IL-1β synergizes with type I IFN to mediate direct antiviral actions and control of WNV infection in cortical neurons.

<p>Cortical neurons were prepared from d15 embryos of WT (closed circles) or <i>Il-1r^−/−</i> (open squares) animals and were assessed for viral load by plaque assay after infection with MOI (1) WNV-TX at 12, 24 and 48 hrs p.i. (<b>A</b>). Quantification of IL-1β (<b>B</b>) or IFN-β (<b>C</b>) from supernatants of mock or WNV MOI 1 infected WT and <i>Il-1r^−/−</i> neurons at 12, 24 and 48 hrs p.i. was assessed by Luminex array (<b>B</b>) or ELISA (<b>C</b>). IL-1β treatment reduces WNV load in cortical neurons. WT cortical neurons were either infected with MOI1 WNV alone (WT no treatment; closed circles) or with WNV MOI1+24 hr pre-treatment of 10 ng/ml recombinant IL-1β (WT 10 ng/ml IL-1β; open squares), (<b>D,E</b>) or with IFN-β (100 IU/ml) or both cytokines together (<b>E</b>) and viral load was assessed by plaque assay (<b>D,E</b>). Western blot analysis for WNV and interferon stimulated genes with IL-1β and IFN-β treatment (<b>F</b>). Data are shown as Mean+/−S.E.M. (<b>A–E</b>) for n = 3 per time-point and are representative of three independent experiments. * p<0.05, ** p<0.005, *** p<0.0005. Dashed line represents the lower limit of detection for each assay.</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

IL-1β Signaling Promotes CNS-Intrinsic Immune Control of West Nile Virus Infection

<div><p>West Nile virus (WNV) is an emerging <em>flavivirus</em> capable of infecting the central nervous system (CNS) and mediating neuronal cell death and tissue destruction. The processes that promote inflammation and encephalitis within the CNS are important for control of WNV disease but, how inflammatory signaling pathways operate to control CNS infection is not defined. Here, we identify IL-1β signaling and the NLRP3 inflammasome as key host restriction factors involved in viral control and CNS disease associated with WNV infection. Individuals presenting with acute WNV infection displayed elevated levels of IL-1β in their plasma over the course of infection, suggesting a role for IL-1β in WNV immunity. Indeed, we found that in a mouse model of infection, WNV induced the acute production of IL-1β <em>in vivo</em>, and that animals lacking the IL-1 receptor or components involved in inflammasome signaling complex exhibited increased susceptibility to WNV pathogenesis. This outcome associated with increased accumulation of virus within the CNS but not peripheral tissues and was further associated with altered kinetics and magnitude of inflammation, reduced quality of the effector CD8⁺ T cell response and reduced anti-viral activity within the CNS. Importantly, we found that WNV infection triggers production of IL-1β from cortical neurons. Furthermore, we found that IL-1β signaling synergizes with type I IFN to suppress WNV replication in neurons, thus implicating antiviral activity of IL-1β within neurons and control of virus replication within the CNS. Our studies thus define the NLRP3 inflammasome pathway and IL-1β signaling as key features controlling WNV infection and immunity in the CNS, and reveal a novel role for IL-1β in antiviral action that restricts virus replication in neurons.</p> </div

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

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

IL-1β signaling is critical for regulating inflammation in the CNS during WNV infection.

<p>Assessment of inflammatory responses in the CNS of WT (closed squares) or <i>Il-1r^−/−</i> (open squares) mice. Leukocyte infiltration into the CNS was assessed by flow cytometry at days 6–10 post infection (p.i.) with WNV-TX (<b>A–E</b>) as derived from total cell numbers. Cells recovered from perfused whole brain were stained with antibodies to CD45 and CD11b and total cell number (<b>B–E</b>) was assessed by flow cytometry. Representative dot-plot analysis for CD11b and CD45 infiltrates and microglia at mock, day 7 and day 10 p.i. is shown (<b>A</b>). Brains were harvested from tissues of WNV-TX02 infected mice at indicated time-points homogenized and assessed for cytokine and chemokine expression by luminex array (<b>F–I</b>). Data are shown as mean +/− S.E.M. for n = 3–4 per time-point and are compiled from 2–3 independent experiments. * p<0.05, **p<0.005.</p

Induction of broad, high frequency, polyfunctional monocyte responses to stimuli.

<p>HIV-infected subjects are shown in circles, HIV-uninfected are shown in triangles. Top Panel: Unstimulated (basal) state, Middle Panel: After oxLDL stimulation, Bottom Panel: After LPS stimulation. To determine the frequency of cells producing one or more cytokines, we performed a sum of the median responses. Mann Whitney U was used for comparison tests, and all comparisons were adjusted for multiple comparisons (p = 0.05/16 comparisons per stimulation condition = p = 0.003), and only p-values meeting or exceeding the p-value threshold are shown. **** p < 0.0001, *** p < 0.001, ** p < 0.003.</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

Global network of putative regulatory interactions between miRNAs and anti-HIV-1 restriction factor mRNAs.

<p>miRNA and mRNA expression data were measured in longitudinal PBMC samples from IFN-α/RBV-treated patients enrolled in the SHCS. Two variables were used to generate the network: 1) inverse expression relationships between a given miRNA-mRNA pair (represented as a dashed line drawn between a miRNA – mRNA pair), and 2) Significant sequence homology between a given miRNA seed region and a restriction factor 3′ UTR (word size 4, alignment length > = 5, e-value<1.0). Significant sequence homology is represented by a solid line. miRNAs are listed in blue; restriction factors are listed in pink.</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