In vivo mechanisms mediating sickle cell vaso-occlusion and organ damage

PROJECT SUMMARY: Previous in vivo studies on this project using a humanized mouse model of sickle cell disease (SCD) have revealed that activated neutrophils play a direct role in vaso-occlusive episodes (VOE) by interacting with circulating erythrocytes. In particular, we have described that aged neutrophils (i.e. those that have spent the most time in circulation) are the most active and this aging phenotype is driven by to the microbiota. Indeed, microbiota depletion markedly reduces aged neutrophil counts, and improves the acute and chronic SCD manifestations. In the past funding cycle, we have evaluated how psychological stress exacerbates VOE by inducing a glucocorticoid response that alters gut permeability, exposing specific commensals (segmented filamentous bacteria), leading to the increased generation of aged neutrophils via IL-17A/G-CSF cytokine induction. In preliminary data supporting this proposal, we have further investigated neuro-immunology in SCD pathogenesis. As pain is a major manifestation of the disease, we have evaluated the impact of nociceptor activation in VOE and organ damage and found, unexpectedly, a protective role of nociceptor activation mediated by the neurotransmitter calcitonin gene-related peptide (CGRP). Activation of nociceptor nerves with capsaicin also reduced VOE and organ damage. In Specific Aim 1, we will investigate the mechanisms by which nociceptors inhibit SCD-associated vaso-occlusion. We will investigate the role of RAMP1 in transducing the CGRP inhibitory signals into neutrophils using genetically deficient mice. We evaluate whether capsaicin improves VOE by activating TRPV1+ neurons vs possible effects on the microbiota. Based on preliminary data implicating histone deacetylases inhibitors in neutrophil aging, we will evaluate in Specific Aim 2 molecular mechanisms orchestrating the neutrophil aging program. We will investigate the molecular basis behind the neutrophil aging program using proteomics analyses. We will also screen for novel markers specific to neutrophil aging using flow cytometry-based approach to complement the mass spectrometry studies of the proteome. In Specific Aim 3, we will dissect how the microbiota promotes organ damage in SCD. Preliminary results in which single antibiotics with narrower spectrum were administered, led us to identify candidate bacteria predicted to produce beneficial or detrimental effects on organ damage in SCD. We will investigate the interplay between these microbial candidates and iron deposition in the mechanism of organ damage. We will also analyze the microbiota of SCD patients (on and off Penicillin or hydroxyurea) with our clinical collaborators and correlate the results with clinical severity. These studies will enhance our understanding of VOE and organ damage and may lead to new clinical trials where the manipulation nociceptor signals or the use of specific probiotic therapy may mitigate SCD pathogenesis.