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Type 2 T helper (TH2) cells are protective against parasitic worm infections but also aggravate allergic inflammation. Although the role of dendritic cells (DCs) in TH2 cell differentiation is well established, the underlying mechanisms are largely unknown. Here, we show that DC induction of TH2 cells depends on membrane-associated RING-CH-1 (MARCH1) ubiquitin ligase. The pro-TH2 effect of MARCH1 relied on lymph node (LN)–resident DCs, which triggered T cell receptor (TCR) signaling and induced GATA-3 expression from naïve CD4+ T cells independent of tissue-driven migratory DCs. Mice with mutations in the ubiquitin acceptor sites of MHCII and CD86, the two substrates of MARCH1, failed to develop TH2 cells. These findings suggest that TH2 cell development depends on ubiquitin-mediated clearance of antigen-presenting and costimulatory molecules by LN-resident DCs and consequent control of TCR signaling.

Severe asthma remains challenging to manage and has limited treatment options. We have previously shown that targeting smooth muscle integrin α5β1 interaction with fibronectin can mitigate the effects of airway hyperresponsiveness by impairing force transmission. In this study, we show that another member of the integrin superfamily, integrin α2β1, is present in airway smooth muscle and capable of regulating force transmission via cellular tethering to the matrix protein collagen I and, to a lesser degree, laminin-111. The addition of an inhibitor of integrin α2β1 impaired IL-13-enhanced contraction in mouse tracheal rings and human bronchial rings and abrogated the exaggerated bronchoconstriction induced by allergen sensitization and challenge. We confirmed that this effect was not due to alterations in classic intracellular myosin light chain phosphorylation regulating muscle shortening. Although IL-13 did not affect surface expression of α2β1, it did increase α2β1-mediated adhesion and the level of expression of an activation-specific epitope on the β1 subunit. We developed a method to simultaneously quantify airway narrowing and muscle shortening using 2-photon microscopy and demonstrated that inhibition of α2β1 mitigated IL-13-enhanced airway narrowing without altering muscle shortening by impairing the tethering of muscle to the surrounding matrix. Our data identified cell matrix tethering as an attractive therapeutic target to mitigate the severity of airway contraction in asthma.

Clinical application of inhaled glucocorticoids (GCs) has been hampered in the case of steroid-resistant severe asthma. To overcome this limitation, we have developed a series of highly potent GCs, including VSGC12, VSG158, and VSG159 based on the structural insight into the glucocorticoid receptor (GR). Particularly, VSG158 exhibits a maximal repression of lung inflammation and is 10 times more potent than the currently most potent clinical GC, Fluticasone Furoate (FF), in a murine model of asthma. More importantly, VSG158 displays a unique property to reduce neutrophilic inflammation in a steroid-resistant airway inflammation model, which is refractory to clinically available GCs, including dexamethasone and FF. VSG158 and VSG159 are able to deliver effective treatments with reduced off-target and side effects. In addition, these GCs also display pharmacokinetic properties that are suitable for the inhalation delivery method for asthma treatment. Taken together, the excellent therapeutic and side-effect profile of these highly potent GCs holds promise for treating steroid-resistant severe asthma.

Th17 cell responses orchestrate immunity against extracellular pathogens but also underlie autoimmune disease pathogenesis. In this study, we uncovered a distinct and critical role for miR-18a in limiting Th17 cell differentiation. miR-18a was the most dynamically upregulated microRNA of the miR-17-92 cluster in activated T cells. miR-18a deficiency enhanced CCR6+ RAR-related orphan receptor (ROR)γt+ Th17 cell differentiation in vitro and increased the number of tissue Th17 cells expressing CCR6, RORγt, and IL-17A in airway inflammation models in vivo. Sequence-specific miR-18 inhibitors increased CCR6 and RORγt expression in mouse and human CD4+ T cells, revealing functional conservation. miR-18a directly targeted Smad4, Hif1a, and Rora, all key transcription factors in the Th17 cell gene-expression program. These findings indicate that activating signals influence the outcome of Th cell differentiation via differential regulation of mature microRNAs within a common cluster.

Treatment options are limited for severe asthma, and the need for additional therapies remains great. Previously, we demonstrated that integrin αvβ6-deficient mice are protected from airway hyperresponsiveness, due in part to increased expression of the murine ortholog of human chymase. Here, we determined that chymase protects against cytokine-enhanced bronchoconstriction by cleaving fibronectin to impair tension transmission in airway smooth muscle (ASM). Additionally, we identified a pathway that can be therapeutically targeted to mitigate the effects of airway hyperresponsiveness. Administration of chymase to human bronchial rings abrogated IL-13-enhanced contraction, and this effect was not due to alterations in calcium homeostasis or myosin light chain phosphorylation. Rather, chymase cleaved fibronectin, inhibited ASM adhesion, and attenuated focal adhesion phosphorylation. Disruption of integrin ligation with an RGD-containing peptide abrogated IL-13-enhanced contraction, with no further effect from chymase. We identified α5β1 as the primary fibronectin-binding integrin in ASM, and α5β1-specific blockade inhibited focal adhesion phosphorylation and IL-13-enhanced contraction, with no additional effect from chymase. Delivery of an α5β1 inhibitor into murine airways abrogated the exaggerated bronchoconstriction induced by allergen sensitization and challenge. Finally, α5β1 blockade enhanced the effect of the bronchodilator isoproterenol on airway relaxation. Our data identify the α5β1 integrin as a potential therapeutic target to mitigate the severity of airway contraction in asthma.

BackgroundEpithelial barrier dysfunction and increased permeability may contribute to antigen sensitization and disease progression in asthma. Claudin-18.1 is the only known lung-specific tight junction protein, but its contribution to airway barrier function or asthma is unclear.ObjectivesWe sought to test the hypotheses that claudin-18 is a determinant of airway epithelial barrier function that is downregulated by IL-13 and that claudin-18 deficiency results in increased aeroantigen sensitization and airway hyperresponsiveness.MethodsClaudin-18.1 mRNA levels were measured in airway epithelial brushings from healthy controls and patients with asthma. In patients with asthma, claudin-18 levels were compared with a three-gene-mean marker of TH2 inflammation. Airway epithelial permeability changes due to claudin-18 deficiency were measured in 16HBE cells and claudin-18 null mice. The effect of IL-13 on claudin expression was determined in primary human airway epithelial cells and in mice. Airway hyperresponsiveness and serum IgE levels were compared in claudin-18 null and wild-type mice following aspergillus sensitization.ResultsEpithelial brushings from patients with asthma (n = 67) had significantly lower claudin-18 mRNA levels than did those from healthy controls (n = 42). Claudin-18 levels were lowest among TH2-high patients with asthma. Loss of claudin-18 was sufficient to impair epithelial barrier function in 16HBE cells and in mouse airways. IL-13 decreased claudin-18 expression in primary human cells and in mice. Claudin-18 null mice had significantly higher serum IgE levels and increased airway responsiveness following intranasal aspergillus sensitization.ConclusionsThese data support the hypothesis that claudin-18 is an essential contributor to the airway epithelial barrier to aeroantigens. Furthermore, TH2 inflammation suppresses claudin-18 expression, potentially promoting sensitization and airway hyperresponsiveness.

Airway mucus in cystic fibrosis (CF) is highly elastic, but themechanism behind this pathology is unclear. We hypothesized that the biophysical properties of CF mucus are altered because of neutrophilic oxidative stress. Using confocal imaging, rheology, and biochemical measures of inflammation and oxidation, we found that CF airway mucus gels have a molecular architecture characterized by a core of mucin covered by a web of DNA and a rheological profile characterized by high elasticity that can be normalized by chemical reduction. We also found that high levels of reactive oxygen species in CF mucus correlated positively and significantly with high concentrations of the oxidized products of cysteine (disulfide cross-links). To directly determine whether oxidation can cross-link mucins to increase mucus elasticity, we exposed induced sputum from healthy subjects to oxidizing stimuli and found a marked and thiol-dependent increase in sputum elasticity. Targeting mucin disulfide cross-links using current thiol-amino structures such as N-acetylcysteine (NAC) requires high drug concentrations to have mucolytic effects. We therefore synthesized a thiol-carbohydrate structure (methyl 6-thio-6-deoxy-α-D-galactopyranoside) and found that it had stronger reducing activity than NAC and more potent and fast-acting mucolytic activity in CF sputum. Thus, oxidation arising from airway inflammation or environmental exposure contributes to pathologic mucus gel formation in the lung, which suggests that it can be targeted by thiol-modified carbohydrates.

The intracellular scaffold protein IQGAP1 supports protein complexes in conjunction with numerous binding partners involved in multiple cellular processes. Here, we determined that IQGAP1 modulates airway smooth muscle contractility. Compared with WT controls, at baseline as well as after immune sensitization and challenge, Iqgap1-/- mice had higher airway responsiveness. Tracheal rings from Iqgap1-/- mice generated greater agonist-induced contractile force, even after removal of the epithelium. RhoA, a regulator of airway smooth muscle contractility, was activated in airway smooth muscle lysates from Iqgap1-/- mice. Likewise, knockdown of IQGAP1 in primary human airway smooth muscle cells increased RhoA activity. Immunoprecipitation studies indicated that IQGAP1 binds to both RhoA and p190A-RhoGAP, a GTPase-activating protein that normally inhibits RhoA activation. Proximity ligation assays in primary airway human smooth muscle cells and mouse tracheal sections revealed colocalization of p190A-RhoGAP and RhoA; however, these proteins did not colocalize in IQGAP1 knockdown cells or in Iqgap1-/- trachea. Compared with healthy controls, human subjects with asthma had decreased IQGAP1 expression in airway biopsies. Together, these data demonstrate that IQGAP1 acts as a scaffold that colocalizes p190A-RhoGAP and RhoA, inactivating RhoA and suppressing airway smooth muscle contraction. Furthermore, our results suggest that IQGAP1 has the potential to modulate airway contraction severity in acute asthma.

S-nitrosoglutathione reductase (GSNOR) is a key denitrosylase and critically important for protecting immune and other cells from nitrosative stress. Pharmacological inhibition of GSNOR is being actively pursued as a therapeutic approach to increase S-nitrosoglutathione levels for the treatment of asthma and cystic fibrosis. In the present study, we employed GSNOR-deficient (GSNOR(-/-)) mice to investigate whether inactivation of GSNOR may increase susceptibility to pulmonary infection by Klebsiella pneumoniae, a common cause of nosocomial pneumonia. We found that compared to wild-type mice, bacterial colony forming units 48 h after intranasal infection with K. pneumoniae were increased over 4-folds in lung and spleen and strikingly, over a 1000-folds in blood of GSNOR(-/-) mice. Lung injury was comparable between infected wild-type and GSNOR(-/-) mice, but inflammation and injury was significantly elevated in spleen of GSNOR(-/-) mice. Whereas all wild-type mice survived 48 h after infection, 10 of 23 GSNOR(-/-) mice died. Thus, GSNOR appears to play a crucial role in controlling pulmonary and systemic infection by K. pneumoniae. Our results suggest that patients treated in clinical trials with inhibitors of GSNOR should be carefully monitored for signs of infection.

Increased airway smooth muscle (ASM) contractility and the development of airway hyperresponsiveness (AHR) are cardinal features of asthma, but the signaling pathways that promote these changes are poorly understood. Tyrosine phosphorylation is tightly regulated by the opposing actions of protein tyrosine kinases and phosphatases, but little is known about whether tyrosine phosphatases influence AHR. Here, we demonstrate that genetic inactivation of receptor-like protein tyrosine phosphatase J (Ptprj), which encodes CD148, protected mice from the development of increased AHR in two different asthma models. Surprisingly, CD148 deficiency minimally affected the inflammatory response to allergen, but significantly altered baseline pulmonary resistance. Mice specifically lacking CD148 in smooth muscle had decreased AHR, and the frequency of calcium oscillations in CD148-deficient ASM was substantially attenuated, suggesting that signaling pathway alterations may underlie ASM contractility. Biochemical analysis of CD148-deficient ASM revealed hyperphosphorylation of the C-terminal inhibitory tyrosine of SRC family kinases (SFKs), implicating CD148 as a critical positive regulator of SFK signaling in ASM. The effect of CD148 deficiency on ASM contractility could be mimicked by treatment of both mouse trachea and human bronchi with specific SFK inhibitors. Our studies identify CD148 and the SFKs it regulates in ASM as potential targets for the treatment of AHR.

Airway obstruction is a hallmark of allergic asthma and is caused primarily by airway smooth muscle (ASM) hypercontractility. Airway inflammation leads to the release of cytokines that enhance ASM contraction by increasing ras homolog gene family, member A (RhoA) activity. The protective mechanisms that prevent or attenuate the increase in RhoA activity have not been well studied. Here, we report that mice lacking the gene that encodes the protein Milk Fat Globule-EGF factor 8 (Mfge8(-/-)) develop exaggerated airway hyperresponsiveness in experimental models of asthma. Mfge8(-/-) ASM had enhanced contraction after treatment with IL-13, IL-17A, or TNF-α. Recombinant Mfge8 reduced contraction in murine and human ASM treated with IL-13. Mfge8 inhibited IL-13-induced NF-κB activation and induction of RhoA. Mfge8 also inhibited rapid activation of RhoA, an effect that was eliminated by an inactivating point mutation in the RGD integrin-binding site in recombinant Mfge8. Human subjects with asthma had decreased Mfge8 expression in airway biopsies compared with healthy controls. These data indicate that Mfge8 binding to integrin receptors on ASM opposes the effect of allergic inflammation on RhoA activity and identify a pathway for specific inhibition of ASM hypercontractility in asthma.

TGFβ activation and signaling have been extensively studied in experimental models of allergen-induced asthma as potential therapeutic targets during chronic or acute phases of the disease. Outcomes of experimental manipulation of TGFβ activity have been variable, in part due to use of different model systems. Using an ovalbumin (OVA)-induced mouse model of asthma, we here show that innate variation within TGFβ1 genetic modifier loci, Tgfbm2 and Tgfbm3, alters disease susceptibility. Specifically, Tgfbm2(129) and Tgfbm3(C57) synergize to reverse accentuated airway hyperresponsiveness (AHR) caused by low TGFβ1 levels in Tgfb1(+/-) mice of the NIH/OlaHsd strain. Moreover, epistatic interaction between Tgfbm2(129) and Tgfbm3(C57) uncouples the inflammatory response to ovalbumin from those of airway remodeling and airway hyperresponsiveness, illustrating independent genetic control of these responses. We conclude that differential inheritance of genetic variants of Tgfbm genes alters biological responses to reduced TGFβ1 signaling in an experimental asthma model. TGFβ antagonists for treatment of lung diseases might therefore give diverse outcomes, dependent on genetic variation.

Mucous cell hyperplasia and airway smooth muscle (ASM) hyperresponsiveness are hallmark features of inflammatory airway diseases, including asthma. Here, we show that the recently identified calcium-activated chloride channel (CaCC) TMEM16A is expressed in the adult airway surface epithelium and ASM. The epithelial expression is increased in asthmatics, particularly in secretory cells. Based on this and the proposed functions of CaCC, we hypothesized that TMEM16A inhibitors would negatively regulate both epithelial mucin secretion and ASM contraction. We used a high-throughput screen to identify small-molecule blockers of TMEM16A-CaCC channels. We show that inhibition of TMEM16A-CaCC significantly impairs mucus secretion in primary human airway surface epithelial cells. Furthermore, inhibition of TMEM16A-CaCC significantly reduces mouse and human ASM contraction in response to cholinergic agonists. TMEM16A-CaCC blockers, including those identified here, may positively impact multiple causes of asthma symptoms.

Exaggerated contraction of airway smooth muscle is the major cause of symptoms in asthma, but the mechanisms that prevent exaggerated contraction are incompletely understood. Here, we showed that integrin α9β1 on airway smooth muscle localizes the polyamine catabolizing enzyme spermidine/spermine N1-acetyltransferase (SSAT) in close proximity to the lipid kinase PIP5K1γ. As PIP5K1γ is the major source of PIP2 in airway smooth muscle and its activity is regulated by higher-order polyamines, this interaction inhibited IP3-dependent airway smooth muscle contraction. Mice lacking integrin α9β1 in smooth muscle had increased airway responsiveness in vivo, and loss or inhibition of integrin α9β1 increased in vitro airway narrowing and airway smooth muscle contraction in murine and human airways. Contraction was enhanced in control airways by the higher-order polyamine spermine or by cell-permeable PIP2, but these interventions had no effect on airways lacking integrin α9β1 or treated with integrin α9β1-blocking antibodies. Enhancement of SSAT activity or knockdown of PIP5K1γ inhibited airway contraction, but only in the presence of functional integrin α9β1. Therefore, integrin α9β1 appears to serve as a brake on airway smooth muscle contraction by recruiting SSAT, which facilitates local catabolism of polyamines and thereby inhibits PIP5K1γ. Targeting key components of this pathway could thus lead to new treatment strategies for asthma.

Emerging evidence suggests that the T helper 17 (T(H)17) subset of αβ T cells contributes to the development of allergic asthma. In this study, we found that mice lacking the αvβ8 integrin on dendritic cells did not generate T(H)17 cells in the lung and were protected from airway hyper-responsiveness in response to house dust mite and ovalbumin sensitization and challenge. Because loss of T(H)17 cells inhibited airway narrowing without any obvious effects on airway inflammation or epithelial morphology, we examined the direct effects of T(H)17 cytokines on mouse and human airway smooth muscle function. Interleukin-17A (IL-17A), but not IL-17F or IL-22, enhanced contractile force generation of airway smooth muscle through an IL-17 receptor A (IL-17RA)-IL-17RC, nuclear factor κ light-chain enhancer of activated B cells (NF-κB)-ras homolog gene family, member A (RhoA)-Rho-associated coiled-coil containing protein kinase 2 (ROCK2) signaling cascade. Mice lacking integrin αvβ8 on dendritic cells showed impaired activation of this pathway after ovalbumin sensitization and challenge, and the diminished contraction of the tracheal rings in these mice was reversed by IL-17A. These data indicate that the IL-17A produced by T(H)17 cells contributes to allergen-induced airway hyper-responsiveness through direct effects on airway smooth muscle.

PurposeThe goal of this study was to test the hypothesis that by controlling intracellular uptake, organic cation transporter 1, Oct1 is a key determinant of the disposition and toxicity of cis-diammine(pyridine)chloroplatinum(II)(CDPCP) and oxaliplatin.MethodsPharmacokinetics, tissue accumulation and toxicity of CDPCP and oxaliplatin were compared between Oct1-/- and wild-type mice.ResultsAfter intravenous administration, hepatic and intestinal accumulation of CDPCP was 2.7-fold and 3.9-fold greater in Oct1 wild-type mice (p < 0.001). Deletion of Oct1 resulted in a significantly decreased clearance (0.444 ± 0.0391 ml/min*kg versus 0.649 ± 0.0807 ml/min*kg in wild-type mice, p < 0.05) and volume distribution (1.90 ± 0.161 L/kg versus 3.37 ± 0.196 L/kg in wild-type mice, p < 0.001). Moreover, Oct1 deletion resulted in more severe off-target toxicities in CDPCP-treated mice. Histologic examination of the liver and measurements of liver function indicated that the level of hepatic toxicity was mild and reversible, but was more apparent in the wild-type mice. In contrast, the effect of Oct1 on the pharmacokinetics and toxicity of oxaliplatin in the mice was minimal.ConclusionsOur study suggests that Oct1 plays an important role in the pharmacokinetics, tissue distribution and toxicity of CDPCP, but not oxaliplatin.

The integrin alpha 9 beta 1 is widely expressed on neutrophils, smooth muscle, hepatocytes, endothelia, and some epithelia. We now show that mice lacking this integrin have a dramatic defect in neutrophil development, with decreased numbers of granulocyte precursors in bone marrow and impaired differentiation of bone marrow cells into granulocytes. In response to granulocyte colony-stimulating factor (G-CSF), alpha 9-deficient bone marrow cells or human bone marrow cells incubated with alpha 9 beta 1-locking antibody demonstrated decreased phosphorylation of signal transducer and activator of transcription 3 and extracellular signal-regulated protein kinase. These effects depended on the alpha 9 subunit cytoplasmic domain, which was required for formation of a physical complex between alpha 9 beta 1 and ligated G-CSF receptor. Integrin alpha 9 beta 1 was required for granulopoiesis and played a permissive role in the G-CSF-signaling pathway, suggesting that this integrin could play an important role in disorders of granulocyte development and other conditions characterized by defective G-CSF signaling.

Nippostrongylus brasiliensis infection and ovalbumin-induced allergic lung pathology are highly interleukin (IL)-4/IL-13 dependent, but the contributions of IL-4/IL-13 from adaptive (T helper [Th]2 cells) and innate (eosinophil, basophils, and mast cells) immune cells remain unknown. Although required for immunoglobulin (Ig)E induction, IL-4/IL-13 from Th2 cells was not required for worm expulsion, tissue inflammation, or airway hyperreactivity. In contrast, innate hematopoietic cell-derived IL-4/IL-13 was dispensable for Th2 cell differentiation in lymph nodes but required for effector cell recruitment and tissue responses. Eosinophils were not required for primary immune responses. Thus, components of type 2 immunity mediated by IL-4/IL-13 are partitioned between T cell-dependent IgE and an innate non-eosinophil tissue component, suggesting new strategies for interventions in allergic immunity.

Apoptosis is a critical process in normal mammary gland development and the rapid clearance of apoptotic cells prevents tissue injury associated with the release of intracellular antigens from dying cells. Milk fat globule-EGF-factor 8 (Mfge8) is a milk glycoprotein that is abundantly expressed in the mammary gland epithelium and has been shown to facilitate the clearance of apoptotic lymphocytes by splenic macrophages. We report that mice with disruption of Mfge8 had normal mammary gland development until involution. However, abnormal mammary gland remodeling was observed postlactation in Mfge8 mutant mice. During early involution, Mfge8 mutant mice had increased numbers of apoptotic cells within the mammary gland associated with a delay in alveolar collapse and fat cell repopulation. As involution progressed, Mfge8 mutants developed inflammation as assessed by CD45 and CD11b staining of mammary gland tissue sections. With additional pregnancies, Mfge8 mutant mice developed progressive dilatation of the mammary gland ductal network. These data demonstrate that Mfge8 regulates the clearance of apoptotic epithelial cells during mammary gland involution and that the absence of Mfge8 leads to inflammation and abnormal mammary gland remodeling.

Daily phagocytosis by the retinal pigment epithelium (RPE) of spent photoreceptor outer segment fragments is critical for vision. In the retina, early morning circadian photoreceptor rod shedding precedes synchronized uptake of shed photoreceptor particles by RPE cells. In vitro, RPE cells use the integrin receptor alphavbeta5 for particle binding. Here, we tested RPE phagocytosis and retinal function in beta5 integrin--deficient mice, which specifically lack alphavbeta5 receptors. Retinal photoresponses severely declined with age in beta5-/- mice, whose RPE accumulated autofluorescent storage bodies that are hallmarks of human retinal aging and disease. beta5-/- RPE in culture failed to take up isolated photoreceptor particles. beta5-/- RPE in vivo retained basal uptake levels but lacked the burst of phagocytic activity that followed circadian photoreceptor shedding in wild-type RPE. Rhythmic activation of focal adhesion and Mer tyrosine kinases that mediate wild-type retinal phagocytosis was also completely absent in beta5-/- retina. These results demonstrate an essential role for alphavbeta5 integrin receptors and their downstream signaling pathways in synchronizing retinal phagocytosis. Furthermore, they identify the beta5-/- integrin mouse strain as a new animal model of age-related retinal dysfunction.

Daily phagocytosis by the retinal pigment epithelium (RPE) of spent photoreceptor outer segment fragments is critical for vision. In the retina, early morning circadian photoreceptor rod shedding precedes synchronized uptake of shed photoreceptor particles by RPE cells. In vitro, RPE cells use the integrin receptor alphavbeta5 for particle binding. Here, we tested RPE phagocytosis and retinal function in beta5 integrin--deficient mice, which specifically lack alphavbeta5 receptors. Retinal photoresponses severely declined with age in beta5-/- mice, whose RPE accumulated autofluorescent storage bodies that are hallmarks of human retinal aging and disease. beta5-/- RPE in culture failed to take up isolated photoreceptor particles. beta5-/- RPE in vivo retained basal uptake levels but lacked the burst of phagocytic activity that followed circadian photoreceptor shedding in wild-type RPE. Rhythmic activation of focal adhesion and Mer tyrosine kinases that mediate wild-type retinal phagocytosis was also completely absent in beta5-/- retina. These results demonstrate an essential role for alphavbeta5 integrin receptors and their downstream signaling pathways in synchronizing retinal phagocytosis. Furthermore, they identify the beta5-/- integrin mouse strain as a new animal model of age-related retinal dysfunction.

Severe asthma remains challenging to manage and has limited treatment options. We have previously shown that targeting smooth muscle integrin [[alpha].sub.5][[beta].sub.1] interaction with fibronectin can mitigate the effects of airway hyperresponsiveness by impairing force transmission. In this study, we show that another member of the integrin superfamily, integrin [[alpha].sub.2][[beta].sub.1], is present in airway smooth muscle and capable of regulating force transmission via cellular tethering to the matrix protein collagen I and, to a lesser degree, laminin-111. The addition of an inhibitor of integrin [[alpha].sub.2][[beta].sub.1] impaired IL-13- enhanced contraction in mouse tracheal rings and human bronchial rings and abrogated the exaggerated bronchoconstriction induced by allergen sensitization and challenge. We confirmed that this effect was not due to alterations in classic intracellular myosin light chain phosphorylation regulating muscle shortening. Although IL-13 did not affect surface expression of [[alpha].sub.2][[beta].sub.1], it did increase [[alpha].sub.2][[beta].sub.1]-mediated adhesion and the level of expression of an activation-specific epitope on the [[beta].sub.1] subunit. We developed a method to simultaneously quantify airway narrowing and muscle shortening using 2-photon microscopy and demonstrated that inhibition of [[alpha].sub.2][[beta].sub.1] mitigated IL-13-enhanced airway narrowing without altering muscle shortening by impairing the tethering of muscle to the surrounding matrix. Our data identified cell matrix tethering as an attractive therapeutic target to mitigate the severity of airway contraction in asthma., Introduction Asthma is a chronic inflammatory disease characterized by airway hyperresponsiveness, inflammation, and remodeling (1-3). Exaggerated airway narrowing in response to stimuli is the hallmark of airway hyperresponsiveness and is [...]

Allergic asthma is the most common form of asthma, affecting more than 10 million Americans. Although it is clear that mast cells have a key role in the pathogenesis of allergic asthma, the mechanisms by which they regulate airway narrowing in vivo remain to be elucidated. Here we report that mice lacking αvβ6 integrin are protected from exaggerated airway narrowing in a model of allergic asthma. Expression microarrays of the airway epithelium revealed mast cell proteases among the most prominent differentially expressed genes, with expression of mouse mast cell protease 1 (mMCP-1) induced by allergen challenge in WT mice and expression of mMCP-4, -5, and -6 increased at baseline in β6-deficient mice. These findings were most likely explained by loss of TGF-βactivation, since the epithelial integrin αvβ6 is a critical activator of latent TGF-β, and in vitro-differentiated mast cells showed TGF-β-dependent expression of mMCP-1 and suppression of mMCP-4 and -6. In vitro, mMCP-1 increased contractility of murine tracheal rings, an effect that depended on intact airway epithelium, whereas mMCP-4 inhibited IL-13-induced epithelial-independent enhancement of contractility. These results suggest that intraepithelial activation of TGF-βby the αvβ6 integrin regulates airway responsive-ness by modulating mast cell protease expression and that these proteases and their proteolytic substrates could be novel targets for improved treatment of allergic asthma., Introduction Mast cells are mainly known for their harmful effects during allergic inflammation and contribute to asthma in several ways (1). Mast cell activation induces degranulation and release of a [...]

The cytokine transforming growth factor-β (TGF-β) is an important negative regulator of adaptive immunity (1-3). TGF-β is secreted by cells as an inactive precursor that must be activated to exert [...]

Author(s): David G. Morris [1, 2]; Xiaozhu Huang [1]; Naftali Kaminski [3]; Yanli Wang [1]; Steven D. Shapiro [4]; Gregory Dolganov [2]; Adam Glick [5]; Dean Sheppard (corresponding author) [1, [...]

Vascular endothelial growth factor (VEGF) promotes vascular permeability (VP) and neovascularization, and is required for development. We find that VEGF-stimulated Src activity in chick embryo blood vessels induces the coupling of focal adhesion kinase (FAK) to integrin [alpha]v[beta]5, a critical event in VEGF-mediated signaling and biological responsiveness. In contrast, FAK is constitutively associated with [beta]1 and [beta]3 integrins in the presence or absence of growth factors. In cultured endothelial cells, VEGF, but not basic fibroblast growth factor, promotes the Src-mediated phosphorylation of FAK on tyrosine 861, which contributes to the formation of a FAK/[alpha]v[beta]5 signaling complex. Moreover, formation of this FAK/[alpha]v[beta]5 complex is significantly reduced in [pp60.sup.c-src] -deficient mice. Supporting these results, mice deficient in either [pp60.sup.c-src] or integrin [beta]5, but not integrin [beta]3, have a reduced VP response to VEGF. This FAK/[alpha]v[beta]5 complex was also detected in epidermal growth factor-stimulated epithelial cells, suggesting a function for this complex outside the endothelium. Our findings indicate that Src can coordinate specific growth factor and extracellular matrix inputs by recruiting integrin [alpha]v[beta]5 into a FAK-containing signaling complex during growth factor-mediated biological responses.

Type 2 T helper (TH2) cells are protective against parasitic worm infections but also aggravate allergic inflammation. Although the role of dendritic cells (DCs) in TH2 cell differentiation is well established, the underlying mechanisms are largely unknown. Here, we show that DC induction of TH2 cells depends on membrane-associated RING-CH-1 (MARCH1) ubiquitin ligase. The pro-TH2 effect of MARCH1 relied on lymph node (LN)–resident DCs, which triggered T cell receptor (TCR) signaling and induced GATA-3 expression from naïve CD4+ T cells independent of tissue-driven migratory DCs. Mice with mutations in the ubiquitin acceptor sites of MHCII and CD86, the two substrates of MARCH1, failed to develop TH2 cells. These findings suggest that TH2 cell development depends on ubiquitin-mediated clearance of antigen-presenting and costimulatory molecules by LN-resident DCs and consequent control of TCR signaling. [ABSTRACT FROM AUTHOR]

Abstract Background Asthma is characterized by type 2 T-helper cell (Th2) inflammation, goblet cell hyperplasia, airway hyperreactivity, and airway fibrosis. Monocyte chemoattractant protein-1 (MCP-1 or CCL2) and its receptor, CCR2, have been shown to play important roles in the development of Th2 inflammation. CCR2-deficient mice have been found to have altered inflammatory and physiologic responses in some models of experimental allergic asthma, but the role of CCR2 in contributing to inflammation and airway hyperreactivity appears to vary considerably between models. Furthermore, MCP-1-deficient mice have not previously been studied in models of experimental allergic asthma. Methods To test whether MCP-1 and CCR2 are each required for the development of experimental allergic asthma, we applied an Aspergillus antigen-induced model of Th2 cytokine-driven allergic asthma associated with airway fibrosis to mice deficient in either MCP-1 or CCR2. Previous studies with live Aspergillus conidia instilled into the lung revealed that MCP-1 and CCR2 play a role in anti-fungal responses; in contrast, we used a non-viable Aspergillus antigen preparation known to induce a robust eosinophilic inflammatory response. Results We found that wild-type C57BL/6 mice developed eosinophilic airway inflammation, goblet cell hyperplasia, airway hyperreactivity, elevations in serum IgE, and airway fibrosis in response to airway challenge with Aspergillus antigen. Surprisingly, mice deficient in either MCP-1 or CCR2 had responses to Aspergillus antigen similar to those seen in wild-type mice, including production of Th2 cytokines. Conclusion We conclude that robust Th2-mediated lung pathology can occur even in the complete absence of MCP-1 or CCR2.