8 research outputs found
IL-1Ξ² signaling is critical for regulating CD8<sup>+</sup> 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 6β10 p.i. WT (closed circles) or <i>Il-1r<sup>β/β</sup></i>(open squares) and staining for CD3, CD8 and WNV-NS4b tetramer was performed. Quantitation of CD3<sup>+</sup>/CD8+ T lymphocytes (<b>A</b>), CD3<sup>+</sup>/CD8<sup>+</sup>/NS4b<sup>+</sup> (<b>B</b>) as derived from total cell numbers or frequency of CD3<sup>+</sup>/CD8<sup>+</sup>/NS4b<sup>+</sup> cells (<b>C</b>). Brains were harvested from day 8 or 10 p.i. and stimulated with 1 Β΅M 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-Ξ³ and TNF-Ξ± 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<sup>β/β</sup></i> (open box) (<b>E,I</b>). Quantification of IFN-Ξ³ secretion by ELISA. Total brain cells (2.5e5) were stimulated with NS4b for 24 hrs and IFN-Ξ³ was quantified by ELISA for WT (closed circles) and <i>Il-1r<sup>β/β</sup></i> (open squares) cells (<b>F</b>). Data are shown as mean +/β S.E.M. for nβ=β3β4 mice per time-point (<b>AβE, H,I</b>) or nβ=β3β7 (<b>F</b>) and are representative of 2β3 independent experiments. *p<0.05, ** p<0.005.</p
IL-1Ξ² signaling and the NRLP3 inflammasome are required for immunity against WNV.
<p>6β10 wk old age matched WT (closed circles; nβ=β26) or <i>Il-1r<sup>β/β</sup></i> (open squares; nβ=β14) (<b>A</b>) or <i>Caspase-1<sup>β/β</sup></i> (open circles; nβ=β12) and <i>Nlrp3<sup>β/β</sup></i> (closed square; nβ=β12) (<b>D</b>) or WT (closed circles, nβ=β5) and <i>Nlrc4<sup>β/β</sup></i> (open circles; nβ=β4) (<b>E</b>) or WT (closed circles, nβ=β5) and <i>Myd88<sup>β/β</sup></i> (closed square; nβ=β5) (<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 14β16 days (<b>A, DβF</b>). Infected WT (closed circles) or <i>Il-1r<sup>β/β</sup></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-1Ξ² 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-1rβ/β</i> (bottom panels) sections (<b>A</b>). Brain areas as indicated in figure. Original magnification 600Γ. In all regions of the WT brain, the sections are histologically unremarkable whereas lesions were readily apparent in the <i>Il1r</i><sup>β/β</sup> tissues. Brainstem: black arrows indicate perivascular regions; note the mildly increased cellularity in the <i>Il1r</i><sup>β/β</sup> 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<sup>β/β</sup></i> (bottom panel) sections. Positive signal is as indicated by brown staining; original magnification for all panels, 200Γ. Mac-2 <sup>+</sup> 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<sup>β/β</sup></i> (data not shown). (<b>C</b>) Quantification of MAC-2 signal to tissue ratio.</p
IL-1Ξ² signaling mediates CNS-intrinsic control of WNV.
<p>WT (closed circles) or <i>Il-1r<sup>β/β</sup></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>AβC</b>). Viral load was assessed at day 2 or day 4 p.i. in brain (<b>A</b>) homogenates by plaque assay. IFN-Ξ² 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-1Ξ² 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+/β S.E.M. and is displayed as nβ=β3β7 mice per time-point from two independent pooled experiments (<b>A</b>) or nβ=β3β4 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<sup>β/β</sup></i> brain sections (bottom panels). (<b>E</b>) Quantification of panel D.</p
IL-1Ξ² is produced during acute West Nile Virus infection in humans.
<p>IL-1 in plasma of WNV infected (nβ=β43) and control subjects (nβ=β21). Plasma from blood donors testing positive for WNV RNA was collected at time-points from 7 to 180 days post-Index and was compared to control (cntrl) samples. Samples were then assessed for IL-1Ξ² (<b>A</b>) or IL-1ra (<b>B</b>). IL-1Ξ² correlates inversely with WNV load. The level of WNV RNA was measured by real-time RT-PCR for days 7, 14, 21, 30, and 42 post-index (<b>C, left axis</b>). Levels of IL-1Ξ² at days 7, 21, and 42 post-index are displayed (<b>C, right axis</b>). Middle bars represent the median for each group. * p<0.05, ** p<0.005, *** p<0.0005 values are reflective of significance compared to control samples. ns refers to not-significant. P value-inset (p<0.0001 (<b>A</b>), pβ=β0.003 (<b>B</b>)) is reflective of trend analysis comparing the trend in WNV<sup>+</sup> blood donors over time post-infection. Dashed lines represent the minimum detectable concentration of the assay.</p
IL-1Ξ² signaling is required for CNS-specific protective immunity against WNV.
<p>Examination of <i>in vivo</i> IL-1 cytokine expression and viral load in WT and <i>Il-1r<sup>β/β</sup></i> animals. 6β10 wk old WT mice were infected s.c. with 100 PFU WNV-TX02 or mock infected. Viral loads were assessed from WT (closed circles) or <i>Il-1r<sup>β/β</sup></i> mice (open squares) by plaque assay from spleen (<b>B</b>) and brain (<b>E</b>) lysates or by Taqman based qRT-PCR using specific primers and probes to WNV for DLN (<b>A</b>) and Serum (<b>C</b>). Cytokine expression for IL-1Ξ± and IL-1Ξ² was assessed by Luminex array for Brain lysates (<b>D</b>) or IFN-Ξ² levels by ELISA (<b>F</b>) Data are shown as the mean +/β S.E.M. for nβ=β3β6 mice per time-point. *p<0.05, *** p<0.0005. Dashed lines represent the lower limit of detection for each assay. BLD denotes below limit of detection.</p
IL-1Ξ² 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<sup>β/β</sup></i> (open squares) mice. Leukocyte infiltration into the CNS was assessed by flow cytometry at days 6β10 post infection (p.i.) with WNV-TX (<b>AβE</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>BβE</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>FβI</b>). Data are shown as mean +/β S.E.M. for nβ=β3β4 per time-point and are compiled from 2β3 independent experiments. * p<0.05, **p<0.005.</p