Project Grant K99AR084572

STAPHYLOCOCCUS AUREUS INDUCED ITCH AND NEURO-IMMUNE SIGNALING IN SKIN INFLAMMATION AND ATOPIC DERMATITIS - PROJECT SUMMARY ITCH IS AN UNPLEASANT SENSATION THAT ELICITS THE DESIRE OR THE REFLEX TO SCRATCH. CHRONIC ITCH IS A CHARACTERISTIC SYMPTOM OF ATOPIC DERMATITIS (AD), ONE OF THE MOST COMMON INFLAMMATORY SKIN DISEASES AND A SIGNIFICANT CAUSE OF PATIENT SUFFERING. ANOTHER HALLMARK OF AD IS MICROBIAL DYSBIOSIS WITH INCREASED STAPHYLOCOCCUS AUREUS ON THE SKIN. OVER 90% OF AD SKIN LESIONS ARE POSITIVE FOR S. AUREUS. HERE, I PROPOSE TO INVESTIGATE THE ROLE OF S. AUREUS- INDUCED ITCH AND NEURO-IMMUNE SIGNALING IN DRIVING SKIN BARRIER DAMAGE AND IMMUNE RESPONSES. MY PRELIMINARY DATA SHOW THAT EPICUTANEOUS EXPOSURE TO S. AUREUS INDUCES ROBUST ITCH BEHAVIORS (SPONTANEOUS ITCH AND ALLOKNESIS) AND RESULTING SKIN PATHOLOGY IN MICE. I IDENTIFIED THE SERINE PROTEASE V8 (SSPA) AS A CRITICAL FACTOR IN MEDIATING BOTH ITCH AND DERMATITIS. V8 DIRECTLY ACTIVATES ITCH NEURONS THROUGH ITS RECEPTOR PROTEINASE-ACTIVATED RECEPTOR 1 (PAR1). I HYPOTHESIZE THAT S. AUREUS AND V8 PROTEASE-INDUCED ITCH TRIGGER SKIN INFLAMMATION AND IMPACT NEUROIMMUNE SIGNALING. IN SPECIFIC AIM 1, I WILL DEFINE THE CONTRIBUTION OF V8 PROTEASE, NEURONS, AND ITCH TO SKIN IMMUNE RESPONSES. I WILL INOCULATE MICE WITH WILDTYPE OR ISOGENIC V8-DEFICIENT (SSPA) S. AUREUS STRAINS AND PERFORM DETAILED FLOW CYTOMETRY TO PROFILE THE SKIN IMMUNE POPULATION. I WILL UTILIZE GENETIC APPROACHES TO ABLATE SPECIFIC SKIN-INNERVATING NEURONS (TRPV1+ AND MRGPRD+) TO ASSAY THEIR ROLE IN ITCH AND INFLAMMATION. I WILL TREAT MICE WITH THE PAR1 ANTAGONIST VORAPAXAR TO DETERMINE HOW ITCH AND SCRATCHING DRIVE SKIN IMMUNE RESPONSES. IN SPECIFIC AIM 2, I WILL CHARACTERIZE THE NEURONAL RESPONSE TO S. AUREUS AND V8 PROTEASE. ITCH IS MEDIATED BY DORSAL ROOT GANGLIA (DRG) SENSORY NEURONS. I WILL PERFORM RNA-SEQUENCING OF DRGS TO IDENTIFY TRANSCRIPTIONAL CHANGES FOLLOWING S. AUREUS EXPOSURE. I WILL USE REPORTER MICE (NAV1.8-CRE/TDTOMATO) TO LABEL SENSORY NEURONS AND FOR WHOLE MOUNT CONFOCAL MICROSCOPY TO QUANTIFY EPIDERMAL NEURON DENSITY IN NAIVE AND S. AUREUS EXPOSED MICE. I WILL CULTURE DRG NEURONS WITH S. AUREUS OR PURIFIED V8 PROTEASE AND PERFORM ELISAS TO MEASURE SECRETED NEUROPEPTIDES. I HYPOTHESIZE THAT S. AUREUS AND V8-INDUCED ITCH/SCRATCHING CONTRIBUTE SIGNIFICANTLY TO SKIN PATHOLOGY IN AD. IN SPECIFIC AIM 3, I WILL UTILIZE TMEM79-/- MOUSE MODEL OF AD TO INVESTIGATE MICROBE-NEURON INTERACTIONS. I WILL COLONIZE TMEM79-/- MICE WITH WT AND SSPA S. AUREUS AND MEASURE ITCH AND DERMATITIS. I WILL SURGICALLY DENERVATE THE BACK SKIN OF TMEM79-/- MICE AND PERFORM FLOW CYTOMETRY AND MULTIPLEX ELISA TO IDENTIFY IMMUNE CHANGES. I WILL PERFORM 16S SEQUENCING OF SKIN SAMPLES FROM CONTROL AND DENERVATED TMEM79-/- MICE TO DETERMINE THE ROLE OF NEURONS IN REGULATING THE SKIN MICROBIOME IN AD SKIN. I WILL ALSO DETERMINE WHETHER S. AUREUS COLONIZATION CHANGES IN DENERVATED MICE. THIS WORK WILL REVEAL NOVEL MOLECULAR CROSSTALK BETWEEN S. AUREUS, NEURONS, AND IMMUNE CELLS IN ITCH AND AD. THE THREE AIMS OF THIS STUDY LEVERAGE MY UNIQUE SKILLSETS AND TRAINING BY COMBINING MICROBIOLOGICAL, NEUROBIOLOGICAL, AND IMMUNOLOGICAL APPROACHES. GIVEN THE IMPORTANCE OF ITCH IN AD AND OTHER SKIN DISEASES, INVESTIGATING HOW MICROBE-NEURON CROSSTALK DRIVE ITCH AND DERMATITIS COULD TRANSFORM OUR UNDERSTANDING OF HOST-MICROBE INTERACTIONS AT THE SKIN BARRIER.