Characterization of peripheral immune dysregulation and brain neuroimmune pathophysiology in the Df(16)A+/- Mouse Model of 22q11.2 Deletion Syndrome, a high genetic risk factor for schizophrenia

Abstract

Schizophrenia (SCZ) is a severe and chronic neurodevelopmental disorder that imposes a profound global health burden. Among its complex genetic underpinnings, the 22q11.2 Deletion Syndrome (22q11.2DS) stands out as the highest-known genetic risk factor, with approximately 25-30% of affected individuals developing a psychotic disorder. Recent research in SCZ has undergone a paradigm shift, moving beyond purely neurocentric models to embrace growing evidence that implicates immune dysregulation and neuroinflammation as critical etiological processes. While 22q11.2DS is known to confer a complex immunological phenotype, including foundational T-cell deficits and a high incidence of autoimmune disorders, the functional consequences of this genetic 'first hit' on immune reactivity and central nervous system (CNS) vulnerability is not well understood. Therefore, the primary objective of this thesis was to systematically characterize immune dysregulation in the Df(16)A+/- mouse model of 22q11.2DS to test the hypothesis that the genetic deletion programs a dysfunctional response to subsequent inflammatory challenges, thereby providing a plausible biological substrate for neuropsychiatric risk.

This investigation utilized the Df(16)A+/- mouse model, which carries a chromosomal deletion syntenic to the human 22q11.2DS critical region. Our experimental strategy involved a comprehensive profiling of the peripheral and central immune systems at a homeostatic baseline and following provocation with distinct immune stimuli, including a sustained Toll-like receptor 7 (TLR7) agonist (Aldara) and an acute Toll-like receptor 3 (TLR3) agonist (LMW-Poly(I:C)). Given the limited characterization of the Df(16)A+/- immune profile and the incompletely elucidated mechanisms linking 22q11.2DS to psychiatric risk, this thesis employed both hypothesis-driven and exploratory analytical frameworks to systematically characterize the immune phenotype. Primary analytical techniques included multi-plex immunoassays (Luminex), multi-parameter flow cytometry, and quantitative immunohistochemistry. This central analysis was supported by the development of a high-throughput, objective morphometric pipeline to robustly quantify glial reactivity and neuronal cytoarchitecture within the hippocampus.

Our results demonstrate that the Df(16)A+/- genotype is not immunologically silent but confers a "first hit" of subtle, multi-system dysregulation. Peripherally, this is defined by a "central deficit, peripheral compensation" model, with foundational T-cell developmental deficits in the bone marrow and thymus that are compensated for in circulating blood and spleen populations. This cellular alteration is mirrored by a rewired baseline molecular milieu, characterized by increased inter-animal heterogeneity and a less interconnected systemic cytokine network. Centrally, this state corresponds to a latent glial vulnerability, defined by a subtle but significant reduction in the morphological complexity of hippocampal microglia, anatomically co-localized to the dorsal CA1 and dentate gyrus.

Subsequent immune challenges revealed this vulnerable baseline dictates a "second hit" of a profoundly dysfunctional and uncoupled neuro-immune response. The systemic reaction to the TLR7 challenge was both attenuated in magnitude, with blunted splenomegaly and pro-inflammatory cytokine upregulation, and qualitatively dysregulated, exemplified by the paradoxical downregulation of IFN-alpha and the collapse of the organized inflammatory network. Critically, this peripheral impairment was associated with a complete uncoupling of the glial inflammatory cascade in the dorsal hippocampus: wildtype mice mounted a coordinated neuroinflammatory response, whereas Df(16)A+/- mice exhibited a dissociated cascade where microglia adopted a morphologically reactive state in the complete absence of a canonical astrocyte (GFAP+) response. This microglial response was itself dysfunctional, occurring without the expected upregulation of the functional protein Iba1. This glial uncoupling was directly associated with the neuronal findings: Df(16)A+/- mice exhibited an altered parvalbumin-positive (PV+) interneuron response, lacking the significant change in cell counts observed in wildtype animals following the challenge. Findings from an acute TLR3 challenge, qualified by a severe physiological confound of hypothermia, also pointed towards a rewired inflammatory program by revealing a paradoxical central myeloid response.

The central conclusion of this thesis is that the 22q11.2DS-relevant genetic deletion establishes a "first hit" of homeostatic instability that dictates a functionally impaired "second hit" response to immune challenges. This work links this high-impact genetic risk factor to a specific CNS vulnerability, defined by an uncoupled neuroinflammatory cascade, where microglia activate but astrocytes fail to respond, and a corresponding genotype-specific PV+ interneuron response, where the significant change in cell counts seen in wildtype animals was absent in the Df(16)A+/- mice. This integrated phenotype provides a novel and plausible biological substrate for the increased neuropsychiatric risk observed in the human 22q11.2DS population.