhanced GPCR-induced Ca(2+) mobilization in ASM. PCLSs from naive Rgs5(-/-) mice contracted maximally at baseline independently of allergen challenge. RGS5 deficiency had little effect on the parameters of allergic inflammation, including cell counts in bronchoalveolar lavage fluid, mucin production, ASM mass, and subepithelial collagen deposition. Unexpectedly, induced IL-13 and IL-33 levels were much lower in challenged lungs from Rgs5(-/-) mice relative to those seen in wild-type mice. CONCLUSION: Loss of RGS5 confers spontaneous AHR in mice in the absence of allergic inflammation. Because it is selectively expressed in ASM within the lung and does not promote inflammation, RGS5 might be a therapeutic target for asthma.

The Bronchoprotective Effects of Dual Pharmacology, Muscarinic Receptor Antagonist and ß2 Adrenergic Receptor Agonist Navafenterol in Human Small Airways.

Bronchodilators and anti-inflammatory agents are the mainstream treatments in chronic obstructive and pulmonary disease (COPD) and asthma. The combination of ß2 adrenergic receptor (ß2AR) agonists and muscarinic antagonists shows superior bronchoprotective effects compared to these agents individually. Navafenterol (AZD8871) is a single-molecule, dual pharmacology agent combining muscarinic antagonist and ß2AR agonist functions, currently in development as a COPD therapeutic. In precision-cut human lung slices (hPCLS), we investigated the bronchoprotective effect of navafenterol against two non-muscarinic contractile agonists, histamine and thromboxane A2 (TxA2) analog (U46619). Navafenterol pre-treatment significantly attenuated histamine-induced bronchoconstriction and ß2AR antagonist propranolol reversed this inhibitory effect. TxA2 analog-induced bronchoconstriction was attenuated by navafenterol pre-treatment, albeit to a lesser magnitude than that of histamine-induced bronchoconstriction. Propranolol completely reversed the inhibitory effect of navafenterol on TxA2 analog-induced bronchoconstriction. In the presence of histamine or TxA2 analog, navafenterol exhibits bronchoprotective effect in human airways and it is primarily mediated by ß2AR agonism of navafenterol.

Trichostatin A abrogates airway constriction, but not inflammation, in murine and human asthma models.

Histone deacetylase (HDAC) inhibitors may offer novel approaches in the treatment of asthma. We postulate that trichostatin A (TSA), a Class 1 and 2 inhibitor of HDAC, inhibits airway hyperresponsiveness in antigen-challenged mice. Mice were sensitized and challenged with Aspergillus fumigatus antigen (AF) and treated with TSA, dexamethasone, or vehicle. Lung resistance (R(L)) and dynamic compliance were measured, and bronchial alveolar lavage fluid (BALF) was analyzed for numbers of leukocytes and concentrations of cytokines. Human precision-cut lung slices (PCLS) were treated with TSA and their agonist-induced bronchoconstriction was measured, and TSA-treated human airway smooth muscle (ASM) cells were evaluated for the agonist-induced activation of Rho and intracellular release of Ca(2+). The activity of HDAC in murine lungs was enhanced by antigen and abrogated by TSA. TSA also inhibited methacholine (Mch)-induced increases in R(L) and decreases in dynamic compliance in naive control mice and in AF-sensitized and -challenged mice. Total cell counts, concentrations of IL-4, and numbers of eosinophils in BALF were unchanged in mice treated with TSA or vehicle, whereas dexamethasone inhibited the numbers of eosinophils in BALF and concentrations of IL-4. TSA inhibited the carbachol-induced contraction of PCLS. Treatment with TSA inhibited the intracellular release of Ca(2+) in ASM cells in response to histamine, without affecting the activation of Rho. The inhibition of HDAC abrogates airway hyperresponsiveness to Mch in both naive and antigen-challenged mice. TSA inhibits the agonist-induced contraction of PCLS and mobilization of Ca(2+) in ASM cells. Thus, HDAC inhibitors demonstrate a mechanism of action distinct from that of anti-inflammatory agents such as steroids, and represent a promising therapeutic agent for airway disease.

Steroid-induced fibroblast growth factors drive an epithelial-mesenchymal inflammatory axis in severe asthma.

Asthma and inflammatory airway diseases restrict airflow in the lung, compromising gas exchange and lung function. Inhaled corticosteroids (ICSs) can reduce inflammation, control symptoms, and improve lung function; however, a growing number of patients with severe asthma do not benefit from ICS. Using bronchial airway epithelial brushings from patients with severe asthma or primary human cells, we delineated a corticosteroid-driven fibroblast growth factor (FGF)-dependent inflammatory axis, with FGF-responsive fibroblasts promoting downstream granulocyte colony-stimulating factor (G-CSF) production, hyaluronan secretion, and neutrophilic inflammation. Allergen challenge studies in mice demonstrate that the ICS, fluticasone propionate, inhibited type 2-driven eosinophilia but induced a concomitant increase in FGFs, G-CSF, hyaluronan, and neutrophil infiltration. We developed a model of steroid-induced neutrophilic inflammation mediated, in part, by induction of an FGF-dependent epithelial-mesenchymal axis, which may explain why some individuals do not benefit from ICS. In further proof-of-concept experiments, we found that combination therapy with pan-FGF receptor inhibitors and corticosteroids prevented both eosinophilic and steroid-induced neutrophilic inflammation. Together, these results establish FGFs as therapeutic targets for severe asthma patients who do not benefit from ICS.

Oncostatin M expression induced by bacterial triggers drives airway inflammatory and mucus secretion in severe asthma.

Exacerbations of symptoms represent an unmet need for people with asthma. Bacterial dysbiosis and opportunistic bacterial infections have been observed in, and may contribute to, more severe asthma. However, the molecular mechanisms driving these exacerbations remain unclear. We show here that bacterial lipopolysaccharide (LPS) induces oncostatin M (OSM) and that airway biopsies from patients with severe asthma present with an OSM-driven transcriptional profile. This profile correlates with activation of inflammatory and mucus-producing pathways. Using primary human lung tissue or human epithelial and mesenchymal cells, we demonstrate that OSM is necessary and sufficient to drive pathophysiological features observed in severe asthma after exposure to LPS or Klebsiella pneumoniae. These findings were further supported through blockade of OSM with an OSM-specific antibody. Single-cell RNA sequencing from human lung biopsies identified macrophages as a source of OSM. Additional studies using Osm-deficient murine macrophages demonstrated that macrophage-derived OSM translates LPS signals into asthma-associated pathologies. Together, these data provide rationale for inhibiting OSM to prevent bacterial-associated progression and exacerbation of severe asthma.

An inherent dysfunction in soluble guanylyl cyclase is present in the airway of severe asthmatics and is associated with aberrant redox enzyme expression and compromised NO-cGMP signaling.

A subset of asthmatics develop a severe form of the disease whose etiology involves airway inflammation along with inherent drivers that remain ill-defined. To address this, we studied human airway smooth muscle cells (HASMC), whose relaxation drives airway bronchodilation and whose dysfunction contributes to airway obstruction and hypersensitivity in severe asthma. Because HASMC relaxation can be driven by the NO-soluble guanylyl cyclase (sGC)-cGMP signaling pathway, we questioned if HASMC from severe asthma donors might possess inherent defects in their sGC or in redox enzymes that support sGC function. We analyzed HASMC primary lines derived from 17 severe asthma and 16 normal donors and corresponding lung tissue samples regarding sGC activation by NO or by pharmacologic agonists, and also determined expression levels of sGC α1 and ß1 subunits, supporting redox enzymes, and related proteins. We found a majority of the severe asthma donor HASMC (12/17) and lung samples primarily expressed a dysfunctional sGC that was NO-unresponsive and had low heterodimer content and high Hsp90 association. This sGC phenotype correlated with lower expression levels of the supporting redox enzymes cytochrome b5 reductase, catalase, and thioredoxin-1, and higher expression of heme oxygenases 1 and 2. Together, our work reveals that severe asthmatics are predisposed toward defective NO-sGC-cGMP signaling in their airway smooth muscle due to an inherent sGC dysfunction, which in turn is associated with inherent changes in the cell redox enzymes that impact sGC maturation and function.

Deletion of microsomal prostaglandin E synthase-1 does not alter ozone-induced airway hyper-responsiveness.

Nonsteroidal anti-inflammatory drugs ameliorate pain and fever by inhibiting cyclooxygenase (COX) and suppressing prostanoid formation. Microsomal prostaglandin E synthase-1 (mPGES-1) catalyzes formation of PGE(2) from the COX product PGH(2) and has emerged as a therapeutic target. Inhibition of mPGES-1, however, renders the PGH(2) substrate available for diversion to other PG synthases. To address the possibility that substrate diversion augments formation of PGs that might modulate bronchial tone, we assessed the impact of mPGES-1 deletion in a mouse model of ozone-induced airway hyper-responsiveness. Ozone exposure increased total lung resistance to inhaled methacholine in wild-type mice. Deletion of mPGES-1 had little effect on total lung resistance in either naive or ozone-exposed animals. The carbachol-induced narrowing of luminal diameter in intrapulmonary airways of lung slices from acute ozone-exposed mice was also unaltered by mPGES-1 deletion. Likewise, although concentrations of PGE(2) were reduced in bronchoalveolar lavage fluid, whereas 6-keto-PGF(1alpha), PGD(2), and PGF(2alpha), all were increased, deletion of mPGES-1 failed to influence cell trafficking into the airways of either naive or ozone-exposed animals. Despite biochemical evidence of PGH(2) substrate diversion to potential bronchomodulator PGs, deletion of mPGES-1 had little effect on ozone-induced airway inflammation or airway hyper-responsiveness. Pharmacologically targeting mPGES-1 may not predispose patients at risk to airway dysfunction.

Inhibition of myristoylated alanine-rich C kinase substrate (MARCKS) protein inhibits ozone-induced airway neutrophilia and inflammation.

Evidence suggests inhibition of leukocyte trafficking mitigates, in part, ozone-induced inflammation. In the present study, the authors postulated that inhibition of myristoylated alanine-rich C kinase substrate (MARCKS), an 82-kDa protein with multiple biological roles, could inhibit ozone-induced leukocyte trafficking and cytokine secretions. BALB/c mice (n = 5/cohort) were exposed to ozone (100 ppb) or forced air (FA) for 4 hours. MARCKS-inhibiting peptides, MANS, BIO-11000, BIO-11006, or scrambled control peptide RNS, were intratracheally administered prior to ozone exposure. Ozone selectively enhanced bronchoalveolar lavage (BAL) levels of killer cells (KCs; 6 +/- 0.9-fold), interleukin-6 (IL-6; 12.7 +/- 1.9-fold), and tumor necrosis factor (TNF; 2.1 +/- 0.5-fold) as compared to cohorts exposed to FA. Additionally, ozone increased BAL neutrophils by 21% +/- 2% with no significant (P > .05) changes in other cell types. MANS, BIO-11000, and BIO-11006 significantly reduced ozone-induced KC secretion by 66% +/- 14%, 47% +/- 15%, and 71.1% +/- 14%, and IL-6 secretion by 69% +/- 12%, 40% +/- 7%, and 86.1% +/- 11%, respectively. Ozone-mediated increases in BAL neutrophils were reduced by MANS (86% +/- 7%) and BIO-11006 (84% +/- 2.5%), but not BIO-11000. These studies identify for the first time the novel potential of MARCKS protein inhibitors in abrogating ozone-induced increases in neutrophils, cytokines, and chemokines in BAL fluid. BIO-11006 is being developed as a treatment for chronic obstructive pulmonary disorder (COPD) and is currently being evaluated in a phase 2 clinical study.

CD33 recruitment inhibits IgE-mediated anaphylaxis and desensitizes mast cells to allergen.

Allergen immunotherapy for patients with allergies begins with weekly escalating doses of allergen under medical supervision to monitor and treat IgE mast cell-mediated anaphylaxis. There is currently no treatment to safely desensitize mast cells to enable robust allergen immunotherapy with therapeutic levels of allergen. Here, we demonstrated that liposomal nanoparticles bearing an allergen and a high-affinity glycan ligand of the inhibitory receptor CD33 profoundly suppressed IgE-mediated activation of mast cells, prevented anaphylaxis in Tg mice with mast cells expressing human CD33, and desensitized mice to subsequent allergen challenge for several days. We showed that high levels of CD33 were consistently expressed on human skin mast cells and that the antigenic liposomes with CD33 ligand prevented IgE-mediated bronchoconstriction in slices of human lung. The results demonstrated the potential of exploiting CD33 to desensitize mast cells to provide a therapeutic window for administering allergen immunotherapy without triggering anaphylaxis.

Elastomeric sensor surfaces for high-throughput single-cell force cytometry.

As cells with aberrant force-generating phenotypes can directly lead to disease, cellular force-generation mechanisms are high-value targets for new therapies. Here, we show that single-cell force sensors embedded in elastomers enable single-cell force measurements with ~100-fold improvement in throughput than was previously possible. The microtechnology is scalable and seamlessly integrates with the multi-well plate format, enabling highly parallelized time-course studies. In this regard, we show that airway smooth muscle cells isolated from fatally asthmatic patients have innately greater and faster force-generation capacity in response to stimulation than healthy control cells. By simultaneously tracing agonist-induced calcium flux and contractility in the same cell, we show that the calcium level is ultimately a poor quantitative predictor of cellular force generation. Finally, by quantifying phagocytic forces in thousands of individual human macrophages, we show that force initiation is a digital response (rather than a proportional one) to the proper immunogen. By combining mechanobiology at the single-cell level with high-throughput capabilities, this microtechnology can support drug-discovery efforts for clinical conditions associated with aberrant cellular force generation.

Publisher Correction: Elastomeric sensor surfaces for high-throughput single-cell force cytometry.

In the version of this Article originally published, in Fig. 1a, all cells in the top schematic were missing, and in the bottom-left schematic showing multiple pattern shapes, two cells were missing in the bottom-right corner. This figure has now been updated in all versions of the Article.