Social Stress Impacts Multicellular Molecular Mechanisms in the Brain Affecting Nociception and Neuroimmune Activity in Sickle Cell Disease

Environment and social determinants of health (SDOH) may affect the whole individual by altering the biology and perception-based stimuli contributing to cognitive dysfunction, pain and poor therapeutic outcomes. Social isolation has been identified as a factor contributing to worsening chronic pain and may interfere with pain treatment. However, the central and peripheral mechanisms underlying the adverse social conditions in sickle cell disease (SCD) are not understood. We therefore examined the mechanisms of social isolation and traumatic experiences by using mouse models of SCD (HbSS) and control human HbA expressing (HbAA) mice. Male HbSS and HbAA mice were singly-housed under ‘isolation’ or in ‘companionship,’ with a female in the same cage, for 3 months. To avoid the confounding effect of pregnancy, female HbSS and HbAA mice were housed in isolation or companionship, with another female. We observed an increase in thermal and mechanical hyperalgesia (P<0.05 for both) and impairment in cognitive function in HbSS mice in isolation vs companionship. Cognitive function was assessed using the novel object recognition (NOR) and object location tests for working and spatial learning and memory, respectively. Male and female HbSS mice in isolation showed significant impairment in NOR (P<0.05) and male mice showed impairment in object location memory (P<0.05) compared to mice in companionship. Spatial transcriptomics of the brain slices in situ of male HbSS mice using multiplexed error-robust fluorescent in situ hybridization (MERFISH) analysis showed molecular alterations at a multicellular level. These molecular signals converged at a significant increase in calmodulin kinase IIα (CaMKIIα), which catalyzes the release of calcium. CaMKIIα expression was increased in endothelium (~56%, P<0.001), astrocytes (~34%, P<0.01), oligodendrocytes (~48%, P<0.01), oligoprogenitor cells (~43%, P=0.084), and microglia (~45%, P=0.056) in isolated vs. companionship mice. Increased calcium activates calpain 1, a proteolytic enzyme which binds to CaMKIIα leading to their translocation to the cell membrane, cellular injury and hyperalgesia; calpain activity was significantly increased in the brain of male HbSS mice in isolation (P<0.05). In the central nervous system (CNS), calpain 1 activity underlies neuronal apoptosis and can exacerbate hyperalgesia. Calpain 1 also plays a major role in sickle pathobiology in the periphery by promoting thrombosis, platelet activation, deformation of red blood cells caused by dehydration, activation of mast cells, and apoptosis in peripheral neurons, which may all contribute to worsening of hyperalgesia. Concomitant with molecular alterations in the brain, spinal cord inflammation was significantly upregulated in female HbSS mice in isolation, indicated by increased levels of interleukin-17 and tumor necrosis factor α (TNFα, P<0.05 for both), and increased elastase activity observed in the spinal cord and plasma (P<0.05 for both). Increased IL17, TNFα and neutrophil activation are hallmark features of SCD, which appear to be regulated by the brain’s perception-based mechanisms. Immunofluorescent staining of spinal cord sections of sickle mice revealed that elastase was associated with neutrophils, whereas IL17 has been associated with T-lymphocytes in osteonecrosis and with renal glomerular cells in kidney injury in SCD. Neutrophil elastase activates protease-activated receptor 2 on nerve fibers which plays a critical role in the development of pain. Stress increases neutrophil activity and organ injury in the periphery via the brain’s motor and fear circuits in wild type mice (Poller et al., Nature 2022, PMID: 35636458). Together, our data show, for the first time, that the neuroimmune mechanisms triggered by social stress in SCD involve the whole body via multicellular changes in the brain. The increased elastase activity in the CNS and the periphery may continue to activate the pro-nociceptive pathways leading to chronic pain and poor response to therapy. Thus, improvement in SDOH and perception-based therapies may reduce pain and/or improve therapeutic outcomes in SCD.