IL-1β signaling is important for limiting neuropathology within the CNS.

<p>Histological analysis from day 10 p.i. sagittally cut brain sections from paraffin embedded tissue. Representative formalin-fixed hematoxylin and eosin-stained sections of day 10 p.i. brains from WT (top panels) and <i>Il-1r−/−</i> (bottom panels) sections (<b>A</b>). Brain areas as indicated in figure. Original magnification 600×. In all regions of the WT brain, the sections are histologically unremarkable whereas lesions were readily apparent in the <i>Il1r</i>^−/− tissues. Brainstem: black arrows indicate perivascular regions; note the mildly increased cellularity in the <i>Il1r</i>^−/− vessel wall and immediate perivascular space. Forebrain: there is a mild focus of acute perivascular hemorrhage (arrow). Midbrain: There is a focus of mild inflammation associated with shrunken and eosinophilic neuron suggestive of neuronal degeneration (white arrow) which was in association with inflammatory infiltrates. Meninges: Black arrow indicates expansion of the meninges with moderate inflammatory infiltrates; compare to WT which is histologically unremarkable. Data are representative of two animals per genotype. (<b>B</b>) Representative day 10 p.i. Mac-2 immunohistochemical stained sections (brain regions are as indicated in the figure) in WT (top panel) or <i>Il-1r^−/−</i> (bottom panel) sections. Positive signal is as indicated by brown staining; original magnification for all panels, 200×. Mac-2 ⁺ cells are present perivascularly (black arrows) and in the adjacent parenchyma (Non-specific staining of the choroid is present (WT top panel hippocampus-thalamus) and <i>Il-1r^−/−</i> (data not shown). (<b>C</b>) Quantification of MAC-2 signal to tissue ratio.</p

Phenotyping IFN-γ secreting T cells in response to stimulation.

<p>The frequency of IFN-γ-producing Tim-3⁻ and Tim-3⁺ CD4⁺ T cells (A) and CD8⁺ T cells (B) collected 30 days post-index from 6 HLA-A02 WNV-infected donors are shown after stimulation in the presence or absence of anti-CD3/anti-CD28 monoclonal antibodies, WNV peptide pool, and SVG9 peptide; Ratios of IFN-γ⁺/IFN-γ⁻ cells within Tim-3⁻ and Tim-3⁺ are shown for CD4⁺ T cells (C) and CD8⁺ T cells (D). The histograms indicate the means and the error bars represent the SEM. **<i>p</i> <0.01, *<i>p</i> <0.05, and *** <i>p</i> <0.001 by <i>t</i>-test.</p

IL-1β signaling mediates CNS-intrinsic control of WNV.

<p>WT (closed circles) or <i>Il-1r^−/−</i> (open squares) animals were infected with 5 pfu WNV-TX via direct intracranial (i.c.) inoculation and were examined for viral load and cytokine responses (<b>A–C</b>). Viral load was assessed at day 2 or day 4 p.i. in brain (<b>A</b>) homogenates by plaque assay. IFN-β secretion was quantified from whole brain lysate from mock or infected at day 2 and day 4 p.i. by ELISA (<b>B</b>). Quantification of IL-1β expression from mock or i.c. infected brains of WT animals was assessed at day 2 and day 4 p.i. by Luminex array (<b>C</b>). Data are shown as Mean+/− S.E.M. and is displayed as n = 3–7 mice per time-point from two independent pooled experiments (<b>A</b>) or n = 3–4 mice (<b>B,C</b>) per time-point, representative or three independent experiments. *p<0.05, ** p<0.005, *** p<0.0005. Dashed line represents the lower limit of detection for each assay. Immunohistochemical analysis day 4 i.c. sagittal cut brain sections from paraffin embedded tissue. Representative WNV-Ag (brown staining; <b>D</b>) in regions as indicated in WT (top panels) or <i>Il-1r^−/−</i> brain sections (bottom panels). (<b>E</b>) Quantification of panel D.</p

WNV+ subject characteristics.

<p>N/A, not available.</p>a<p>Highest number of symptoms reported on either questionnaire.</p>b<p>Female (F) and male (M).</p>c<p>Antibody interpretation at index: positive (+), negative (−), or equivocal (E).</p>d<p>AS is for asymptomatic when peak symptom number ≤3 and S is for symptomatic when peak symptom number ≥4.</p

Gating strategy for measuring CD28 differentiation and CD57 senescence makers on T cells.

<p>The plots show (A) the gating strategy for live CD3⁺ lymphocytes, (B) for CD4⁺ (left) and CD8⁺ (right) T cells. Gates were set on FMO no CD28 (C) and FMO no CD57 (D). Plots are shown for representative (E) West Nile virus (WNV) uninfected controls (WNV−) and (F) WNV infected subjects (WNV+) day 14 post-index donation.</p

IL-1β signaling and the NRLP3 inflammasome are required for immunity against WNV.

<p>6–10 wk old age matched WT (closed circles; n = 26) or <i>Il-1r^−/−</i> (open squares; n = 14) (<b>A</b>) or <i>Caspase-1^−/−</i> (open circles; n = 12) and <i>Nlrp3^−/−</i> (closed square; n = 12) (<b>D</b>) or WT (closed circles, n = 5) and <i>Nlrc4^−/−</i> (open circles; n = 4) (<b>E</b>) or WT (closed circles, n = 5) and <i>Myd88^−/−</i> (closed square; n = 5) (<b>F</b>) animals were infected with 100 PFU WNV-TX via s.c. foot-pad inoculation and monitored for survival over the course of 14–16 days (<b>A, D–F</b>). Infected WT (closed circles) or <i>Il-1r^−/−</i> (open squares) animals from panel A, were monitored daily for weight loss (<b>B</b>) or scored for hind limb paralysis and morbidity (<b>C</b>) to day 16 post infection. *p<0.05, ** p<0.005, *** p<0.0005.</p

IL-1β signaling is critical for regulating CD8⁺ T cell effector activity within the CNS.

<p>Examination of T lymphocyte effector activity in the CNS by flow cytometry. Cell were isolated from the brains of mock or day 6–10 p.i. WT (closed circles) or <i>Il-1r^−/−</i>(open squares) and staining for CD3, CD8 and WNV-NS4b tetramer was performed. Quantitation of CD3⁺/CD8+ T lymphocytes (<b>A</b>), CD3⁺/CD8⁺/NS4b⁺ (<b>B</b>) as derived from total cell numbers or frequency of CD3⁺/CD8⁺/NS4b⁺ cells (<b>C</b>). Brains were harvested from day 8 or 10 p.i. and stimulated with 1 µM WNV-NS4b for 4 hrs (<b>D,E, H,I</b>) and assessed for cytokine expression by intracellular staining. Representative dot-plot analysis for IFN-γ and TNF-α expression at day 10 p.i. in the brain (<b>D</b>) or perforin and granzyme B (<b>H</b>). Data are shown as percent cytokine positive for WT (closed box) or <i>Il-1r^−/−</i> (open box) (<b>E,I</b>). Quantification of IFN-γ secretion by ELISA. Total brain cells (2.5e5) were stimulated with NS4b for 24 hrs and IFN-γ was quantified by ELISA for WT (closed circles) and <i>Il-1r^−/−</i> (open squares) cells (<b>F</b>). Data are shown as mean +/− S.E.M. for n = 3–4 mice per time-point (<b>A–E, H,I</b>) or n = 3–7 (<b>F</b>) and are representative of 2–3 independent experiments. *p<0.05, ** p<0.005.</p

Correlations between miRNA and HIV-1 viral load.

<p>Correlations between miRNA and HIV-1 viral load.</p

miR-422a is modulated by IFN-α treatment.

<p>(A) Expression of miR-422a in PBMC <i>in vivo</i> before, during and after IFN-α/RBV treatment (labeled as “Rx”). Error bars represent SEM. P-value was obtained using a paired t-test and FDR is reported. (B) Effects of IFN-α on the expression of miR-422a in CD4+ T cells <i>in vitro</i>; cells were plated at a million cells per well and treated with either 5 U/ml of IFN-α or media as a negative control. (C) Expression of MLH1 in PBMC <i>in vivo</i> before, during, and after IFN-α/RBV treatment. (D) Expression of TP53 in PBMC <i>in vivo</i> before, during, and after IFN-α/RBV treatment. Error bars represent SEM. P-values were obtained using paired Wilcoxon tests. (E) Correlation between TP53 fold induction and MLH1 fold induction in PBMC <i>in vivo</i> during IFN-α/RBV treatment. P-value was obtained using Spearman’s rank test.</p

Gating strategy for phenotyping IFN-γ secreting T cells in response to stimulation.

<p>The frequencies of Tim-3⁺ and Tim-3⁻ IFN-γ secreting CD4⁺ and CD8⁺ T cells were measured in PBMC collected at day 30 post-index from 6 HLA-A02 WNV-infected donors and incubated with or without anti-CD3/anti-CD28 mAbs, WNV peptide pool, and SVG9 tetramer. Tim-3⁻ and Tim-3⁺ CD4⁺ and CD8⁺ T cells were analyzed for IFN-γ secretion. The gates were set using fluorescence minus one controls. Dot-plots for CD8⁺ T cells from all 6 WNV+ subjects in different stimulation conditions are shown.</p