Microglia as a Target of Immunomodulation in Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections (PANDAS)

Abstract

PANDAS, or Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections, is a neuropsychiatric condition characterized by the abrupt onset of diverse neuropsychiatric manifestations following streptococcal infections in children. It's believed to involve immune dysregulation and autoimmune targeting of the brain basal ganglia. However, the precise mechanisms remain unclear. Considering the role of microglia in neuroinflammation, evidence for their activation in PET imaging of PANDAS patients, and increased number of CD68+/Iba1+ activated microglia in mice inoculated with group A Streptococcus (GAS), we hypothesized that as a consequence of disrupted blood-brain barrier (BBB), circulating factors modulate microglial functions towards a pro-inflammatory phenotype. Human iPSC-derived microglia were generated and exposed to 5% serum from PANDAS patients or healthy controls in the presence or absence of Polymyxin B (PMX; endotoxin scavenger) and assessed for nitric oxide (NOx) production, immunophenotyped with flow cytometry (TMEM119, TREM2, CD68, Iba-1, P2Y12, CX3CR1) and bulk RNA-seq analysis. Immortalized human brain endothelial hCMEC/D3 cells were used for ECIS (Electric Cell-substrate Impedance Sensing) analysis of blood brain barrier integrity following serum treatment from PANDAS patients or healthy controls. Treatment of iPSC-derived microglia with PANDAS serum led to a significant increase in NOx production, which was blunted by approx. 30% by PMX. Immunophenotyping revealed a significant reduction in homeostatic markers TMEM119 and TREM2, and expansion of CD68+ and Iba-1+ microglia associated as an inflammatory response. It also showed significant reduction of P2RY12 and increase of CX3CR1. Regulation of TMEM119, TREM2, and P2RY12 was endotoxin-independent, while PMX only partially blunted the increase in CD68, Iba-1, and CX3CR1. RNAseq revealed significant effects of PANDAS sera on microglial gene expression pattern, with significant changes in genes associated with M1 polarization, cytokine-cytokine receptor signaling pathway, steroid synthesis pathway, and NLR (NOD-like Receptor)-, TLR (Toll-like Receptor)-, and IL-17 signaling pathways, systemic lupus erythematosus (SLE)-related genes and steroid biosynthesis pathways. BBB modeling using ECIS and human endothelial cells revealed alterations in BBB integrity following PANDAS serum treatment. The results suggest that as a result of BBB disruption in PANDAS, associated systemic inflammatory mediators and/or circulating metabolites modulate the phenotype and function of the brain microglia, a mechanism that likely contributes to neuroinflammation and the observed symptoms. The observed contribution of endotoxin may also suggest a dysfunction of intestinal barrier which may compound the systemic immune responses in PANDAS.