Pathway-instructed therapeutic selection of ruxolitinib reduces neuroinflammation in fungal postinfectious inflammatory syndrome

Abstract

Therapies to reduce neuroinflammation following resolution of acute central nervous system (CNS) infections are urgently needed, particularly for patients with non-HIV-associated cryptococcal meningoencephalitis complicated by a postinfectious inflammatory response syndrome (cPIIRS). To identify druggable targets in cPIIRS, patient cerebral spinal fluid samples underwent transcriptional analysis, revealing a Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway dominance in neuroinflammatory gene signatures. MurinecPIIRS models recapitulated this pathway predominance and treatment with the JAK inhibitor ruxolitinib, confirmed a mechanistic requirement for this pathway in disease pathology. Ruxolitinib treatment improved markers of neuronal damage, reduced activated T cell and myeloid cells, and improved weight. On the basis of these findings, we conducted a first-in-human ruxolitinib treatment of patients with cPIIRS (NCT00001352). Ruxolitinib treatment of six patients led to demonstrated tolerability, reductions in inflammatory biomarkers and activated immune cells, and improved brain imaging. These results advocate for pathway-instructed therapeutics in neuroinflammatory diseases and endorse JAK inhibitors in further clinical studies of cPIIRS.

Fig. 1.. Transcriptional pathway analysis identifies the JAK/STAT inflammatory pathway as predominant in the CSF of patients with cPIIRS.

(A to F) Transcriptional analysis of the CFS of patients with cPIIRS (at diagnosis) and non-cPIIRS donor CSF was performed using a NanoString multiplex neuroinflammation panel (n = 4 patients and 3 donors from one experiment). (A) PCA of normalized read counts of 757 total transcripts associated with neuroinflammation for the CSF of patients with cPIIRS and non-cPIIRS donor CSF. (B) Venn diagram indicating the number of DEGs (as described in Methods) in CSF known to be STAT1 (pink) or STAT3 (purple) dependent. Underlined are the total DEGs for each comparison. (C) Scatterplot of NanoString analysis comparing gene expression profiles of CSF of patients with cPIIRS to non-cPIIRS donor CSF. Highlighted are STAT1 (pink)/STAT3 (purple)–dependent and independent (light blue) DEGs. (D) Top 14 significantly enriched biological pathways identified in the CSF of patients with cPIIRS (MSigDB Hallmark 2020). (E and F) Pathway analysis using DEGs between the CSF of patients with cPIIRS and non-cPIIRS donor CSF was performed using Enrichr TRRUST Transcription Factors 2019 Database (E) and the Enrichr Transcription Factor PPI to construct networks (F). (G to I) CSF cells from a patient (at cPIIRS diagnosis, before treatment) were subjected to scRNA-seq. (G) UMAP plot of CSF cell scRNA-seq data showing cell-type clusters used for subsequent analysis (n = 5070). (H) Dotplot depicting selected marker genes in cell clusters. The dot size corresponds to the percentage of cells expressing the gene, and the color indicates the average per cell gene expression. (I) Feature plots of STAT1/STAT3–dependent genes, JAK/STAT genes, and cytokine/cognate cytokine receptor genes using colors to indicate gene expression (log₂UMI counts) levels in the UMAP embedding from (G). The number and percent of cells with detected expression for each gene are reported at the bottom of each feature plot.

Fig. 2.. Transcriptional pathway analysis identifies the JAK/STAT inflammatory pathway as predominant in a cPIIRS mouse model and disease improvement with ruxolitinib.

(A) Graphical schema for the mouse cPIIRS model and daily treatment with ruxolitinib. d, days. (B to G) Gene transcription of mouse brains at 21 dpi was analyzed using a NanoString Neuroinflammation panel (n = 4 mice per group). (B) PCA of mouse brain NanoString results. (C) Venn diagram showing overlapping DEGs in the mouse brain, including STAT1 (pink)–dependent, STAT3 (purple)–dependent, and total (underlined) DEGs. (D) Scatterplots comparing gene expression profiles in mouse brains. Highlighted are STAT1 (pink)– and STAT3 (purple)–dependent DEGs. (E) Top 14 significantly enriched biological pathways identified in the mouse brain (MSigDB Hallmark 2020). (F) Top upstream regulators of DEGs in mouse brains (IPA). Highlighted are JAK/STAT pathway–specific (pink) and consensus downstream JAK/STAT targets (blue). (G) Top IPA-predicted upstream regulators of DEGs in humans (patients with cPIIRS versus non-cPIIRS donors) and mice (Cn infection versus naïve). (H and I) Ruxolitinib concentrations in the mouse brain at 21 dpi (H) and serum (I) (n = 3 mice per group). (J to N) Immunoblot analysis of phospho-STAT1 (Tyr⁷⁰¹) [(J) and (K)], phospho-STAT3 (Tyr⁷⁰⁵) [(J) and (L)], total STAT1 [(J) and (M)], and total STAT3 [(J) and (N)] levels in mouse brains (n = 4 to 8 mice per group; two experiments). (O to T) Brain weight (21 dpi) (O), brain fungal burden (21 dpi) (P), mouse weights (throughout infection) (Q), spleen size (21 dpi) (R), spleen weight (21 dpi) (S), and splenocytes (21 dpi) (T) were enumerated. CFU, colony-forming units. (U) Graphical schema for the mouse cPIIRS model with AMB/ruxolitinib treatment. (V to X) Brain fungal burden (21 dpi) (V), mouse weights (W), and brain weight (21 dpi) (X) were enumerated. Error bars indicate SD from two independent experiments (n = 8 to 20 mice per time point). Student’s t test, with P values indicated above each comparison. Graphical schemas were created with BioRender.com.

Fig. 3.. JAK/STAT–mediated inflammatory neurotoxicity is mitigated by ruxolitinib in a mouse model of cPIIRS.

(A) IFM of brain sections stained with antibodies to β-III tubulin (green) was used to assess cryptococcal lesion size as indicated (yellow arrows). Scale bar, 100 μm. (B) Summary statistics of brain lesion sizes from Fig. 2A. (C) IFM of the brain sections stained with antibodies to β-III tubulin (green), Syt-7 (red), and 4′,6-diamidino-2-phenylindole (DAPI; blue). Cryptococcal lesion indicated (white arrow). Scale bars, 10 μm. (D) Summary statistics of the MFI quantitation of Syt-7 from Fig. 2C. (E) IFM of brain sections stained with β-III tubulin (green), cleaved caspase-3 (red), CD45 (pink), and DAPI (blue). Cryptococcal lesion indicated (white arrow). Scale bars, 10 μm. (F and G) Summary statistics of cleaved caspase-3 MFI (F) and CD45⁺ cells/lesion (G) from Fig. 2E. Data shown are representative of two independent experiments (n = 3 mice per group) +/− SEM, and at least 10 fields were examined for each sample. Statistical significance was determined using the linear regression models fit via GEE with P values indicated over each comparison.

Fig. 4.. Ruxolitinib reduces accumulation of activated T cells and inflammatory myeloid cell populations and suppresses neuroinflammatory gene expression during murine cPIIRS.

(A) At 21 dpi, predicted brain cell-type abundance was imputed from whole-brain mRNA obtained as in Fig. 2 (B to D) (n = 4 mice per group). (B to M) At 21 dpi, brains were harvested, brain-infiltrating lymphocytes (BIL) were isolated, and flow cytometry was performed to characterize live brain mononuclear cell populations. Representative flow plots with the proportion of indicated gated population (named above plot) and absolute number of brain cell population were enumerated for CD45^hi and CD45^int cells [(B) and (C)], CD3⁺ and CD4⁺ T cells [(D) to (F)], CD44^hiCD62L^lo effector CD4 T cells (G), Ly6C⁺ myeloid cells expressing MHC II [(H) to (J)], microglia expressing MHC II and Ly6C [(K) and (L)], and MHC II⁺/Ly6C⁺ activated microglia [(K) and (M)]. Flow cytometry data shown are the means ± SD from two independent experiments with 10 to 20 mice per time point, and statistical significance was determined by unpaired Student’s t test, with P values indicated above each comparison. (N) Top 14 significantly enriched biological pathways identified in the brains of Cn-infected mice compared to ruxolitinib-treated/Cn-infected mice (MSigDB Hallmark 2020). JAK/STAT–specific pathways highlighted in pink. (O) Scatterplots comparing gene expression profiles in mouse brains. Highlighted are the IFN-γ response (top panels; light blue), cell proliferation and DNA replication (middle panels; yellow), and cytokine signaling pathways (bottom panels; green) (n = 4 mice per group). (P) Heatmap of cytokine and chemokine levels in whole-brain homogenates at 21 dpi. Data from two independent experiments with n = 6 mice per treatment group and arranged in order of the highest to lowest value for Cn-infected/untreated mice. The pink asterisk indicates statistical significance (P < 0.05) between infected groups.

Fig. 5.. Ruxolitinib improves cellular and soluble markers of CSF inflammation and neurological findings in patients with cPIIRS.

Lumbar punctures and brain MRI imaging studies were performed before ruxolitinib treatment (Pre) and at 1-month postruxolitinib treatment initiation (Post) in six patients, maintaining a constant dose of corticosteroids throughout ruxolitinib treatment (n = 6 patients with 7 treatment courses). (A) Concentrations of ruxolitinib in patient CSF and plasma. The line indicates the median value for four patients. (B) WBC counts, (C) CSF CD45+ cell counts, (D) CSF protein, and (E) glucose/serum ratio were assessed as described in Methods. (F to P) CSF T cell activation (HLA-DR+ expression) on CD4 [(F) and (G)] and CD8 T cells [(H) and (I)], CD56+ NK cells [(J) and (K)], and CD14+ monocytes [(L) and (M)], including both mature [(N) and (O)] and innate monocytes [(N) and (P)], was measured by flow cytometry at the baseline (postpulse, before ruxolitinib treatment) and 1 month following ruxolitinib initiation. Representative and summary data are shown. (Q) Concentrations of indicated soluble cytokines were measured from the CSF as described in Methods. (R) Patient 1: Axial postcontrast fluid-attenuated inversion recovery (FLAIR) images of the brain reveal meningeal enhancement observed in multiple convexity sulci at the baseline. Postruxolitinib, meningeal enhancement has decreased (open white arrow). Postcontrast T1-weighted images demonstrating abnormal enhancement (solid red arrow) along the cerebellar folia bilaterally, which resolved 1 month postruxolitinib. (S) Patient 6: Axial postcontrast FLAIR and postcontrast T1-weighted images of the brain reveal meningeal enhancement in convexity sulci (red arrows) along with edema in the adjacent cortical regions (open white arrow) at the baseline. Postruxolitinib, the meningeal enhancement improved with the resolution of edematous changes in the adjacent parenchyma (open white arrow). (T) Aggregate MRI scores (n = 7). CSF parameters and MRI scores analyzed using the Wilcoxon-matched pair signed-rank test, with P values indicated above each comparison.