The helminth TGF-ß mimic TGM4 is a modular ligand that binds CD44, CD49d and TGF-ß receptors to preferentially target myeloid cells.

The murine helminth parasite Heligmosomoides polygyrus expresses a family of modular proteins which, replicating the functional activity of the immunomodulatory cytokine TGF-ß, have been named TGM (TGF-ß Μimic). Multiple domains bind to different receptors, including TGF-ß receptors TßRI (ALK5) and TßRII through domains 1-3, and prototypic family member TGM1 binds the cell surface co-receptor CD44 through domains 4-5. This allows TGM1 to induce T lymphocyte Foxp3 expression, characteristic of regulatory (Treg) cells, and to activate a range of TGF-ß-responsive cell types. In contrast, a related protein, TGM4, targets a much more restricted cell repertoire, primarily acting on myeloid cells, with less potent effects on T cells and lacking activity on other TGF-ß-responsive cell types. TGM4 binds avidly to myeloid cells by flow cytometry, and can outcompete TGM1 for cell binding. Analysis of receptor binding in comparison to TGM1 reveals a 10-fold higher affinity than TGM1 for TGFßR-I (TßRI), but a 100-fold lower affinity for TßRII through Domain 3. Consequently, TGM4 is more dependent on co-receptor binding; in addition to CD44, TGM4 also engages CD49d (Itga4) through Domains 1-3, as well as CD206 and Neuropilin-1 through Domains 4 and 5. TGM4 was found to effectively modulate macrophage populations, inhibiting lipopolysaccharide-driven inflammatory cytokine production and boosting interleukin (IL)-4-stimulated responses such as Arginase-1 in vitro and in vivo. These results reveal that the modular nature of TGMs has allowed the fine tuning of the binding affinities of the TßR- and co-receptor binding domains to establish cell specificity for TGF-ß signalling in a manner that cannot be attained by the mammalian cytokine.

GxcM-Fbp17/RacC-WASP signaling regulates polarized cortex assembly in migrating cells via Arp2/3.

The actin-rich cortex plays a fundamental role in many cellular processes. Its architecture and molecular composition vary across cell types and physiological states. The full complement of actin assembly factors driving cortex formation and how their activities are spatiotemporally regulated remain to be fully elucidated. Using Dictyostelium as a model for polarized and rapidly migrating cells, we show that GxcM, a RhoGEF localized specifically in the rear of migrating cells, functions together with F-BAR protein Fbp17, a small GTPase RacC, and the actin nucleation-promoting factor WASP to coordinately promote Arp2/3 complex-mediated cortical actin assembly. Overactivation of this signaling cascade leads to excessive actin polymerization in the rear cortex, whereas its disruption causes defects in cortical integrity and function. Therefore, apart from its well-defined role in the formation of the protrusions at the cell front, the Arp2/3 complex-based actin carries out a previously unappreciated function in building the rear cortical subcompartment in rapidly migrating cells.

TGM6, a helminth secretory product, mimics TGF-ß binding to TßRII to antagonize TGF-ß signaling in fibroblasts.

The murine helminth parasite Heligmosomoides polygyrus expresses a family of proteins structurally related to TGF-ß Mimic 1 (TGM1), a secreted five domain protein that activates the TGF-ß pathway and converts naïve T lymphocytes to immunosuppressive Tregs. TGM1 signals through the TGF-ß type I and type II receptors, TßRI and TßRII, with domains 1-2 and 3 binding TßRI and TßRII, respectively, and domains 4-5 binding CD44, a co-receptor abundant on T cells. TGM6 is a homologue of TGM1 that is co-expressed with TGM1, but lacks domains 1 and 2. Herein, we show that TGM6 binds TßRII through domain 3, but does not bind TßRI, or other type I or type II receptors of the TGF-ß family. In TGF-ß reporter assays in fibroblasts, TGM6, but not truncated TGM6 lacking domains 4 and 5, potently inhibits TGF-ß- and TGM1-induced signaling, consistent with its ability to bind TßRII but not TßRI or other receptors of the TGF-ß family. However, TGM6 does not bind CD44 and is unable to inhibit TGF-ß and TGM1 signaling in T cells. To understand how TGM6 binds TßRII, the X-ray crystal structure of the TGM6 domain 3 bound to TßRII was determined at 1.4 Å. This showed that TGM6 domain 3 binds TßRII through an interface remarkably similar to the TGF-ß:TßRII interface. These results suggest that TGM6 has adapted its domain structure and sequence to mimic TGF-ß binding to TßRII and function as a potent TGF-ß and TGM1 antagonist in fibroblasts. The coexpression of TGM6, along with the immunosuppressive TGMs that activate the TGF-ß pathway, may prevent tissue damage caused by the parasite as it progresses through its life cycle from the intestinal lumen to submucosal tissues and back again.

Corrigendum: Increased expression of LASI LncRNA regulates the cigarette smoke and COPD associated airway inflammation and mucous cell hyperplasia.

[This corrects the article DOI: 10.3389/fimmu.2022.803362.].

Increased Expression of LASI lncRNA Regulates the Cigarette Smoke and COPD Associated Airway Inflammation and Mucous Cell Hyperplasia.

Research Impact: Cigarette smoke (CS) exposure is strongly associated with chronic obstructive pulmonary disease (COPD). In respiratory airways, CS exposure disrupts airway barrier functions, mucous/phlegm production, and basic immune responses of airway epithelial cells. Based on our recent identification of a specific immunomodulatory long noncoding RNA (lncRNA), we investigated its role in CS-induced responses in bronchial airways of cynomolgus macaque model of CS-induced COPD and in former smokers with and without COPD. The lncRNA was significantly upregulated in CS-induced macaque airways and in COPD airways that exhibited higher mucus expression and goblet cell hyperplasia. Experimental models of cells derived from COPD subjects recapitulated the augmented inflammation and mucus expression following the smoke challenge. Blocking of lncRNA expression in cell culture setting suppressed the smoke-induced and COPD-associated dysregulated mucoinflammatory response suggesting that this airway specific immunomodulatory lncRNA may represent a novel target to mitigate the smoke-mediated inflammation and mucus hyperexpression. Rationale: In conducting airways, CS disrupts airway epithelial functions, mucociliary clearances, and innate immune responses that are primarily orchestrated by human bronchial epithelial cells (HBECs). Mucus hypersecretion and dysregulated immune response are the hallmarks of chronic bronchitis (CB) that is often exacerbated by CS. Notably, we recently identified a long noncoding RNA (lncRNA) antisense to ICAM-1 (LASI) that mediates airway epithelial responses. Objective: To investigate the role of LASI lncRNA in CS-induced airway inflammation and mucin hyperexpression in an animal model of COPD, and in HBECs and lung tissues from former smokers with and without COPD. To interrogate LASI lncRNA role in CS-mediated airway mucoinflammatory responses by targeted gene editing. Methods: Small airway tissue sections from cynomolgus macaques exposed to long-term mainstream CS, and those from former smokers with and without COPD were analyzed. The structured-illumination imaging, RNA fluorescence in-situ hybridization (FISH), and qRT-PCR were used to characterize lncRNA expression and the expression of inflammatory factors and airway mucins in a cell culture model of CS extract (CSE) exposure using HBECs from COPD (CHBEs) in comparison with cells from normal control (NHBEs) subjects. The protein levels of mucin MUC5AC, and inflammatory factors ICAM-1, and IL-6 were determined using specific ELISAs. RNA silencing was used to block LASI lncRNA expression and lentivirus encoding LASI lncRNA was used to achieve LASI overexpression (LASI-OE). Results: Compared to controls, LASI lncRNA was upregulated in CS-exposed macaques and in COPD smoker airways, correlating with mucus hyperexpression and mucus cell hyperplasia in severe COPD airways. At baseline, the unstimulated CHBEs showed increased LASI lncRNA expression with higher expression of secretory mucin MUC5AC, and inflammatory factors, ICAM-1, and IL-6 compared to NHBEs. CSE exposure of CHBEs resulted in augmented inflammation and mucus expression compared to controls. While RNA silencing-mediated LASI knockdown suppressed the mucoinflammatory response, cells overexpressing LASI lncRNA showed elevated mRNA levels of inflammatory factors. Conclusions: Altogether, LASI lncRNA may represent a novel target to control the smoke-mediated dysregulation in airway responses and COPD exacerbations.

Leep1 interacts with PIP3 and the Scar/WAVE complex to regulate cell migration and macropinocytosis.

Polarity is essential for diverse functions in many cell types. Establishing polarity requires targeting a network of specific signaling and cytoskeleton molecules to different subregions of the cell, yet the full complement of polarity regulators and how their activities are integrated over space and time to form morphologically and functionally distinct domains remain to be uncovered. Here, by using the model system Dictyostelium and exploiting the characteristic chemoattractant-stimulated translocation of polarly distributed molecules, we developed a proteomic screening approach, through which we identified a leucine-rich repeat domain-containing protein we named Leep1 as a novel polarity regulator. We combined imaging, biochemical, and phenotypic analyses to demonstrate that Leep1 localizes selectively at the leading edge of cells by binding to PIP3, where it modulates pseudopod and macropinocytic cup dynamics by negatively regulating the Scar/WAVE complex. The spatiotemporal coordination of PIP3 signaling, Leep1, and the Scar/WAVE complex provides a cellular mechanism for organizing protrusive structures at the leading edge.

Evolution of Reproductive Division of Labor - Lessons Learned From the Social Amoeba Dictyostelium discoideum During Its Multicellular Development.

The origin of multicellular life from unicellular beings is an epochal step in the evolution of eukaryotes. There are several factors influencing cell fate choices during differentiation and morphogenesis of an organism. Genetic make-up of two cells that unite and fertilize is the key factor to signal the formation of various cell-types in due course of development. Although ploidy of the cell-types determines the genetics of an individual, the role of ploidy in cell fate decisions remains unclear. Dictyostelium serves as a versatile model to study the emergence of multicellular life from unicellular life forms. In this work, we investigate the role played by ploidy status of a cell on cell fate commitments during Dictyostelium development. To answer this question, we created Dictyostelium cells of different ploidy: haploid parents and derived isogenic diploids, allowing them to undergo development. The diploid strains used in this study were generated using parasexual genetics. The ploidy status of the haploids and diploids were confirmed by microscopy, flow cytometry, and karyotyping. Prior to reconstitution, we labeled the cells by two methods. First, intragenic expression of red fluorescent protein (RFP) and second, staining the amoebae with a vital, fluorescent dye carboxyfluorescein succinimidyl ester (CFSE). RFP labeled haploid cells allowed us to track the haploids in the chimeric aggregates, slugs, and fruiting bodies. The CFSE labeling method allowed us to track both the haploids and the diploids in the chimeric developmental structures. Our findings illustrate that the haploids demonstrate sturdy cell fate commitment starting from the aggregation stage. The haploids remain crowded at the aggregation centers of the haploid-diploid chimeric aggregates. At the slug stage haploids are predominantly occupying the slug posterior, and are visible in the spore population in the fruiting bodies. Our findings show that cell fate decisions during D. discoideum development are highly influenced by the ploidy status of a cell, adding a new aspect to already known factors Here, we report that ploidy status of a cell could also play a crucial role in regulating the cell fate commitments.

Gestational Exposure to Cigarette Smoke Suppresses the Gasotransmitter H2S Biogenesis and the Effects Are Transmitted Transgenerationally.

Rationale: Gestational cigarette smoke (CS) impairs lung angiogenesis and alveolarization, promoting transgenerational development of asthma and bronchopulmonary dysplasia (BPD). Hydrogen sulfide (H2S), a proangiogenic, pro-alveolarization, and anti-asthmatic gasotransmitter is synthesized by cystathionine-γ-lyase (CSE), cystathionine-ß-synthase (CBS), and 3-mercaptopyruvate sulfur transferase (3MST). Objective: Determine if gestational CS exposure affected the expression of H2S synthesizing enzymes in the mouse lung and human placenta. Methods: Mice were exposed throughout gestational period to secondhand CS (SS) at approximating the dose of CS received by a pregnant woman sitting in a smoking bar for 3 h/days during pregnancy. Lungs from 7-days old control and SS-exposed pups and human placenta from mothers who were either non-smokers or smokers during pregnancy were analyzed for expression of the enzymes. Measurements: Mouse lungs and human placentas were examined for the expression of CSE, CBS, and 3MST by immunohistochemical staining, qRT-PCR and/or Western blot (WB) analyses. Results: Compared to controls, mouse lung exposed gestationally to SS had significantly lower levels of CSE, CBS, and 3MST. Moreover, the SS-induced suppression of CSE and CBS in F1 lungs was transmitted to the F2 generation without significant change in the magnitude of the suppression. These changes were associated with impaired epithelial-mesenchymal transition (EMT)-a process required for normal lung angiogenesis and alveolarization. Additionally, the placentas from mothers who smoked during pregnancy, expressed significantly lower levels of CSE, CBS, and 3MST, and the effects were partially moderated by quitting smoking during the first trimester. Conclusions: Lung H2S synthesizing enzymes are downregulated by gestational CS and the effects are transmitted to F2 progeny. Smoking during pregnancy decreases H2S synthesizing enzymes is human placentas, which may correlate with the increased risk of asthma/BPD in children.

Acetylcholinesterase Inhibitor Pyridostigmine Bromide Attenuates Gut Pathology and Bacterial Dysbiosis in a Murine Model of Ulcerative Colitis.

BACKGROUND: Ulcerative colitis (UC) is a Th2 inflammatory bowel disease characterized by increased IL-5 and IL-13 expression, eosinophilic/neutrophilic infiltration, decreased mucus production, impaired epithelial barrier, and bacterial dysbiosis of the colon. Acetylcholine and nicotine stimulate mucus production and suppress Th2 inflammation through nicotinic receptors in lungs but UC is rarely observed in smokers and the mechanism of the protection is unclear. METHODS: In order to evaluate whether acetylcholine can ameliorate UC-associated pathologies, we employed a mouse model of dextran sodium sulfate (DSS)-induced UC-like conditions, and a group of mice were treated with Pyridostigmine bromide (PB) to increase acetylcholine availability. The effects on colonic tissue morphology, Th2 inflammatory factors, MUC2 mucin, and gut microbiota were analyzed. RESULTS: DSS challenge damaged the murine colonic architecture, reduced the MUC2 mucin and the tight-junction protein ZO-1. The PB treatment significantly attenuated these DSS-induced responses along with the eosinophilic infiltration and the pro-Th2 inflammatory factors. Moreover, PB inhibited the DSS-induced loss of commensal Clostridia and Flavobacteria, and the gain of pathogenic Erysipelotrichia and Fusobacteria. CONCLUSIONS: Together, these data suggest that in colons of a murine model, PB promotes MUC2 synthesis, suppresses Th2 inflammation and attenuates bacterial dysbiosis therefore, PB has a therapeutic potential in UC.

HIV-1 Productively Infects and Integrates in Bronchial Epithelial Cells.

Background: The role of lung epithelial cells in HIV-1-related lung comorbidities remains unclear, and the major hurdle in curing HIV is the persistence of latent HIV reservoirs in people living with HIV (PLWH). The advent of combined antiretroviral therapy has considerably increased the life span; however, the incidence of chronic lung diseases is significantly higher among PLWH. Lung epithelial cells orchestrate the respiratory immune responses and whether these cells are productively infected by HIV-1 is debatable. Methods: Normal human bronchial epithelial cells (NHBEs) grown on air-liquid interface were infected with X4-tropic HIV-1LAV and examined for latency using latency-reversing agents (LRAs). The role of CD4 and CXCR4 HIV coreceptors in NHBEs were tested, and DNA sequencing analysis was used to analyze the genomic integration of HIV proviral genes, Alu-HIVgag-pol, HIV-nef, and HIV-LTR. Lung epithelial sections from HIV-infected humans and SHIV-infected macaques were analyzed by FISH for HIV-gag-pol RNA and epithelial cell-specific immunostaining. Results and Discussion: NHBEs express CD4 and CXCR4 at higher levels than A549 cells. NHBEs are infected with HIV-1 basolaterally, but not apically, by X4-tropic HIV-1LAV in a CXCR4/CD4-dependent manner leading to HIV-p24 antigen production; however, NHBEs are induced to express CCR5 by IL-13 treatment. In the presence of cART, HIV-1 induces latency and integration of HIV provirus in the cellular DNA, which is rescued by the LRAs (endotoxin/vorinostat). Furthermore, lung epithelial cells from HIV-infected humans and SHIV-infected macaques contain HIV-specific RNA transcripts. Thus, lung epithelial cells are targeted by HIV-1 and could serve as potential HIV reservoirs that may contribute to the respiratory comorbidities in PLWH.

Distinct Interaction Sites of Rac GTPase with WAVE Regulatory Complex Have Non-redundant Functions in Vivo.

Cell migration often involves the formation of sheet-like lamellipodia generated by branched actin filaments. The branches are initiated when Arp2/3 complex [1] is activated by WAVE regulatory complex (WRC) downstream of small GTPases of the Rac family [2]. Recent structural studies defined two independent Rac binding sites on WRC within the Sra-1/PIR121 subunit of the pentameric WRC [3, 4], but the functions of these sites in vivo have remained unknown. Here we dissect the mechanism of WRC activation and the in vivo relevance of distinct Rac binding sites on Sra-1, using CRISPR/Cas9-mediated gene disruption of Sra-1 and its paralog PIR121 in murine B16-F1 cells combined with Sra-1 mutant rescue. We show that the A site, positioned adjacent to the binding region of WAVE-WCA mediating actin and Arp2/3 complex binding, is the main site for allosteric activation of WRC. In contrast, the D site toward the C terminus is dispensable for WRC activation but required for optimal lamellipodium morphology and function. These results were confirmed in evolutionarily distant Dictyostelium cells. Moreover, the phenotype seen in D site mutants was recapitulated in Rac1 E31 and F37 mutants; we conclude these residues are important for Rac-D site interaction. Finally, constitutively activated WRC was able to induce lamellipodia even after both Rac interaction sites were lost, showing that Rac interaction is not essential for membrane recruitment. Our data establish that physical interaction with Rac is required for WRC activation, in particular through the A site, but is not mandatory for WRC accumulation in the lamellipodium.

Cigarette smoke and HIV synergistically affect lung pathology in cynomolgus macaques.

In the era of combined antiretroviral therapy (cART), lung diseases such as chronic bronchitis (CB) and chronic obstructive pulmonary disease (COPD) are common among persons living with HIV (PLWH), particularly smokers. Although smoking is highly prevalent among PLWH, HIV may be an independent risk factor for lung diseases; however, the role of HIV and cigarette smoke (CS) and their potential interaction in the development of chronic lung diseases among PLWH has not been delineated. To investigate this interaction, cynomolgus macaques were exposed to CS and/or simian-adapted human immunodeficiency virus (SHIV) and treated with cART. The development of CB and the lung functions were evaluated following CS±SHIV treatment. The results showed that in the lung, SHIV was a strong independent risk factor for goblet cell metaplasia/hyperplasia and mucus formation, MUC5AC synthesis, loss of tight junction proteins, and increased expression of Th2 cytokines/transcription factors. In addition, SHIV and CS synergistically reduced lung function and increased extrathoracic tracheal ring thickness. Interestingly, SHIV infection generated significant numbers of HIV-gp120+ epithelial cells (HGECs) in small airways and alveoli, and their numbers doubled in CS+SHIV-infected lungs. We conclude that even with cART, SHIV independently induces CB and pro-COPD changes in the lung, and the effects are exacerbated by CS.

Gestational Exposure to Sidestream (Secondhand) Cigarette Smoke Promotes Transgenerational Epigenetic Transmission of Exacerbated Allergic Asthma and Bronchopulmonary Dysplasia.

Embryonic development is highly sensitive to xenobiotic toxicity and in utero exposure to environmental toxins affects physiological responses of the progeny. In the United States, the prevalence of allergic asthma (AA) is inexplicably rising and in utero exposure to cigarette smoke increases the risk of AA and bronchopulmonary dysplasia (BPD) in children and animal models. We reported that gestational exposure to sidestream cigarette smoke (SS), or secondhand smoke, promoted nicotinic acetylcholine receptor-dependent exacerbation of AA and BPD in mice. Recently, perinatal nicotine injections in rats were reported to induce peroxisome proliferator-activated receptor γ-dependent transgenerational transmission of asthma. Herein, we show that first generation and second generation progeny from gestationally SS-exposed mice exhibit exacerbated AA and BPD that is not dependent on the decrease in peroxisome proliferator-activated receptor γ levels. Lungs from these mice show strong eosinophilic infiltration, excessive Th2 polarization, marked airway hyperresponsiveness, alveolar simplification, decreased lung compliance, and decreased lung angiogenesis. At the molecular level, these changes are associated with increased RUNX3 expression, alveolar cell apoptosis, and the antiangiogenic factor GAX, and decreased expression of HIF-1α and proangiogenic factors NF-κB and VEGFR2 in the 7-d first generation and second generation lungs. Moreover, the lungs from these mice exhibit lower levels of microRNA (miR)-130a and increased levels of miR-16 and miR-221. These miRs regulate HIF-1α-regulated apoptotic, angiogenic, and immune pathways. Thus the intergenerational effects of gestational SS involve epigenetic regulation of HIF-1α through specific miRs contributing to increased incidence of AA and BPD in the progenies.

HIF-1α Plays a Critical Role in the Gestational Sidestream Smoke-Induced Bronchopulmonary Dysplasia in Mice.

RATIONALE: Smoking during pregnancy increases the risk of bronchopulmonary dysplasia (BPD) and, in mice, gestational exposure to sidestream cigarette smoke (SS) induces BPD-like condition characterized by alveolar simplification, impaired angiogenesis, and suppressed surfactant protein production. Normal fetal development occurs in a hypoxic environment and nicotinic acetylcholine receptors (nAChRs) regulate the hypoxia-inducible factor (HIF)-1α that controls apoptosis and angiogenesis. To understand SS-induced BPD, we hypothesized that gestational SS affected alveolar development through HIF-1α. METHODS: Pregnant BALB/c mice were exposed to air (control) or SS throughout the gestational period and the 7-day-old lungs of the progeny were examined. RESULTS: Gestational SS increased apoptosis of alveolar and airway epithelial cells. This response was associated with increased alveolar volumes, higher levels of proapoptotic factors (FOXO3a, HIPK2, p53, BIM, BIK, and BAX) and the antiangiogenic factor (GAX), and lower levels of antiapoptotic factors (Akt-PI3K, NF-κB, HIF-1α, and Bcl-2) in the lung. Although gestational SS increased the cells containing the proangiogenic bombesin-like-peptide, it markedly decreased the expression of its receptor GRPR in the lung. The effects of SS on apoptosis were attenuated by the nAChR antagonist mecamylamine. CONCLUSIONS: Gestational SS-induced BPD is potentially regulated by nAChRs and associated with downregulation of HIF-1α, increased apoptosis of epithelial cells, and increased alveolar volumes. Thus, in mice, exposure to sidestream tobacco smoke during pregnancy promotes BPD-like condition that is potentially mediated through the nAChR/HIF-1α pathway.

HIV gp120 induces mucus formation in human bronchial epithelial cells through CXCR4/α7-nicotinic acetylcholine receptors.

Lung diseases such as chronic obstructive pulmonary disease (COPD), asthma, and lung infections are major causes of morbidity and mortality among HIV-infected patients even in the era of antiretroviral therapy (ART). Many of these diseases are strongly associated with smoking and smoking is more common among HIV-infected than uninfected people; however, HIV is an independent risk factor for chronic bronchitis, COPD, and asthma. The mechanism by which HIV promotes these diseases is unclear. Excessive airway mucus formation is a characteristic of these diseases and contributes to airway obstruction and lung infections. HIV gp120 plays a critical role in several HIV-related pathologies and we investigated whether HIV gp120 promoted airway mucus formation in normal human bronchial epithelial (NHBE) cells. We found that NHBE cells expressed the HIV-coreceptor CXCR4 but not CCR5 and produced mucus in response to CXCR4-tropic gp120. The gp120-induced mucus formation was blocked by the inhibitors of CXCR4, α7-nicotinic acetylcholine receptor (α7-nAChR), and γ-aminobutyric acid (GABA)AR but not the antagonists of CCR5 and epithelial growth factor receptor (EGFR). These results identify two distinct pathways (α7-nAChR-GABAAR and EGFR) for airway mucus formation and demonstrate for the first time that HIV-gp120 induces and regulates mucus formation in the airway epithelial cells through the CXCR4-α7-nAChR-GABAAR pathway. Interestingly, lung sections from HIV ± ART and simian immunodeficiency virus (SIV) ± ART have significantly more mucus and gp120-immunoreactivity than control lung sections from humans and macaques, respectively. Thus, even after ART, lungs from HIV-infected patients contain significant amounts of gp120 and mucus that may contribute to the higher incidence of obstructive pulmonary diseases in this population.

Gestational exposure of mice to secondhand cigarette smoke causes bronchopulmonary dysplasia blocked by the nicotinic receptor antagonist mecamylamine.

BACKGROUND: Cigarette smoke (CS) exposure during gestation may increase the risk of bronchopulmonary dysplasia (BPD)-a developmental lung condition primarily seen in neonates that is characterized by hypoalveolarization, decreased angiogenesis, and diminished surfactant protein production and may increase the risk of chronic obstructive pulmonary disease. OBJECTIVE: We investigated whether gestational exposure to secondhand CS (SS) induced BPD and sought to ascertain the role of nicotinic acetylcholine receptors (nAChRs) in this response. METHODS: We exposed BALB/c and C57BL/6 mice to filtered air (control) or SS throughout the gestation period or postnatally up to 10 weeks. Lungs were examined at 7 days, 10 weeks, and 8 months after birth. RESULTS: Gestational but not postnatal exposure to SS caused a typical BPD-like condition: suppressed angiogenesis [decreased vascular endothelial growth factor (VEGF), VEGF receptor, and CD34/CD31 (hematopoietic progenitor cell marker/endothelial cell marker)], irreversible hypoalveolarization, and significantly decreased levels of Clara cells, Clara cell secretory protein, and surfactant proteins B and C, without affecting airway ciliated cells. Importantly, concomitant exposure to SS and the nAChR antagonist mecamylamine during gestation blocked the development of BPD. CONCLUSIONS: Gestational exposure to SS irreversibly disrupts lung development leading to a BPD-like condition with hypoalveolarization, decreased angiogenesis, and diminished lung secretory function. Nicotinic receptors are critical in the induction of gestational SS-induced BPD, and the use of nAChR antagonists during pregnancy may block CS-induced BPD.

Role of nicotinic receptors and acetylcholine in mucous cell metaplasia, hyperplasia, and airway mucus formation in vitro and in vivo.

BACKGROUND: Airway mucus hypersecretion is a key pathophysiologic feature in a number of lung diseases. Cigarette smoke/nicotine and allergens are strong stimulators of airway mucus; however, the mechanism of mucus modulation is unclear. OBJECTIVES: We sought to characterize the pathway by which cigarette smoke/nicotine regulates airway mucus and identify agents that decrease airway mucus. METHODS: IL-13 and γ-aminobutyric acid type A receptors (GABA(A)Rs) are implicated in airway mucus. We examined the role of IL-13 and GABA(A)Rs in nicotine-induced mucus formation in normal human bronchial epithelial (NHBE) and A549 cells and secondhand cigarette smoke-induced, ovalbumin-induced, or both mucus formation in vivo. RESULTS: Nicotine promotes mucus formation in NHBE cells; however, the nicotine-induced mucus formation is independent of IL-13 but sensitive to the GABA(A)R antagonist picrotoxin. Airway epithelial cells express α7-, α9-, and α10-nicotinic acetylcholine receptors (nAChRs), and specific inhibition or knockdown of α7- but not α9/α10-nAChRs abrogates mucus formation in response to nicotine and IL-13. Moreover, addition of acetylcholine or inhibition of its degradation increases mucus in NHBE cells. Nicotinic but not muscarinic receptor antagonists block allergen- or nicotine/cigarette smoke-induced airway mucus formation in NHBE cells, murine airways, or both. CONCLUSIONS: Nicotine-induced airway mucus formation is independent of IL-13, and α7-nAChRs are critical in airway mucous cell metaplasia/hyperplasia and mucus production in response to various promucoid agents, including IL-13. In the absence of nicotine, acetylcholine might be the biological ligand for α7-nAChRs to trigger airway mucus formation. α7-nAChRs are downstream of IL-13 but upstream of GABA(A)Rα2 in the MUC5AC pathway. Acetylcholine and α7-nAChRs might serve as therapeutic targets to control airway mucus.

Inhalation of sulfur mustard causes long-term T cell-dependent inflammation: possible role of Th17 cells in chronic lung pathology.

Sulfur mustard (SM) is a highly toxic chemical warfare agent that remains a threat to human health. The immediate symptoms of pulmonary distress may develop into chronic lung injury characterized by progressive lung fibrosis, the major cause of morbidity among the surviving SM victims. Although SM has been intensely investigated, little is known about the mechanism(s) by which SM induces chronic lung pathology. Increasing evidence suggests that IL-17(+) cells are critical in fibrosis, including lung fibrotic diseases. In this study we exposed F344 rats and cynomolgus monkeys to SM via inhalation and determined the molecular and cellular milieu in their lungs at various times after SM exposure. In rats, SM induced a burst of pro-inflammatory cytokines/chemokines within 72 h, including IL-1ß, TNF-α, IL-2, IL-6, CCL2, CCL3, CCL11, and CXCL1 that was associated with neutrophilic infiltration into the lung. At 2 wks and beyond (chronic phase), lymphocytic infiltration and continued elevated expression of cytokines/chemokines were sustained. TGF-ß, which was undetectable in the acute phase, was strongly upregulated in the chronic phase; these conditions persisted until the animals were sacrificed. The chronic phase was also associated with myofibroblast proliferation, collagen deposition, and presence of IL-17(+) cells. At ≥30 days, SM inhalation promoted the accumulation of IL-17(+) cells in the inflamed areas of monkey lungs. Thus, SM inhalation causes acute and chronic inflammatory responses; the latter is characterized by the presence of TGF-ß, fibrosis, and IL-17(+) cells in the lung. IL-17(+) cells likely play an important role in the pathogenesis of SM-induced lung injury.

Prenatal secondhand cigarette smoke promotes Th2 polarization and impairs goblet cell differentiation and airway mucus formation.

Parental, particularly maternal, smoking increases the risk for childhood allergic asthma and infection. Similarly, in a murine allergic asthma model, prenatal plus early postnatal exposure to secondhand cigarette smoke (SS) exacerbates airways hyperreactivity and Th2 responses in the lung. However, the mechanism and contribution of prenatal versus early postnatal SS exposure on allergic asthma remain unresolved. To identify the effects of prenatal and/or early postnatal SS on allergic asthma, BALB/c dams and their offspring were exposed gestationally and/or 8-10 wk postbirth to filtered air or SS. Prenatal, but not postnatal, SS strongly increased methacholine and allergen (Aspergillus)-induced airway resistance, Th2 cytokine levels, and atopy and activated the Th2-polarizing pathway GATA3/Lck/ERK1/2/STAT6. Either prenatal and/or early postnatal SS downregulated the Th1-specific transcription factor T-bet and, surprisingly, despite high levels of IL-4/IL-13, dramatically blocked the allergen-induced mucous cell metaplasia, airway mucus formation, and the expression of mucus-related genes/proteins: Muc5ac, γ-aminobutyric acid A receptors, and SAM pointed domain-containing Ets-like factor. Given that SS/nicotine exposure of normal adult mice promotes mucus formation, the results suggested that fetal and neonatal lung are highly sensitive to cigarette smoke. Thus, although the gestational SS promotes Th2 polarization/allergic asthma, it may also impair and/or delay the development of fetal and neonatal lung, affecting mucociliary clearance and Th1 responses. Together, this may explain the increased susceptibility of children from smoking parents to allergic asthma and childhood respiratory infections.

Fibrogenic and redox-related but not proinflammatory genes are upregulated in Lewis rat model of chronic silicosis.

Silicosis, a fibrotic granulomatous lung disease, may occur through accidental high-dose or occupational inhalation of silica, leading to acute/accelerated and chronic silicosis, respectively. While chronic silicosis has a long asymptomatic latency, lung inflammation and apoptosis are hallmarks of acute silicosis. In animal models, histiocytic granulomas develop within days after high-dose intratracheal (IT) silica instillation. However, following chronic inhalation of occupationally relevant doses of silica, discrete granulomas resembling human silicosis arise months after the final exposure without significant lung inflammation/apoptosis. To identify molecular events associated with chronic silicosis, lung RNA samples from controls or subchronic silica-exposed rats were analyzed by Affymetrix at 28 wk after silica exposures. Results suggested a significant upregulation of 144 genes and downregulation of 7 genes. The upregulated genes included complement cascade, chemokines/chemokine receptors, G-protein signaling components, metalloproteases, and genes associated with oxidative stress. To examine the kinetics of gene expression relevant to silicosis, quantitative polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA), Luminex-bead assays, Western blotting, and/or zymography were performed on lung tissues from 4 d, 28 wk, and intermediate times after subchronic silica exposure and compared with 14-d acute silicosis samples. Results indicated that genes regulating fibrosis (secreted phosphoprotein-1, Ccl2, and Ccl7), redox enzymes (superoxide dismutase-2 and arginase-1), and the enzymatic activities of matrix metalloproteinases 2 and 9 were upregulated in acute and chronic silicosis models. However, proinflammatory cytokines were strongly upregulated only in acute silicosis. Thus, inflammatory cytokines are associated with acute but not chronic silicosis. Data suggest that genes regulating fibrosis, oxidative stress, and metalloproteases may contribute to both acute and chronic silicosis.