BTK is an essential molecule in the B-cell signaling , and its deficiency results in agammaglobulinemia in males. BTK is also expressed in myeloid cells and neutrophils and plays a crucial role in immune host defense. The development of effective small molecules that potently inhibit BTK and actively have been used in clinical trials for Multiple Sclerosis persuade us to further investigate the role of this pathway in CNS immune compartment.

Patients with BTK deficiency experience recurrent systemic infections. IVIg and advancements in antimicrobials led patients survive into adulthood. However, a subset of patients develop progressive neurodegenerative syndromes later in life with significant fatality. The pathogenesis of this rapidly progressive dementia has been attributed to chronic viral encephalitis.

 using iPSC derived technology we explore the role of BTK deficiency in microglia. Himan iPSC was made from healthy donor and BTK deletion introduced in isogenic iPSCs using CRSPR-CAS9 technology. BTK sufficient and deficient iPSCs differentiated towards microglia.

Both BTK-deficient and sufficient iPSCs successfully differentiated and expressed  TMEM119, TREM2, and IBA1.  BTK-deficient iMicroglia were smaller and tended to cluster.  Using a zymosan-based phagocytosis assay as a screening tool to evaluate phagocytosis in microglia, we found that BTK-deficient iMicroglia had reduced phagocytosis compared to controls. To assess cytokine production, BTK-sufficient and BTK-deficient iMicroglia were stimulated with LPS and supernatant cytokine levels were measured using a 47-plex Luminex assay. The results showed a significant reduction in pro-inflammatory cytokines (IL-6, TNF-α and IL-1β) and type I (IFNα) interferon in BTK-deficient microglia. Furthermore, chemokines associated with Th1/Tc1 responses, including CXCL10 (IP-10) and CCL2 (MCP-1), were markedly decreased, whereas CCL22 (MDC), a chemokine that promotes Th2/Tc2 response, was significantly elevated.

These findings suggest that BTK deficiency/inhibition may impair phagocytosis and shift microglia toward an M2-like, anti-inflammatory phenotype, which could compromise Th1/Tc1-mediated immune responses necessary for antiviral effect and at the same time modulate inflammatory profile in the context of neuroinflammatory diseases.