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Regulation of neutrophil endoplasmic reticulum stress response by IRE1a
Project Summary/Abstract: The lungs are exposed to pathogens and environmental toxins that trigger stress and cause numerous respiratory diseases. Effective host defenses against lung infection by bacterial pathogens, including methicillin- resistant Staphylococcus aureus (MRSA), rely on innate immune cells including neutrophils, prominent early responders to sites of infection. If host defenses are ineffective, MRSA causes serious lung infection, resulting in severe morbidity and a significant economic burden on healthcare facilities, where it is endemic. MRSA infections have a mortality rate of up to 14% and an estimated $500 million in healthcare costs in the US alone. Increasing resistance to vancomycin, the last resort antibiotic for MRSA infections, underscore the urgent need for innovative treatment approaches. Although directly targeting pathogens with antibiotics has been a successful approach for treating infections, many pathogens, including MRSA, eventually will become resistant to these drugs. As an alternative, immunomodulatory strategies to enhance host defenses, such as those shown to be effective against cancer cells, have the potential for treating drug-resistant pathogen infections. Recently, we showed that the inositol-requiring enzyme 1-α (IRE1α), an endoplasmic reticulum (ER) stress sensor, is required for clearance of MRSA in a murine skin abscess model, where neutrophils are robustly recruited to the site of infection. Further, IRE1α coordinates signaling events upstream of calcium (Ca2+) mobilization, histone citrullination, and production of mitochondrial reactive oxygen species (mitoROS), all of which are important for neutrophil inflammatory responses including the formation of antimicrobial neutrophil extracellular traps (NETs). Because excessive neutrophil activation and NET release can be detrimental to vital organs, it is not clear whether neutrophil IRE1α-mediated stress responses aid or impede the resolution of infection in the lungs. While IRE1α activation has been linked to the development of lung fibrosis through the regulation of alveolar epithelial- to-mesenchymal transition in the context of chronic inflammatory diseases, its role in pulmonary neutrophil defenses is unknown. Thus, there is a gap in our knowledge of how cellular stress responses modulate pulmonary neutrophil defenses and infection outcomes in the lungs. The overarching goal of this proposal is to elucidate the mechanisms by which neutrophil IRE1α signaling influences production of mitoROS and Ca2+ mobilization to drive NET release, injure lungs, and regulate pulmonary host defense against MRSA. We will accomplish the following Aims: (1) Define the molecular mechanisms underlying IRE1α-mediated mitoROS hyperactivation of human and mouse primary neutrophils and excessive NET release, and (2) Elucidate the role of neutrophil IRE1α signaling in excessive NET release, lung injury, and immunity in vivo using a MRSA pneumonia infection mouse model. These studies will yield mechanistic insight into how IRE1α-driven ER stress responses impact pulmonary neutrophil defenses and lung injury revealing potential targets for anti-microbial immunotherapies.
Calcium signaling in MR1-dependent presentation of Mycobacterium tuberculosis antigens
Project Summary The fundamental role of the immune system is to detect self from non-self. The detection and elimination of microbial infection is critical for human survival. One challenge to the immune system is infection from an intracellular microbe because the microbe masks its presence in a host cell. One strategy of the immune system to detect microbes is the sampling of different kinds of antigens, such as peptides, lipids and glycolipids, by antigen presenting molecules. A fundamentally unique arm of the immune system is MR1, which is an antigen presenting molecule that is intracellular, ubiquitously expressed across tissues, and detects small molecules derived from microbial metabolism. These features suggest that MR1 is poised to detect intracellular microbes. MR1 presents antigens to MR1-restricted T cells. These T cells are highly prevalent in the lungs and can kill infected cells. Because MR1 presents small molecule antigens and adopts an intracellular distribution, the mechanisms governing MR1 sampling of the intracellular environment are distinct from other antigen presenting molecules. These mechanisms remain unknown. Our over-arching hypothesis is that intracellular calcium signaling is important for MR1 antigen presentation. We use Mycobacterium tuberculosis (Mtb) as a model for intracellular infection and have identified calcium-sensitive trafficking proteins and calcium channels important for MR1 antigen presentation. Aim 1 of this study will determine the mechanism of two-pore channel 1 in MR1- dependent antigen presentation, with a focus on endoplasmic reticulum-endosome contact sites. Aim 2 will determine the role of specific calcium-sensitive Synaptotagmins and their binding partners. Aim 3 will determine the mechanism behind augmented MR1 antigen presentation following modulation of the of the cystic fibrosis transmembrane conductance regulator. Successful completion of these Aims has the potential to lead to new MR1-based immunotherapies.
Airway Epithelial Defense Mechanisms in Combating STAT3-Deficiency-Related Lung Infections
Airway Epithelial Defense Mechanisms in Combating STAT3-Deficiency-Related Lung Infections Signal transducer and activator of transcription 3 (STAT3) regulates the expression of genes essential for various cellular processes, including survival, proliferation, differentiation, self-renewal, angiogenesis, and immune response. Abnormal and persistent STAT3 activation is detected in diverse human cancers, driving multiple pro- oncogenic functions. Multiple antitumor drug development targets the inhibition of STAT3 to treat various types of cancer. Unfortunately, downregulated STAT3 significantly increases host susceptibility to recurrent infections, especially pneumonia. Additionally, individuals with genetic polymorphisms associated with lower STAT3 expression are more susceptible to severe tuberculosis. Furthermore, patients with autosomal dominant hyper- IgE syndrome (AD-HIES), also known as Job Syndrome, which is caused by de novo STAT3 mutations and substantially decreased STAT3 expression, have a significantly increased susceptibility to bacterial and fungal infections, with high mortality rates and a shortened life span often associated with Pseudomonas aeruginosa infections. Gram-negative bacteria, particularly P. aeruginosa, are opportunistic pathogens that frequently cause hospital-acquired infections. The problems are worsened by the emerging P. aeruginosa with multidrug resistance (MDR), especially in patients with repeated antibiotic treatments, such as Job Syndrome sufferers. Notably, airway epithelial cell-derived proteins play a significant role in the antimicrobial milieu, promoting effective host defense against invading pathogens. One of the most critical STAT3-regulated antimicrobial molecules is bactericidal permeability-increasing protein fold A1 (BPIFA1, also known as SPLUNC1), a multifunctional innate immunity molecule and indispensable host defense protein that is abundantly secreted in the lungs. This application aims to elucidate how STAT3 deficiency impairs host epithelial defense against microbial infections and whether BPIFA1-mediated innate immune responses can sufficiently restore effective antimicrobial protection to prevent pneumonia. The long-term objective is to advance our understanding of the respiratory innate immune response, particularly in relation to epithelial cell-specific antimicrobial defense. We characterized BPIFA1 as an airway lining fluid protein secreted apically in the airway lumen and in primary human airway epithelial cultures. In this study, we hypothesize that mucosal BPIFA1 is an essential antimicrobial protein that plays a critical role in host defense against microbial infections in STAT3-deficiency- associated pneumonia. Our proposed studies will assess innate immunity mechanisms regulating the antimicrobial activity of the airway epithelium in STAT3 deficiency-associated lung infections. By focusing on the crucial epithelial-derived protein product, BPIFA1, our study will provide an alternative treatment for respiratory infections by augmenting native host defense mechanisms in high-risk individuals, including AD-HIES, cancer, and immunocompromised patients.
The multiciliation cycle: a variant cell cycle coordinating centriole biogenesis and ciliogenesis
Project summary/Abstract Differentiating multiciliated cells line the mammalian airway and are critical for protecting the lungs from inhaled pathogens and particulates. Multiciliated cells have a distinct architecture from other cell types, having hundreds of centrioles, each of which matures into a basal body and nucleates a motile cilium. Defects in multiciliation cause a form of Primary Ciliary Dyskinesia (PCD), a lung disease. Most cells generate two centrioles and one cilium per cell cycle. We found that differentiating multiciliated cells redeploy cell cycle regulators into a novel cell cycle variant, which we refer to as the multiciliation cycle, to break these rules, generate hundreds of centrioles and cilia, and coordinate their differentiation. The multiciliation cycle redeploys many mitotic cell cycle regulators, including cyclin-dependent kinases (CDKs) and their cognate cyclins. For example, Cyclin D1-CDK4/6, regulators of mitotic G1 to S progression, is required for multiciliated cell fate initiation and entry into the multiciliation cycle. While we have focused on lung multiciliated cells, others have found that cell cycle regulators similarly participate in multiciliation of ependymal cells of the brain. Some cells, such as mammalian trophoblast giant cells, employ cell cycle variants like the endocycle to bypass mitosis. We propose that the multiciliation cycle is another cell cycle variant that augments some aspects of the canonical cell cycle, such as centriole synthesis, and blocks others, such as DNA replication. During the multiciliation cycle, E2F7, a transcriptional regulator of canonical S to G2 progression, is expressed at high levels. During multiciliated cell differentiation, E2F7 directly dampens expression of genes encoding DNA replication machinery and terminates the S phase-like gene expression program. Loss of E2F7 causes a reacquisition of DNA synthesis in multiciliated cells and dysregulation of multiciliation cycle progression, disrupting centriole maturation and ciliogenesis. We propose that multiciliated cell differentiation is coordinated by an alternative cell cycle that organizes, instead of cell proliferation, the steps of cell differentiation. In this project, we investigate how the multiciliation cycle redeploys the mitotic cell cycle regulatory framework to generate many centrioles without undergoing DNA synthesis or cytokinesis. More specifically, we seek to uncover how CDKs and cyclins are regulated to control the amount and timing of basal body synthesis, how Retinoblastoma (RB) protein controls the transcriptional program of multiciliation, and how E2Fs advance the multiciliation cycle. This work will test the hypothesis that multiciliation is organized by a variant cell cycle that uncouples centriole synthesis from DNA replication and mitosis. We propose that his variant cell cycle orchestrates progression through sequential phases required to construct the multiciliated cells that protect the lungs.
The retrotrapezoid nucleus: an integrative and interoceptive hub in neural control of breathing
In this presentation, we will discuss the cellular and molecular properties of the retrotrapezoid nucleus (RTN), an integrative and interoceptive control node for the respiratory motor system. We will present the molecular profiling that has allowed definitive identification of a cluster of tonically active neurons that provide a requisite drive to the respiratory central pattern generator (CPG) and other pre-motor neurons. We will discuss the ionic basis for steady pacemaker-like firing, including by a large subthreshold oscillation; and for neuromodulatory influences on RTN activity, including by arousal state-dependent neurotransmitters and CO2/H+. The CO2/H+-dependent modulation of RTN excitability represents the sensory component of a homeostatic system by which the brain regulates breathing to maintain blood gases and tissue pH; it relies on two intrinsic molecular proton detectors, both a proton-activated G protein-coupled receptor (GPR4) and a proton-inhibited background K+ channel (TASK-2). We will also discuss downstream neurotransmitter signaling to the respiratory CPG, focusing especially on a newly-identified peptidergic modulation of the preBötzinger complex that becomes activated following birth and the initiation of air breathing. Finally, we will suggest how the cellular and molecular properties of RTN neurons identified in rodent models may contribute to understanding human respiratory disorders, such as congenital central hypoventilation syndrome (CCHS) and sudden infant death syndrome (SIDS).
Neuro-immune interactions in pain and host defense
The Chiu laboratory focuses on neuro-immune interactions in pain, itch, and tissue inflammation. Dr. Chiu’s research has uncovered molecular interactions between the nervous system, the immune system and microbes that modulates host defense. He has found that sensory neurons can directly detect bacterial pathogens and their toxins to produce pain. Neurons in turn release neuropeptides that modulate immune cells in host defense. These interactions occur at major tissue barriers in the body including the gut, skin and lungs. In this talk, he will discuss these major neuro-immune interactions and how understanding them could lead to novel approaches to treat pain or inflammation.
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