No abstract

Cognitive impairment is a frequent manifestation of neuropsychiatric systemic lupus erythematosus (NPSLE), present in up to 80% of patients and leading to a diminished quality of life. We have developed a model of lupus-like cognitive impairment which is initiated when anti-DNA, anti-N-methyl D-aspartate receptor (NMDAR) cross-reactive antibodies, which are present in 30% of SLE patients, penetrate the hippocampus. This leads to immediate, self-limited excitotoxic death of CA1 pyramidal neurons followed by a significant loss of dendritic arborization in the remaining CA1 neurons and impaired spatial memory. Both microglia and C1q are required for dendritic loss. Here we show that this pattern of hippocampal injury creates a maladaptive equilibrium that is sustained for at least one year. It requires HMGB1 secretion by neurons to bind RAGE, a receptor for HMGB1 expressed on microglia, and leads to decreased expression of microglial LAIR-1, an inhibitory receptor for C1q. The angiotensin converting enzyme (ACE) inhibitor captopril, which can restore a healthy equilibrium, microglial quiescence, and intact spatial memory, leads to upregulation of LAIR-1. This paradigm highlights HMGB1:RAGE and C1q:LAIR-1 interactions as pivotal pathways in the microglial-neuronal interplay that defines a physiologic versus a maladaptive equilibrium.

Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by the presence of anti‐nuclear antibodies as well as a broad array of clinical manifestations spanning nearly every organ system. Neuropsychiatric syndromes affect over one half of SLE patients, presenting most commonly as cognitive dysfunction or headache. The pathophysiology of neuropsychiatric lupus is not fully understood; however, a critical mechanism has been identified, which involves passage of inflammatory cytokines and anti‐brain antibodies through the blood‐brain barrier. We previously developed a mouse model to study the effects of anti‐dsDNA antibodies that cross‐react with the NMDA receptor (NMDAR), termed DNRAbs, which are present in approximately 30% of SLE patients and whose presence in the CSF is associated with non‐focal CNS manifestations of neuropsychiatric lupus. In this mouse model, non‐spontaneously autoimmune mice are immunized with DWEYS peptide, a DNA mimotope that elicits production of DNRAb antibodies, which act as positive allosteric modulators of the NMDAR. DNRAbs only enter the brain when the blood‐brain barrier is porous; so, we administer LPS, which breaches the blood‐brain barrier in the hippocampus. In this model, we discovered two stages of brain injury. In the first stage, lasting up to a week, we observed excitotoxic neuronal death, secondary to DNRAb‐mediated NMDAR activation. In the second stage, lasting months, we identified an inflammatory homeostasis, consisting of microglial activation, loss of dendritic arborization in surviving hippocampal neurons, and neuronal secretion of HMGB1, a chromatin protein that can be secreted to act as a damage‐associated molecular pattern (DAMP). Once secreted by neurons, HMGB1 interacts with two important receptors —RAGE and TLR4 — inducing a cascade of downstream inflammatory events. In addition, HMGB1 binds to the GluN2B subunit of NMDARs and the complement component C1q, forming a molecular bridge that facilitates synaptic pruning. Here we show that HMGB1 acts directly on microglia, where it activates microglia through RAGE and TLR4 signaling. We found that HMGB1 stimulates microglia to secrete IFNa, transcriptionally upregulating interferon regulatory factors, including IRF7. We also show that IFNa secreted by microglia acts in an autocrine fashion, regulating IFN‐response genes such as MX1; IFNa also induces C3 and C1q transcription in microglia. HMGB1 also stimulates microglia to secrete TNFa and IL‐1b, which enhances the inflammatory milieu in surrounding neurons. In addition, we found that the ACE‐inhibitor captopril reverses dendritic pruning in DWEYS‐immunized mice­­ — an essential component of the second stage of DNRAb‐related damage — by regulating expression of the inhibitory receptor LAIR1 in microglia. Whereas captopril reverses dendritic pruning in DWEYS‐immunized wildtype mice, captopril has no such effect on LAIR1 knockout mice. We are currently analyzing RNA sequencing data from microglia isolated from captopril‐treated, DWEYS immunized mice, t...

The coronavirus disease 2019 (COVID-19) response necessitated innovations and a series of regulatory deviations that also affected laboratory-developed tests (LDTs). To examine real-world consequences and specify regulatory paradigm shifts, legislative proposals were aligned on a common timeline with Emergency Use Authorization (EUA) of LDTs and the US Food and Drug Administration (FDA)-orchestrated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) labeling update study. The initial EUA adoption by LDT developers shows that the FDA can have oversight over LDTs. We used efficiency-corrected microcosting of our EUA PCR assay to estimate the national cost of the labeling update study to $0.3 to $1.4 million US dollars. Labeling update study performance data showed lower average detection limits in commercial in vitro diagnostic (IVD) assays versus LDTs (32,000 ± 75,000 versus 71,000 ± 147,000 nucleic acid amplification tests/mL; P = 0.04); however, comparison also shows that FDA review of IVD assays and LDTs did not prevent differences between initial and labeling update performance (IVD assay, P < 0.0001; LDT, P = 0.003). The regulatory shifts re-emphasized that both commercial tests and LDTs rely heavily on laboratory competence and procedures; however, lack of performance data on authorized tests, when clinically implemented, precludes assessment of the benefit related to regulatory review. Temporary regulatory deviations during the pandemic and regulatory science tools (ie, reference material) have generated valuable real-world evidence to inform pending legislation regarding LDT regulation.