Isaac Chiu - US grants

DESCRIPTION (provided by applicant): The sensory nervous system innervates mucosal tissues that are highly exposed to pathogenic organisms. Pain is a major symptom of patients suffering from infectious diseases. During tissue inflammation, nociceptors, the neurons which react to intense/damaging stimuli, are excited, leading to a state of hyper-algesia. The role of pain-sensing fibers in host-pathogen defense has not been elucidated. In this study, we propose that the nervous system plays an active role in the detection and defense against bacterial infection. We find that nociceptor neurons can directly recognize and respond to two major bacterial ligands, N-formyl-Met-Leu-Phe (fMLF) and Lipopolysaccharide (LPS). Nociceptor neurons express the co-receptors for LPS, CD14 and Toll-like Receptor 4 (TLR4). Therefore, sensory neurons may share similar pathogen recognition pathways as the innate immune system. In Specific Aim 1 and Aim 2, I will investigate the receptor signaling pathways that mediate the recognition of fMLF and LPS by pain-sensing neurons. Upon stimulation, peripheral nociceptor fibers release neuro-peptides including CGRP and substance P, which act on endothelial and smooth muscle cells to induce vaso-dilation. This leads to the influx of inflammatory mediators and recruitment of innate immune cells. The role of this "neurogenic inflammation" has not ben analyzed in the context of infection. We propose that pain mediated inflammation plays an important role in limiting the invasion and spread of pathogens. In Specific Aim 3, I will investigate the relevance of neurogenic inflammation in the defense against bacterial infection. We will generate C- fiber nociceptor neuron deficient mice, which lack the ability to produce neuropeptides, and neuronal conditional knockout mice for MyD88, the downstream signaling adaptor for all Toll-like Receptors. These mice will be tested in two clinically relevant models of LPS induced endotoxemia and Streptococcus Pneumoniae bacterial infection. A direct pathogen detection mechanism by sensory neurons indicates remarkable similarities between innate immunity and nociception. Activation of pain neurons may directly efectuate the host inflammatory response to pathogens. Elucidating the molecular pathways that mediate this process will benefit the future treatment of pain and infectious disease. PUBLIC HEALTH RELEVANCE: Noxious stimuli detecting, (nociceptor) sensory neurons innervate tissues that are highly exposed to pathogenic organisms. In this study, we propose a role for the nervous system in directly detecting bacterial ligands and modulating the induction of host protective neurogenic inflammation. A role for nociceptor sensory neurons in the detection and defense against pathogens holds implications for the future treatment of pain and infectious diseases.

DESCRIPTION (provided by applicant): Pain is a major component of inflammation, but the role of pain-sensing nociceptor neurons in the regulating the inflammatory process is not well understood. It is increasingly clear that molecular and cellular interactions between the nervous system and immune system play important roles in development, homeostasis, and tissue inflammation. Nociceptor neurons are specialized to detect noxious/harmful stimuli and upon activation, transduce the sensation of pain. Nociceptor neurons densely innervate peripheral tissues including the skin, lungs, and gastrointestinal tract, tissues that are often exposed to pathogenic infection. Bacterial infection often produces significant pain. Our previous work demonstrated that S. aureus directly activates nociceptor neurons through release of N-formyl peptides and the pore-forming toxin ?emolysin, thereby producing pain. Upon activation, these nociceptor neurons release neuropeptides that are able to directly act on the immune system. Thus, like the immune system, the nervous system is able to directly detect and respond to pathogens. We hypothesize that nociceptor neurons may play an important and previously unappreciated role in host defense against pathogens. The goal of this study is to determine the contribution of nociceptor neurons and their neuropeptides in modulating the function of both innate and adaptive immune cells during bacterial host defense. We will utilize specific genetic and pharmacological tools to silence or activate nociceptor neuron activity with microbiological and immunological analyses to ascertain the significance of neuro-immune communication in host defense against S. aureus infection. These experiments will test the role of nociceptor neurons in the regulation of: 1) Neutrophil and monocyte mediated clearance of S. aureus and dendritic cell activation during infection. 2) T cell responses to S. aureus including priming in lymph nodes and effector function at the site of infection. These analyses will determine the role of nociceptor neurons in regulating immunity and inflammation, a relatively unexplored and potentially significant area of research. These studies have the potential to transform our understanding of host-pathogen interactions and of neuro-immune mechanisms in bacterial host defense.

PROJECT ABSTRACT Pain is a fundamental protective neuronal signal for organisms to avoid danger. Nociceptors are the specific subset of peripheral sensory neurons that detect harmful/noxious stimuli and transmit pain signals to the brain. Chronic pain is a major socio-economic burden, but the underlying molecular mechanisms are not well understood. I previously found that bacterial pathogens produced pain by directly activating nociceptor neurons during infection. Moreover, I found that nociceptors played a role in suppressing local immune cell recruitment and lymphadenopathy. These findings raise the possibility that the nervous system can play a direct participatory role in host defense. Nociceptor neurons densely innervate the skin and gut, which are heavily colonized by commensal bacteria. However, the bidirectional crosstalk between the tissue-resident microbes with the sensory nervous system is poorly understood. In this NIH Director's New Innovator Award, I test the hypothesis that molecular interactions between the host microbiota and nociceptor neurons play a key role in governing pain production and the composition of the microbiota. This research is motivated by basic questions about the role of host-microbe interactions that will help us gain insights into mammalian physiology: i) Do specific commensal gut or skin bacterial species (pathobionts or symbionts) set the threshold for nociceptor neuron activity and development of chronic pain? ii) Can we identify specific bacterial molecular mediators that modulate nociceptor neural activity and pain? iii) Do nociceptor-associated ligands in spicy foods (e.g. capsaicin, mustard oil) have a significant impact on the composition and quality of the microbiota? Iv) Do nociceptor neurons produce molecular mediators that directly impact the microbiota or tissue-resident immune cells? To address these questions, I will combine neurobiological, immunological, and microbiological approaches to analyze the reciprocal interactions between nociceptor neurons and the resident microbiota. Germ-free and bacterial monocolonization experiments will determine if distinct symbiotic or pathobiotic commensal bacterial strains influence the development of pain. Neuronal calcium flux, multi-electrode array analysis, and protein chemical techniques will define the bacterial mediators that modulate nociceptor activity. These analyses will lead to the identification of potential novel molecular mediators of pain. Conversely, I will analyze if nociceptor activity in vivo plays a role in regulating the host microbiota. Using transgenic, pharmacological, and optogenetic strategies to specifically deplete, activate or silence nociceptors, I will ascertain whether sensory neurons modulate the composition of the skin and gut microbiota. Based on my foundational training in Immunology and Neurobiology, along with the ability to carry out cross-disciplinary scientific approaches, I am uniquely qualified to lead this effort. I have demonstrated a willingness to challenge convential paradigms, focusing my research on questions that have potential impacts on human health. With this proposed NIH Director's New Innovator Award, I will carry out studies to produce novel insights into host- microbe interactions facilitating the development of treatments for chronic pain and microbial dysbiosis.

PROJECT SUMMARY Pain (Dolor) is one of the four cardinal signs of inflammation, and often accompanies bacterial infections. Nociceptor neurons are the peripheral sensory neurons that mediate pain, which densely innervate barrier tissues including the skin and soft tissues that are exposed to pathogens. Streptococcus pyogenes is a leading cause of necrotizing fasciitis, an invasive and life-threatening form of infection, in which pain occurs early and ?out of proportion? with other symptoms. We hypothesize that pain plays a major causative role in the pathogenesis of S. pyogenes, by inducing neural mediated suppression of innate immune cell recruitment and killing of bacteria. Here, we will determine: 1) The molecular mechanisms of pain during S. pyogenes infection, with a focus on streptococcal pore-forming toxins streptolysin S (SLS) and streptolysin O (SLO), and 2) The role of nociceptors and pain signaling in regulating neutrophil function and host defense against S. pyogenes. We test the hypothesis that targeting pain signaling would lead to enhanced innate immune responses. Given the importance of pain in the diagnosis of necrotizing fasciitis, and the widespread use of analgesics, our findings connecting pain to S. pyogenes host defense could have important clinical implications. The two aims of this study leverage the complementary skills of Dr. Chiu and Dr. Wessels, combining neurobiological, immunological, and microbiological approaches to investigate the role of pain in host defense. In Specific Aim 1, we will determine the critical molecular mechanisms of pain production during S. pyogenes infection by two clinical isolates. We use isogenic mutant bacterial strains and plasmid complementation strategies together with neurobehavioral assays to determine whether SLS and SLO mediate S. pyogenes-induced pain. We will determine whether the inflammasome and IL-1? are involved in neuronal recognition of pore-forming toxins or pain signaling. In addition, we determine the effectiveness of non-steroidal anti-inflammatory drugs (NSAIDs) in impacting pain perception during S. pyogenes infection. In Specific Aim 2, we will determine how targeted ablation of TRPV1+ nociceptors using RTX treatment or Trpv1-cre/DTA mice enhances host defenses against S. pyogenes. We will determine how temporally and spatially controlled modulation of TRPV1+ neural activity using optogenetic or DREADD strategies affects the outcome of S. pyogenes infection. We will also utilize opioids to block central pain perception or Botulinum toxin to block peripheral pain signaling to determine which neural component modulates immune responses against S. pyogenes. In our analysis, we elucidate the role of nociceptor-derived neuropeptides such as CGRP in suppressing neutrophil phagocytosis and killing of S. pyogenes. This study analyzes a significant link between induction of pain signaling, neural blockade of innate immunity, and the potentiation of S. pyogenes bacterial pathogenesis. Targeting pain and neuro-immune suppression could lead to novel approaches to treatment of this and other invasive bacterial infections.

PROJECT SUMMARY Nociceptor neurons are peripheral sensory neurons that densely innervate the gastrointestinal tract, detecting noxious/harmful stimuli to mediate protective neural reflexes including pain. However, the role of nociceptor neurons or their molecular mediators in regulating gut physiology, barrier protection, and host defense is not well understood. Here we hypothesize that specific gut-innervating nociceptors signal to epithelial cells to modulate gut barrier defenses at homeostasis and during bacterial invasion. Our preliminary data show that nociceptor neurons and the nociceptor neuropeptide calcitonin gene-related peptide (CGRP) signal to intestinal goblet cells and microfold (M) cells, two types of gut epithelial cells that mediate barrier protection and host defenses. Recent molecular phenotyping of DRG neurons show that multiple types of nociceptors innervate the gut. In this project, we will utilize targeted genetic and molecular approaches to: 1) Determine whether Nav1.8+ nociceptor neurons or their specific subsets (CGRP+, MRGPRD+) are necessary for maintenance of gut epithelial cell function during homeostasis, and barrier protection against the enteric pathogens Salmonella Typhimurium or Citrobacter rodentium; 2) Determine whether chemogenetic (DREADD) or dietary ligand activation of nociceptor neurons is sufficient to induce changes in gut epithelial cells and barrier function; 3) Define the role of the nociceptor neuropeptide CGRP-RAMP1 axis in regulating barrier epithelial (goblet cells, M cells) and immune cells in modulating barrier host defenses. We aim to use innovative and interdisciplinary approaches from neurobiology, gastroenterology, and immunology to interrogate the role of nociceptor neurons in gut barrier function and enteric host defense. The team includes experts in nociceptor neurons and neuroimmunology (Chiu lab), and gut microbial and immune responses (Huh lab). A role for the nervous system in regulating gastrointestinal barrier defenses could lead to novel approaches to treat autoimmune and inflammatory diseases. A greater understanding of how neurons signal to epithelial cells and immune cells the gastrointestinal tract could lead to novel insights in tissue barrier homeostasis. Our findings could also have relevance to disease conditions where gut barrier dysfunction occur such as autoimmune or inflammatory diseases. Targeting neurons or CGRP signaling could therefore lead to novel approaches to enhance barrier integrity to treat gastrointestinal diseases.