Roles of Th17 cytokines in microglial and neurovascular responses to recurrent intranasal Streptococcus pyogenes infections
By: Charlotte Remy Wayne
Publisher: [publisher not identified]
Published: 2022
Place of publication: [New York, N.Y.?]
Language: eng
Description: Streptococcus pyogenes infections can give rise to a diverse array of long-term secondary sequelae, including those in the brain characterized by both motor and neuropsychiatric disorders: Sydenham’s chorea and Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus infections (PANDAS). These conditions are thought to be mediated by neuroinflammatory responses and autoantibody entry into the brain, but the mechanisms are not well understood.Previous work by our laboratory has demonstrated that recurrent intranasal S. pyogenes (Group A Streptococcus, or GAS) inoculations in mice cause infiltration of CD4 T cells into the anterior brain, disruption of the blood-brain barrier (BBB), increased numbers of activated myeloid cells and degradation of excitatory synapses leading to neural circuitry deficits. However, the molecular mechanisms underlying these phenotypes have not been fully explored. To understand how the neurovasculature and myeloid cells respond to recurrent GAS infections at the transcriptome level, I profiled cells from mouse olfactory bulb (OB) and nasal lymphoid tissue by single-cell RNA sequencing (scRNAseq).

I found marked shifts in both endothelial cell and microglia populations at the transcriptome level after GAS infections, including downregulation of BBB-associated transcripts by endothelial cells (ECs), and increased production of inflammatory cytokines and chemokines, type I interferon response, and antigen presentation genes by microglia (Chapter 3). I validated several differentially expressed genes using flow cytometry, immunosorbant assays, RNA fluorescence in situ hybridization (FISH), and multiplexed error-robust FISH (MERFISH). Single-cell spatial transcriptomics of the OB revealed regional heterogeneity among microglial responses to GAS, possibly driven by proximity to infiltrating T cells. Analysis of transgenic CX3CR1/TMEM119 dual myeloid reporter mice confirmed that perivascular and meningeal macrophage numbers increase in response to GAS, but, unlike in other neuroinflammatory diseases, few macrophages infiltrate the brain parenchyma. Our laboratory has previously shown that Th17 cells are critical for BBB damage and activated microglia in response to repeated intranasal GAS infections, but the contribution of T helper (Th) 17 cell-derived cytokines in this process, as well as the transcriptional effects of Th17 cells on endothelial cells and microglia are unknown.

To expand on these findings, I performed scRNAseq on retinoic acid-related orphan receptor γt (RORγt) mutant mice (Chapter 4) which showed a significant rescue in BBB-associated genes (e.g. Mfsd2a, Itm2a and Itih5) in endothelial cells. Chemokine production and type I interferon gene expression by microglia was also significantly rescued in RORγt mutants; surprisingly antigen presentation by microglia in response to GAS was exacerbated, at both the gene and protein level. Interleukin (IL)-17A is a major cytokine produced by Th17 cells. To examine the role of IL-17A in disease pathogenesis, I treated wild-type mice with an IL-17A neutralizing antibody during the course of GAS infections (Chapter 4). This treatment was sufficient to recapitulate the transcriptional effects on microglia and endothelial cells, as well as rescue BBB permeability previously found in RORγt mutants, indicating that IL-17A may play a critical role in transcriptional responses of endothelial cells and microglia to recurrent GAS infections in vivo.

However, IL-17A did not disrupt tight junctions or induce transcytosis on ECs in vitro, suggesting that its effects on ECs in vivo are indirect. Th17 cells are capable of considerable phenotypic plasticity in response to chronic inflammation. To understand this process during recurrent GAS infections, I performed a time course analysis of CD4 T cell subsets after two, three, four and five infections (Chapter 5). This analysis revealed that proportions of “pathogenic” interferon γ-expressing Th17 cells increased over time, as did the number of CD4 T cells expressing granulocyte-macrophage colony stimulating factor (GM-CSF), a cytokine with pleiotropic effects on autoimmunity. Moreover, I determined that RORγt mutants have decreased proportions of GM-CSF+ CD4 T cells in their nasal mucosa, raising the question of whether GM-CSF may also contribute to CNS pathology (BBB permeability or microglial activation) in addition to IL-17A. To address this question, I generated mice deficient for GM-CSF in T cells and found that conditional deletion of GM-CSF in CD4+ cells partly rescued type I interferon and antigen presentation responses in microglia by scRNAseq, but did not rescue BBB leakage, suggesting that GM-CSF and IL-17A have distinct roles in the neurovascular and neuroinflammatory responses to GAS.

To relate the findings in mice to the human disease, in Chapter 6 we performed cytokine profiling in sera from PANDAS/PANS patients at the acute phase of the disease using a multiplex bead-based immunoassay. We found that many chemokines and cytokines produced by activated microglia or macrophages in the mouse model were also highly elevated in the sera of PANDAS/PANS patients. These findings suggest an important link to the human disorder both to understand disease mechanisms in humans and to use them as future clinical biomarkers for diagnosis and treatment monitoring.
Notes: Department: Cellular, Molecular and Biomedical Studies.

Thesis advisor: Dritan Agalliu.

Thesis (Ph.D.)--Columbia University, 2022.