Lupus autoantibodies initiate neuroinflammation sustained by continuous HMGB1:RAGE signaling and reversed by increased LAIR-1 expression.

Cognitive impairment is a frequent manifestation of neuropsychiatric systemic lupus erythematosus, present in up to 80% of patients and leading to a diminished quality of life. In the present study, we used a model of lupus-like cognitive impairment that is initiated when antibodies that crossreact with excitatory neuronal receptors penetrate the hippocampus, causing immediate, self-limited, excitotoxic death of hippocampal neurons, which is then followed by a significant loss of dendritic complexity in surviving neurons. This injury creates a maladaptive equilibrium that is sustained in mice for at least 1 year. We identified a feedforward loop of microglial activation and microglia-dependent synapse elimination dependent on neuronal secretion of high mobility group box 1 protein (HMGB1) which binds the receptor for advanced glycation end products (RAGE) and leads to microglial secretion of C1q, upregulation of interleukin-10 with consequent downregulation of leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1), an inhibitory receptor for C1q. Treatment with a centrally acting angiotensin-converting enzyme inhibitor or with an angiotensin-receptor blocker restored a healthy equilibrium, microglial quiescence and intact spatial memory.

Interpersonal violence exposure and inflammation during adolescence and young adulthood.

Exposure to violence increases young peoples' risk of developing mental and physical health problems. Chronic stress-related upregulation of innate immune system activity and the development of low-grade inflammation may partially underlie this health risk. However, much of the previous research has been limited to cross-sectional studies utilizing between-person analytic designs, susceptible to confounding by unmeasured factors. In this six-wave panel study of N=157 female adolescents and young adults, we tested within-person associations between interpersonal violence exposure and multiple measures of inflammatory activity. Ex vivo culture studies suggested that participants' immune cells were more reactive to microbial stimulation and less sensitive to inhibition by glucocorticoids after violence. Numbers of circulating monocyte cells increased after violence, but serum levels of interleukin-6 and c-reactive protein did not. Findings from this within-person analysis suggest that violence exposure up-regulates innate immune system activity during adolescence and young adulthood in ways that may increase mental and physical health risk.

Resting-State Functional Connectivity of the Central Executive Network Moderates the Relationship Between Neighborhood Violence and Proinflammatory Phenotype in Children.

BACKGROUND: Neighborhood violence increases children's risk for a variety of health problems. Yet, little is known about biological pathways involved or neural mechanisms that might render children more or less vulnerable. Here, we address these questions by considering whether neighborhood violence is associated with the expression of a proinflammatory phenotype and whether this relationship is moderated by resting-state functional connectivity (rsFC) of the central executive network (CEN). METHODS: The study involved 217 children (13.9 years old; 66.4% female; 36.9% Black; 30.9% Latinx), enrolled in eighth grade and reassessed 2 years later. At time 1, geocoding was used to estimate murder frequency in children's neighborhoods, and functional magnetic resonance imaging was used to characterize CEN rsFC. At both visits, children gave antecubital blood for ex vivo studies, where leukocytes were incubated with stimulators and inhibitors of inflammation, and cytokine production was measured. RESULTS: Consistent with our hypotheses, the relationship between neighborhood murder and inflammatory activity was moderated by CEN rsFC. Among children with lower rsFC, neighborhood violence covaried with a proinflammatory phenotype, reflected in larger cytokine responses to triggering stimuli and lower sensitivity to inhibitory agents. These associations were generally not apparent for children with higher rsFC, although occasionally they ran in the opposite direction. The same patterns were apparent 2 years later. CONCLUSIONS: These results advance the understanding of neighborhood violence and its relationship with processes involved in the initiation and resolution of inflammation. They also deepen understanding of variability in children's immunologic responses to stress and suggest that the CEN may be a neurobiological contributor to resilience.

Out of the Laboratory and Into the Field: Validation of Portable Cell Culture Protocols.

OBJECTIVE: Field-based research on inflammation and health is typically limited to baseline measures of circulating cytokines or acute-phase proteins, whereas laboratory-based studies can pursue a more dynamic approach with ex vivo cell culture methods. The laboratory infrastructure required for culturing leukocytes limits application in community-based settings, which in turn limits scientific understandings of how psychosocial, behavioral, and contextual factors influence the regulation of inflammation. We aim to address this gap by validating two "field-friendly" cell culture protocols, one using a small volume of venous whole blood and another using finger-stick capillary whole blood. METHODS: We evaluated the performance of both protocols against a standard laboratory-based protocol using matched venous and capillary blood samples collected from young adults (n = 24). Samples were incubated with lipopolysaccharide and hydrocortisone, and the production of proinflammatory cytokines interleukin 1ß, interleukin 6, and tumor necrosis factor α was measured in response. RESULTS: Comparisons indicate a high level of agreement in responses across the protocols and culture conditions. The overall correlation in results was 0.88 between the standard and small-volume protocols and 0.86 between the standard and capillary blood protocols. Repeatability for the small-volume and capillary blood protocols was high, with mean coefficients of variation across five replicates of 6.2% and 5.4%, respectively. CONCLUSIONS: These results demonstrate the feasibility of culturing cells and quantifying the inflammatory response to challenge outside the laboratory, with a wide range of potential applications in biobehavioral research in community-based and remote field settings.

Programmed cell removal by calreticulin in tissue homeostasis and cancer.

Macrophage-mediated programmed cell removal (PrCR) is a process essential for the clearance of unwanted (damaged, dysfunctional, aged, or harmful) cells. The detection and recognition of appropriate target cells by macrophages is a critical step for successful PrCR, but its molecular mechanisms have not been delineated. Here using the models of tissue turnover, cancer immunosurveillance, and hematopoietic stem cells, we show that unwanted cells such as aging neutrophils and living cancer cells are susceptible to "labeling" by secreted calreticulin (CRT) from macrophages, enabling their clearance through PrCR. Importantly, we identified asialoglycans on the target cells to which CRT binds to regulate PrCR, and the availability of such CRT-binding sites on cancer cells correlated with the prognosis of patients in various malignancies. Our study reveals a general mechanism of target cell recognition by macrophages, which is the key for the removal of unwanted cells by PrCR in physiological and pathophysiological processes.

Macrophages eat cancer cells using their own calreticulin as a guide: roles of TLR and Btk.

Macrophage-mediated programmed cell removal (PrCR) is an important mechanism of eliminating diseased and damaged cells before programmed cell death. The induction of PrCR by eat-me signals on tumor cells is countered by don't-eat-me signals such as CD47, which binds macrophage signal-regulatory protein α to inhibit phagocytosis. Blockade of CD47 on tumor cells leads to phagocytosis by macrophages. Here we demonstrate that the activation of Toll-like receptor (TLR) signaling pathways in macrophages synergizes with blocking CD47 on tumor cells to enhance PrCR. Bruton's tyrosine kinase (Btk) mediates TLR signaling in macrophages. Calreticulin, previously shown to be an eat-me signal on cancer cells, is activated in macrophages for secretion and cell-surface exposure by TLR and Btk to target cancer cells for phagocytosis, even if the cancer cells themselves do not express calreticulin.

Therapeutic antibody targeting of CD47 eliminates human acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy and constitutes 15% of adult leukemias. Although overall prognosis for pediatric ALL is favorable, high-risk pediatric patients and most adult patients have significantly worse outcomes. Multiagent chemotherapy is standard of care for both pediatric and adult ALL, but is associated with systemic toxicity and long-term side effects and is relatively ineffective against certain ALL subtypes. Recent efforts have focused on the development of targeted therapies for ALL including monoclonal antibodies. Here, we report the identification of CD47, a protein that inhibits phagocytosis, as an antibody target in standard and high-risk ALL. CD47 was found to be more highly expressed on a subset of human ALL patient samples compared with normal cell counterparts and to be an independent predictor of survival and disease refractoriness in several ALL patient cohorts. In addition, a blocking monoclonal antibody against CD47 enabled phagocytosis of ALL cells by macrophages in vitro and inhibited tumor engraftment in vivo. Significantly, anti-CD47 antibody eliminated ALL in the peripheral blood, bone marrow, spleen, and liver of mice engrafted with primary human ALL. These data provide preclinical support for the development of an anti-CD47 antibody therapy for treatment of human ALL.

Calreticulin is the dominant pro-phagocytic signal on multiple human cancers and is counterbalanced by CD47.

Under normal physiological conditions, cellular homeostasis is partly regulated by a balance of pro- and anti-phagocytic signals. CD47, which prevents cancer cell phagocytosis by the innate immune system, is highly expressed on several human cancers including acute myeloid leukemia, non-Hodgkin's lymphoma, and bladder cancer. Blocking CD47 with a monoclonal antibody results in phagocytosis of cancer cells and leads to in vivo tumor elimination, yet normal cells remain mostly unaffected. Thus, we postulated that cancer cells must also display a potent pro-phagocytic signal. Here, we identified calreticulin as a pro-phagocytic signal that was highly expressed on the surface of several human cancers, but was minimally expressed on most normal cells. Increased CD47 expression correlated with high amounts of calreticulin on cancer cells and was necessary for protection from calreticulin-mediated phagocytosis. Blocking the interaction of target cell calreticulin with its receptor, low-density lipoprotein receptor-related protein, on phagocytic cells prevented anti-CD47 antibody-mediated phagocytosis. Furthermore, increased calreticulin expression was an adverse prognostic factor in diverse tumors including neuroblastoma, bladder cancer, and non-Hodgkin's lymphoma. These findings identify calreticulin as the dominant pro-phagocytic signal on several human cancers, provide an explanation for the selective targeting of tumor cells by anti-CD47 antibody, and highlight the balance between pro- and anti-phagocytic signals in the immune evasion of cancer.