Functional roles of chemokines in response to viral infection of the CNS.

<p>Potential roles of chemokines in attracting innate immune cells (<b>A</b>) and lymphocytes (<b>B</b>) into the CNS during acute viral infection. The cartoons emphasize several key points derived from recent studies focusing on experimental infection with neurotropic viruses. (<b>A</b>) Early (days 1–3) after viral infection, activated and/or virally infected astrocytes, microglia, and endothelial cells secrete chemokines that serve to attract myeloid cells to the CNS. Among the earliest cells to respond to viral infection, neutrophils are recruited into the CNS by virtue of CXCR2 responding to ligands expressed within the CNS (e.g., CXCL1). Monocytes are also attracted into the CNS via the chemokine CCL5 and its receptor CCR5. Neutrophils and monocytes participate in the degradation of the blood–brain barrier (BBB), in part through the release of the matrix metalloproteinase MMP-9, and therefore ensure successive infiltration of virus-specific lymphocytes into the CNS. (<b>B</b>) During the acute stage of disease, astrocytes, microglia, neurons, and endothelial cells continue to secrete chemokines, serving to attract activated T lymphocytes, NK cells, and monocytes into the CNS. CD8+ and CD4+ T lymphocytes bearing the receptor CXCR3 and/or CCR5 are attracted by the chemokines CXCL10 and CCL5, respectively, and mediate viral control through direct cytolytic activity and/or cytokine secretion. CXCL12, which signals through CXCR4, may, however, sequester T lymphocytes within the perivascular space and regulate penetration of the parenchyma, thus inhibiting efficient viral clearance.</p

IFNαβR deletion prior to secondary viral infection does not limit LCMV-specific T cell attrition or the expression of MHC Class I & Qa-1 in vivo.

<p>(A) LCMV immune UBC-CreERT2⁺ and UBC-Cre-ERT2^-IFNαβR^fl/fl mice were tamoxifen injected and rechallenged with LCMV or given a sham injection. LCMV-specific (B) CD8⁺ and (C) CD4⁺ T cells were quantified via standard intracellular cytokine staining at the indicated times p.i. (D & E) Qa-1 and (F & G) MHC Class I expression was measured upon D^bGP_33-41⁺ (left column) and D^bNP_396-404⁺ (right column). Shown in panels D & F are representative histograms (gated on D^bGP_33-41⁺ CD8⁺ T cells). Data in B, C, E, & G are summations of 2 independent experiments (n = 4–7). Significance was determined via two-way ANOVA with Sidak correction (**** p<0.0001, *** p<0.001, ** p<0.01, * p<0.05).</p

Widespread and efficient deletion of IFNαβR in vivo following tamoxifen activation of Cre-ERT2.

<p>(A) LCMV immune UBC-Cre-ERT2⁺IFNαβR^fl/fl and UBC-Cre-ERT2^-IFNαβR^fl/fl mice were injected with tamoxifen, and the indicated analyses were carried out. (B) The efficiency of deletion at the DNA level was assessed by PCR analysis of genomic DNA extracted from the indicated tissues; M = markers; Splns = splenocytes; LN = lymph nodes; x = empty lane. 1144bp = floxed allele, 309bp = deleted allele. (C & D) The efficiency of Cre activation in T cells of LCMV-immune mice was determined. Cre⁺ or Cre^- IFNαβR^f/fzsGreen^+/wt mice were injected with tamoxifen and, two weeks later, splenocytes were harvested and incubated with each of the four indicated peptides, and virus-specific T cells were identified using standard intracellular cytokine staining for IFNγ. zsGreen expression also was evaluated. The red numbers indicate zsGreen⁺ cells as a percentage of all CD8⁺ T cells (No Peptide groups) or of virus-specific (i.e, IFNγ⁺) cells (peptide-stimulated groups). (C) Gated on CD8⁺ T cells and (D) gated on CD4⁺ T cells. (E) The efficiency with which Cre activation resulted in ablation of IFNαβR biological function was determined, by measuring Stat 1 phosphorylation after <i>in vitro</i> incubation with IFNβ. ZsGreen+CD8⁺ T cells from Cre⁺ mice were compared to CD8⁺ T cells from Cre^- animals. Grey histograms = isotype control antibody. Mouse numbers: C & D, n = 6–7; E, n = 4.</p

Reporter-negative (IFNαβR-intact) CD8+ or CD4+ memory T cells do not preferentially expand during the recall response.

<p>As described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005861#ppat.1005861.g003" target="_blank">Fig 3A</a>, LCMV-immune Cre reporter (zsGreen) mice were generated, treated with tamoxifen, and subjected to secondary LCMV infection. Mice were sacrificed, and virus-specific CD8⁺ and CD4⁺ T cells were enumerated using intracellular cytokine staining, and evaluated for zsGreen expression, days 0, 2, 5 & 23 following the secondary infection. Representative epitope-specific responses in individual mice are shown. Cells were harvested at day 23 following secondary infection, and are gated on (A) CD8⁺ T cells or (B) CD4⁺ T cells. Numbers represent the proportion of the gated cells in each quadrant. (C-F) Cumulative data showing the proportion of zsGreen-positive cells at the indicated time points over the course of the recall response, in each of the four epitope-specific T cell populations. (G) At the indicated time points, splenocytes from Cre⁺ mice were exposed in vitro to IFNβ, and levels of pSTAT expression were evaluated. Representative plots are shown, gated on CD8 and zsGreen. The rightmost panel is a positive control, showing the responsiveness to IFNβ of day 23 post-secondary CD8⁺ T cells from Cre^- animals. Grey histograms = isotype control antibody.</p

Functional type I and II interferons are expressed in response to secondary viral infection.

<p>LCMV-immune mice were rechallenged with 2x10⁶ PFU LCMV-Arm and (A) IFNα and (B) IFNγ levels in the plasma were assessed via ELISA or multiplex, respectively (n = 5 per time point). (C) Type I and type II ISG expression was quantified by PCR array in the spleens of LCMV-immune mice at 12 hours p.i., and the data were normalized to equivalent analyses of sham infected mice (sham n = 6, 12 hrs n = 4).</p

Tamoxifen-treated naïve Cre⁺IFNαβR^f/f mount primary responses that are similar to those reported for conventional IFNαβR knockout animals.

<p>(A) LCMV naive UBC-Cre-ERT2⁺IFNαβR^fl/fl and UBC-Cre-ERT2^-IFNαβR^fl/fl mice were injected with tamoxifen and ~9 weeks later were given a primary infection with LCMV. Seven days p.i., virus specific CD8⁺ (B & C) and CD4⁺ (D & E) T cells were identified and quantified via standard intracellular staining. For each cell type, representative contour plots from individual mice are shown (B & D), followed by cumulative data from all animals (C & E). (F) LCMV vRNA was quantified within the spleens of Cre⁺ and Cre^- mice (n = 2).</p

Secondary expansion, long term maintenance, and qualitative changes of memory CD8+ and CD4+ T cells are not reliant upon T1IFN signaling.

<p>LCMV-immune UBC-Cre-ERT2+ and UBC-Cre-ERT2-IFNαβR^fl/fl mice were injected with tamoxifen and later rechallenged with LCMV. (A-C) CD8⁺ and (D) CD4⁺ T cells specific for the indicated LCMV immunodominant epitope were identified and quantified within the spleen at the indicated times post infection, using standard intracellular cytokine staining. Data are a summation of five independent experiments; each dot represents an individual mouse (at each time point, n = 4–11 per group). (E & F) The ability of CD8⁺ (E) and CD4⁺ (F) memory T cells to produce IFNγ, TNF, and IL-2 was examined using a standard ICCS assay at days 2, 5, 14, and 23 following secondary challenge. The loss of multifunctionality in memory cells soon after secondary infection, and its gradual restoration, are shown by the relative proportion, at each time point, of cells that are capable of producing one, two or three cytokines (black, grey and white segments, respectively).</p

Reduced T1IFN-inducible gene expression does not limit viral control within the spleen or liver during secondary viral infection.

<p>(A) Type I and II ISG expression at 1 day post-secondary LCMV infection was assessed via PCR array, in LCMV-immune tamoxifen-treated Cre⁺ and Cre^- IFNαβR^f/f mice (Cre⁺ n = 5, Cre^- n = 6). Relative expression (Cre^- /Cre⁺) is shown for each ISG, together with statistical significance. (B & C) LCMV RNA was quantified by qPCR in the spleens (B) and livers (C) of Cre+ and Cre-IFNαβR^fl/fl mice at the indicated times following secondary challenge. Data are summations of two (A & C) and five (B) independent experiments. Each dot in panel B & C represents an individual mouse.</p