Golgi Apparatus in Airway Secretory Cells

https://openworks.mdanderson.org/sumexp22/1068/thumbnail.jp

Airway Epithelial Innate Immunity

Besides providing an essential protective barrier, airway epithelial cells directly sense pathogens and respond defensively. This is a frontline component of the innate immune system with specificity for different pathogen classes. It occurs in the context of numerous interactions with leukocytes, but here we focus on intrinsic epithelial mechanisms. Type 1 immune responses are directed primarily at intracellular pathogens, particularly viruses. Prominent stimuli include microbial nucleic acids and interferons released from neighboring epithelial cells. Epithelial responses revolve around changes in the expression of interferon-sensitive genes (ISGs) that interfere with viral replication, as well as the further induction of interferons that signal in autocrine and paracrine manners. Type 2 immune responses are directed primarily at helminths and fungi. Prominent pathogen stimuli include proteases and chitin, and important responses include mucin hypersecretion and chitinase release. Type 3 immune responses are directed primarily at extracellular microbial pathogens, including bacteria and fungi, as well as viruses during their extracellular phase of infection. Prominent microbial stimuli include bacterial wall components, such as lipopeptides and endotoxin, as well as microbial nucleic acids. Key responses are the release of reactive oxygen species (ROS) and antimicrobial peptides (AMPs). For all three types of response, paracrine signaling to neighboring epithelial cells induces resistance to infection over a wide field. Often, the epithelial effector molecules themselves also have signaling properties, in addition to the release of inflammatory cytokines that boost local innate immunity. Together, these epithelial mechanisms provide a powerful first line of pathogen defense, recruit leukocytes, and instruct adaptive immune responses

Munc18b is an essential gene in mice whose expression is limiting for secretion by airway epithelial and mast cells

Airway mucin secretion and MC (mast cell) degranulation must be tightly controlled for homoeostasis of the lungs and immune system respectively. We found the exocytic protein Munc18b to be highly expressed in mouse airway epithelial cells and MCs, and localized to the apical pole of airway secretory cells. To address its functions, we created a mouse with a severely hypomorphic Munc18b allele such that protein expression in heterozygotes was reduced by∼50%. Homozygous mutant mice were not viable, but heterozygotes showed a ∼50% reduction in stimulated release of mucin from epithelial cells and granule contents from MCs. The defect in MCs affected only regulated secretion and not constitutive or transporter-mediated secretion. The severity of passive cutaneous anaphylaxiswas also reduced by ∼50%, showing that reduction of Munc18b expression results in an attenuation of physiological responses dependent on MC degranulation. The Munc18b promoter is controlled by INR (initiator), Sp1 (specificity protein 1), Ets, CRE (cAMP-response element), GRE (glucocorticoid-response element), GATA and E-box elements in airway epithelial cells; however, protein levels did not change during mucous metaplasia induced by allergic inflammation. Taken together, the results of the present study identifyMunc18b as an essential gene that is a limiting component of the exocytic machinery of epithelial cells and MCs

Synergistic TLR2/6 and TLR9 Activation Protects Mice against Lethal Influenza Pneumonia

Lower respiratory tract infections caused by influenza A continue to exact unacceptable worldwide mortality, and recent epidemics have emphasized the importance of preventative and containment strategies. We have previously reported that induction of the lungs' intrinsic defenses by aerosolized treatments can protect mice against otherwise lethal challenges with influenza A virus. More recently, we identified a combination of Toll like receptor (TLR) agonists that can be aerosolized to protect mice against bacterial pneumonia. Here, we tested whether this combination of synthetic TLR agonists could enhance the survival of mice infected with influenza A/HK/8/68 (H3N2) or A/California/04/2009 (H1N1) influenza A viruses. We report that the TLR treatment enhanced survival whether given before or after the infectious challenge, and that protection tended to correlate with reductions in viral titer 4 d after infection. Surprisingly, protection was not associated with induction of interferon gene expression. Together, these studies suggest that synergistic TLR interactions can protect against influenza virus infections by mechanisms that may provide the basis for novel therapeutics

Chronic Exposure to Beta-Blockers Attenuates Inflammation and Mucin Content in a Murine Asthma Model

Single-dose administration of beta-adrenoceptor agonists produces bronchodilation and inhibits airway hyperresponsiveness (AHR), and is the standard treatment for the acute relief of asthma. However, chronic repetitive administration of beta-adrenoceptor agonists may increase AHR, airway inflammation, and risk of death. Based upon the paradigm shift that occurred with the use of beta-blockers in congestive heart failure, we previously determined that chronic administration of beta-blockers decreased AHR in a murine model of asthma. To elucidate the mechanisms for the beneficial effects of beta-blockers, we examined the effects of chronic administration of several beta-adrenoceptor ligands in a murine model of allergic asthma. Administration of beta-blockers resulted in a reduction in total cell counts, eosinophils, and the cytokines IL-13, IL-10, IL-5, and TGF-β1 in bronchoalveolar lavage, and attenuated epithelial mucin content and morphologic changes. The differences in mucin content also occurred if the beta-blockers were administered only during the ovalbumin challenge phase, but administration of beta-blockers for 7 days was not as effective as administration for 28 days. These results indicate that in a murine model of asthma, chronic administration of beta-blockers reduces inflammation and mucous metaplasia, cardinal features of asthma that may contribute to airflow obstruction and AHR. Similar to heart failure, our results provide a second disease model in which beta-blockers producing an acutely detrimental effect may provide a therapeutically beneficial effect with chronic administration

Augmented Lung Inflammation Protects against Influenza A Pneumonia

Influenza pneumonia causes high mortality every year, and pandemic episodes kill millions of people. Influenza-related mortality has been variously ascribed to an ineffective host response that fails to limit viral replication, an excessive host inflammatory response that results in lung injury and impairment of gas exchange, or to bacterial superinfection. We sought to determine whether lung inflammation promoted or impaired host survival in influenza pneumonia.To distinguish among these possible causes of influenza-related death, we induced robust lung inflammation by exposing mice to an aerosolized bacterial lysate prior to challenge with live virus. The treatment induced expression of the inflammatory cytokines IL-6 and TNF in bronchoalveolar lavage fluid 8- and 40-fold greater, respectively, than that caused by lethal influenza infection. Yet, this augmented inflammation was associated with striking resistance to host mortality (0% vs 90% survival, p = 0.0001) and reduced viral titers (p = 0.004). Bacterial superinfection of virus infected lungs was not observed. When mice were repeatedly exposed to the bacterial lysate, as would be clinically desirable during an influenza epidemic, there was no tachyphylaxis of the induced viral resistance. When the bacterial lysate was administered after the viral challenge, there was still some mortality benefit, and when ribavirin was added to the aerosolized bacterial lysate, host survival was synergistically improved (0% vs 93.3% survival, p<0.0001).Together, these data indicate that innate immune resistance to influenza can be effectively stimulated, and suggest that ineffective rather than excessive inflammation is the major cause of mortality in influenza pneumonia

SNAP23 is selectively expressed in airway secretory cells and mediates baseline and stimulated mucin secretion

Airway mucin secretion is important pathophysiologically and as a model of polarized epithelial regulated exocytosis. We find the trafficking protein, SNAP23 (23-kDa paralogue of synaptosome-associated protein of 25 kDa), selectively expressed in secretory cells compared with ciliated and basal cells of airway epithelium by immunohistochemistry and FACS, suggesting that SNAP23 functions in regulated but not constitutive epithelial secretion. Heterozygous SNAP23 deletant mutant mice show spontaneous accumulation of intracellular mucin, indicating a defect in baseline secretion. However mucins are released from perfused tracheas of mutant and wild-type (WT) mice at the same rate, suggesting that increased intracellular stores balance reduced release efficiency to yield a fully compensated baseline steady state. In contrast, acute stimulated release of intracellular mucin from mutant mice is impaired whether measured by a static imaging assay 5 min after exposure to the secretagogue ATP or by kinetic analysis of mucins released from perfused tracheas during the first 10 min of ATP exposure. Together, these data indicate that increased intracellular stores cannot fully compensate for the defect in release efficiency during intense stimulation. The lungs of mutant mice develop normally and clear bacteria and instilled polystyrene beads comparable to WT mice, consistent with these functions depending on baseline secretion that is fully compensated

SNAP23 Is Selectively Expressed in Airway Secretory Cells and Mediates Baseline and Stimulated Mucin Secretion

Airway mucin secretion is important pathophysiologically and as a model of polarized epithelial regulated exocytosis. We find the trafficking protein, SNAP23 (23-kDa paralogue of synaptosome-associated protein of 25 kDa), selectively expressed in secretory cells compared with ciliated and basal cells of airway epithelium by immunohistochemistry and FACS, suggesting that SNAP23 functions in regulated but not constitutive epithelial secretion. Heterozygous SNAP23 deletant mutant mice show spontaneous accumulation of intracellular mucin, indicating a defect in baseline secretion. However mucins are released from perfused tracheas of mutant and wild-type (WT) mice at the same rate, suggesting that increased intracellular stores balance reduced release efficiency to yield a fully compensated baseline steady state. In contrast, acute stimulated release of intracellular mucin from mutant mice is impaired whether measured by a static imaging assay 5 min after exposure to the secretagogue ATP or by kinetic analysis of mucins released from perfused tracheas during the first 10 min of ATP exposure. Together, these data indicate that increased intracellular stores cannot fully compensate for the defect in release efficiency during intense stimulation. The lungs of mutant mice develop normally and clear bacteria and instilled polystyrene beads comparable to WT mice, consistent with these functions depending on baseline secretion that is fully compensated

Compositions for stimulation of mammalian innate immune resistance to pathogens

Embodiments of the invention are directed to methods of treating, inhibiting or attenuating a microbial infection in an individual who has or is at risk for developing such an infection, comprising the step of administering an effective amount of a TLR9 agonist and a TLR 2/6 agonist to the individual.Board of Regents, University of Texas Syste