PULMONARY IMMUNE ENVIRONMENT DETERMINES THE MANIFESTATION OF EXPERIMENTAL EMPHYSEMA

The lung is a complex well-designed organ providing a large surface area for exchanging gas to provide oxygen to the body and eliminate carbon dioxide from the circulation. Because of its exposure to the environment, it is required to have active immunologic defense mechanisms to remove hazardous agents and maintain homeostasis. However, an aberration in this pulmonary immune environment can lead to several pathologic developments. Emphysema is characterized by progressive loss of alveolar surface area with a permanent airspace enlargement, and it is one of the major public health issues leading to serious morbidity and mortality. Disparities in individual immune responses interact with host genetic predispositions and environmental factors in a complex manner that impacts the susceptibility to develop emphysema. The overall goal of this thesis was to study immunological factors that are involved in determining the susceptibility to develop emphysema using a simplified elastase-induced murine model. First, we determined the susceptibility to develop emphysema in two common strains of mice. We found BALB/cJ mice to be much more sensitive to exogenous elastase compared to C57BL/6J mice. Based on gene expression analysis, we found different immunologic mechanisms that might underlie the differential progression of elastase-induced emphysema in these two mouse strains. In addition, MMP-producing macrophages (but not neutrophils or lymphocytes) were identified as the critical cells that mediate the extracellular matrix degradation in emphysema. Furthermore, we use genetically engineered mice to study the importance of several cytokine signaling pathways and transcription factors. We found important roles of IL-17A, IFN-, IL-33/ST2/MyD88, STAT6 and STAT3 in activating or modulating the macrophages to become more destructive. Lastly, we also showed that recent viral infections could impact on the severity of emphysema following the acute elastase injury. In conclusion, the intricacy of genetic and environmental factors together influence immune responses in the lungs to determine the susceptibility to develop emphysema. This knowledge provides new insights into the cellular and molecular mechanisms that may be responsible for the heterogeneity observed in human susceptibility to develop emphysema

Instillation and Fixation Methods Useful in Mouse Lung Cancer Research

The ability to instill live agents, cells, or chemicals directly into the lung without injuring or killing the mice is an important tool in lung cancer research. Although there are a number of methods that have been published showing how to intubate mice for pulmonary function measurements, none are without potential problems for rapid tracheal instillation in large cohorts of mice. In the present paper, a simple and quick method is described that enables an investigator to carry out such instillations in an efficient manner. The method does not require any special tools or lighting and can be learned with very little practice. It involves anesthetizing a mouse, making a small incision in the neck to visualize the trachea, and then inserting an intravenous catheter directly. The small incision is quickly closed with tissue adhesive, and the mice are allowed to recover. A skilled student or technician can do instillations at an average rate of 2 min/mouse. Once the cancer is established, there is frequently a need for quantitative histologic analysis of the lungs. Traditionally pathologists usually do not bother to standardize lung inflation during fixation, and analyses are often based on a scoring system that can be quite subjective. While this may sometime be sufficiently adequate for gross estimates of the size of a lung tumor, any proper stereological quantification of lung structure or cells requires a reproducible fixation procedure and subsequent lung volume measurement. Here we describe simple reliable procedures for both fixing the lungs under pressure and then accurately measuring the fixed lung volume. The only requirement is a laboratory balance that is accurate over a range of 1 mg–300 g. The procedures presented here thus could greatly improve the ability to create, treat, and analyze lung cancers in mice

The odorant receptor OR2W3 on airway smooth muscle evokes bronchodilation via a cooperative chemosensory tradeoff between TMEM16A and CFTR.

The recent discovery of sensory (tastant and odorant) G protein-coupled receptors on the smooth muscle of human bronchi suggests unappreciated therapeutic targets in the management of obstructive lung diseases. Here we have characterized the effects of a wide range of volatile odorants on the contractile state of airway smooth muscle (ASM) and uncovered a complex mechanism of odorant-evoked signaling properties that regulate excitation-contraction (E-C) coupling in human ASM cells. Initial studies established multiple odorous molecules capable of increasing intracellular calcium ([Ca2+]i) in ASM cells, some of which were (paradoxically) associated with ASM relaxation. Subsequent studies showed a terpenoid molecule (nerol)-stimulated OR2W3 caused increases in [Ca2+]i and relaxation of ASM cells. Of note, OR2W3-evoked [Ca2+]i mobilization and ASM relaxation required Ca2+ flux through the store-operated calcium entry (SOCE) pathway and accompanied plasma membrane depolarization. This chemosensory odorant receptor response was not mediated by adenylyl cyclase (AC)/cyclic nucleotide-gated (CNG) channels or by protein kinase A (PKA) activity. Instead, ASM olfactory responses to the monoterpene nerol were predominated by the activity of Ca2+-activated chloride channels (TMEM16A), including the cystic fibrosis transmembrane conductance regulator (CFTR) expressed on endo(sarco)plasmic reticulum. These findings demonstrate compartmentalization of Ca2+ signals dictates the odorant receptor OR2W3-induced ASM relaxation and identify a previously unrecognized E-C coupling mechanism that could be exploited in the development of therapeutics to treat obstructive lung diseases

PULMONARY IMMUNE ENVIRONMENT DETERMINES THE MANIFESTATION OF EXPERIMENTAL EMPHYSEMA

The lung is a complex well-designed organ providing a large surface area for exchanging gas to provide oxygen to the body and eliminate carbon dioxide from the circulation. Because of its exposure to the environment, it is required to have active immunologic defense mechanisms to remove hazardous agents and maintain homeostasis. However, an aberration in this pulmonary immune environment can lead to several pathologic developments. Emphysema is characterized by progressive loss of alveolar surface area with a permanent airspace enlargement, and it is one of the major public health issues leading to serious morbidity and mortality. Disparities in individual immune responses interact with host genetic predispositions and environmental factors in a complex manner that impacts the susceptibility to develop emphysema. The overall goal of this thesis was to study immunological factors that are involved in determining the susceptibility to develop emphysema using a simplified elastase-induced murine model. First, we determined the susceptibility to develop emphysema in two common strains of mice. We found BALB/cJ mice to be much more sensitive to exogenous elastase compared to C57BL/6J mice. Based on gene expression analysis, we found different immunologic mechanisms that might underlie the differential progression of elastase-induced emphysema in these two mouse strains. In addition, MMP-producing macrophages (but not neutrophils or lymphocytes) were identified as the critical cells that mediate the extracellular matrix degradation in emphysema. Furthermore, we use genetically engineered mice to study the importance of several cytokine signaling pathways and transcription factors. We found important roles of IL-17A, IFN-, IL-33/ST2/MyD88, STAT6 and STAT3 in activating or modulating the macrophages to become more destructive. Lastly, we also showed that recent viral infections could impact on the severity of emphysema following the acute elastase injury. In conclusion, the intricacy of genetic and environmental factors together influence immune responses in the lungs to determine the susceptibility to develop emphysema. This knowledge provides new insights into the cellular and molecular mechanisms that may be responsible for the heterogeneity observed in human susceptibility to develop emphysema

Instillation and Fixation Methods Useful in Mouse Lung Cancer Research

The ability to instill live agents, cells, or chemicals directly into the lung without injuring or killing the mice is an important tool in lung cancer research. Although there are a number of methods that have been published showing how to intubate mice for pulmonary function measurements, none are without potential problems for rapid tracheal instillation in large cohorts of mice. In the present paper, a simple and quick method is described that enables an investigator to carry out such instillations in an efficient manner. The method does not require any special tools or lighting and can be learned with very little practice. It involves anesthetizing a mouse, making a small incision in the neck to visualize the trachea, and then inserting an intravenous catheter directly. The small incision is quickly closed with tissue adhesive, and the mice are allowed to recover. A skilled student or technician can do instillations at an average rate of 2 min/mouse. Once the cancer is established, there is frequently a need for quantitative histologic analysis of the lungs. Traditionally pathologists usually do not bother to standardize lung inflation during fixation, and analyses are often based on a scoring system that can be quite subjective. While this may sometime be sufficiently adequate for gross estimates of the size of a lung tumor, any proper stereological quantification of lung structure or cells requires a reproducible fixation procedure and subsequent lung volume measurement. Here we describe simple reliable procedures for both fixing the lungs under pressure and then accurately measuring the fixed lung volume. The only requirement is a laboratory balance that is accurate over a range of 1 mg–300 g. The procedures presented here thus could greatly improve the ability to create, treat, and analyze lung cancers in mice

Emerging Role of the Mast Cell–Microbiota Crosstalk in Cutaneous Homeostasis and Immunity

The skin presents a multifaceted microbiome, a balanced coexistence of bacteria, fungi, and viruses. These resident microorganisms are fundamental in upholding skin health by both countering detrimental pathogens and working in tandem with the skin’s immunity. Disruptions in this balance, known as dysbiosis, can lead to disorders like psoriasis and atopic dermatitis. Central to the skin’s defense system are mast cells. These are strategically positioned within the skin layers, primed for rapid response to any potential foreign threats. Recent investigations have started to unravel the complex interplay between these mast cells and the diverse entities within the skin’s microbiome. This relationship, especially during times of both balance and imbalance, is proving to be more integral to skin health than previously recognized. In this review, we illuminate the latest findings on the ties between mast cells and commensal skin microorganisms, shedding light on their combined effects on skin health and maladies

PACAP activates MRGPRX2 on meningeal mast cells to drive migraine-like pain

Abstract Migraine ranks among the most prevalent disorders worldwide, leading to disability and decreased quality of life in patients. Recently, neurogenic inflammation has been recognized as a potential underlying pathology contributing to the migraine pain pathway. Mast cells reside in the meninges and have been implicated in contributing to the pathophysiology of migraine. Here we report for the first time that the mouse Mas-Related G-protein-coupled Receptor B2 (MrgprB2), is expressed on meningeal connective tissue mast cells and contributes to Pituitary Adenylate Cyclase Activating Peptide (PACAP)-induced migraine-like pain behavior. We also found that PACAP was able to dose-dependently lead to enzyme release from human mast cells via activation of MRGPRX2; the human homolog of MrgprB2. Using a transgenic MRGPRX2 mouse, we observed significant increases in PACAP-induced migraine-like pain behavior in MRGPRX2+ mice vs mice lacking the receptor. These results reveal both MrgprB2 and MRGPRX2 as important contributors to neuropeptide-induced migraine pain