The Roles of Two IκB Kinase-related Kinases in Lipopolysaccharide and Double Stranded RNA Signaling and Viral Infection

Viral infection and stimulation with lipopolysaccharide (LPS) or double stranded RNA (dsRNA) induce phosphorylation of interferon (IFN) regulatory factor (IRF)-3 and its translocation to the nucleus, thereby leading to the IFN-β gene induction. Recently, two IκB kinase (IKK)–related kinases, inducible IκB kinase (IKK-i) and TANK-binding kinase 1 (TBK1), were suggested to act as IRF-3 kinases and be involved in IFN-β production in Toll-like receptor (TLR) signaling and viral infection. In this work, we investigated the physiological roles of these kinases by gene targeting. TBK1-deficient embryonic fibroblasts (EFs) showed dramatic decrease in induction of IFN-β and IFN-inducible genes in response to LPS or dsRNA as well as after viral infection. However, dsRNA-induced expression of these genes was residually detected in TBK1-deficient cells and intact in IKK-i–deficient cells, but completely abolished in IKK-i/TBK1 doubly deficient cells. IRF-3 activation, in response not only to dsRNA but also to viral infection, was impaired in TBK1-deficient cells. Together, these results demonstrate that TBK1 as well as, albeit to a lesser extent, IKK-i play a crucial role in the induction of IFN-β and IFN-inducible genes in both TLR-stimulated and virus-infected EFs

Conditional Deletion of TAK1 in T Cells Reveals a Pivotal Role of TCRαβ⁺ Intraepithelial Lymphocytes in Preventing Lymphopenia-Associated Colitis

<div><p>The kinase TAK is required for the development of conventional and regulatory T cells. We previously reported that mice with conditional deletion of TAK1 in T cells (Lck-cre:TAK1^fl/fl mice) exhibited severe T lymphopenia, and were nevertheless predisposed to spontaneous colitis with unknown etiology. Here we focused on the immunopathological mechanism in colitic Lck-cre:TAK1^fl/fl mice. We found that ‘leaky’ CD4⁺ T cells retaining TAK1 acquired inflammatory phenotypes that contribute to disease onset in Lck-cre:TAK1^fl/fl mice. Furthermore, the gut microbiota-triggered signaling was also a key event leading to the pathogenesis. We discovered that Lck-cre:TAK1^fl/fl mice were almost completely devoid of TCRαβ⁺CD8α⁺ intestinal intraepithelial lymphocytes (IELs) and this was largely due to the developmental defect of the thymic precursors by TAK1 deficiency. Remarkably, transfer of TCRαβ⁺CD8α⁺ IELs from wild-type mice ameliorated colitis in Lck-cre:TAK1^fl/fl mice. Taken together, our current study highlighted the emerging role of TAK1 in configuring the gut-specialized T cell subset, which regulates mucosal homeostasis under lymphopenic conditions.</p></div

Administration of TCRαβ⁺CD8α⁺ IELs ameliorates spontaneous colitis in LTAC mice.

<p>Individual T cell subsets sorted from the spleen or the small and large intestine of CD45.1^<b>+</b> C57BL/6 mice were intravenously transferred into 6- to 8-week-old LTAC mice. Eight weeks after transfer, the mice were used for analysis. (A) HE staining with proximal colons. Data are representative of 5 to 6 individual mice. Scale bar, 200 μm. (B) Histological score determined based upon criteria for colitis is depicted in each mouse group. No transfer (–) (n = 5, filled bar), CD8^<b>+</b> T (n = 6, open bar), TCRαβ^<b>+</b>CD8α^<b>+</b> IEL (n = 6, hatched bar), NKT cells (n = 6, vertical striped bar). Data are shown as mean ± standard error of the mean (s.e.m.). In (B), one-way analysis of variance (ANOVA) Bonferroni’s multiple comparison test was performed. Statistical significance is indicated by ***<i>P</i> < 0.001; ns, not significant.</p

Regulatory T cell population in LTAC mice.

<p>(A) Flow cytometry analysis with thymocytes from 6-week-old (w.o.) or 11 w.o. WT and LTAC mice. Staining for CD25 and Foxp3 determined Treg in CD4 single positive cells. The plots are representative of three independent experiments. (B) Foxp3 expression in TCRβ^<b>+</b>CD4^<b>+</b> cells in tissues indicated. The histograms are representative of at least five independent experiments. (C and D) Frequencies (left) and absolute numbers (right) of Foxp3^<b>+</b> cells of TCRβ^<b>+</b>CD4^<b>+</b> cells from the mLNs (C) and cLP (D) of WT (n = 5–8, filled circle) and LTAC mice (n = 5–8, open circle), determined by flow cytometry analysis from (B). Horizontal bars represent mean. (E) Genomic DNAs from the sorted cells indicated, followed by PCR amplification to detect flox or Δ allele in TAK1 genomic locus like <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128761#pone.0128761.g002" target="_blank">Fig 2C</a>. (F) CFSE-labeled CD45.1^<b>+</b>CD4^<b>+</b>CD25^<b>–</b> T cells (Tresp) were cultured with soluble anti-CD3 antibody and T cell-depleted splenocytes in the absence (Tresp alone) or presence of CD4^<b>+</b>CD25^<b>+</b> regulatory T cells (Treg) purified from wild-type or LTAC mice for 3 days (The ratio of Tresp to Treg is 2:1.). The histograms are representative of three independent experiments. In (C) and (D), unpaired <i>t</i> tests were performed. Statistical significance was indicated by **<i>P</i> < 0.01, ***<i>P</i> < 0.001; ns, not significant.</p

TAK1-dependent TCR-driven signaling pathway is essential for the development of TCRαβ⁺ IELs.

<p>(A) Flow cytometry of TCRβ^<b>+</b>CD4^<b>–</b>CD8^<b>–</b>NK1.1^<b>–</b> precursor cells in thymus. The plots are representative of at least three independent experiments. (B) Frequency (left) and absolute number (right) of TCRβ^<b>+</b> after gating out of CD4^<b>+</b>, CD8^<b>+</b> and NK1.1^<b>+</b> cells of WT (n = 8, filled circle) and LTAC mice (n = 9, open circle), determined by flow cytometry analysis from (A). Horizontal bars represent mean. (C) Characterization of cell surface proteins on TCRβ^<b>+</b> thymic precursor cells after depletion of CD4^<b>+</b>, CD8^<b>+</b> and NK1.1^<b>+</b> cells of WT and LTAC mice. The plots are representative of at least three independent experiments. (D, E and F) Thymocytes after depletion of CD4^<b>+</b>, CD8^<b>+</b> and NK1.1^<b>+</b> cells were cultured with the plate-bound anti-CD3 antibody (αCD3) in the absence or presence of IL-2 for 3 days. In (D), forward scatter (FSC) for monitoring cell size and inducible expression of CD25 are depicted as histograms. In (E), inducible expression of CD8α on TCRβ^<b>+</b> precursor cells is shown. The plots are representative of at least three independent experiments. In (F), frequency of TCRβ^<b>+</b>CD8α^<b>+</b> cells in cultured cells from WT (n = 3, filled bar) and LTAC mice (n = 3, open bar) are shown. Data are shown as mean ± s.e.m.. In (B) and (F), unpaired <i>t</i> tests were performed. Statistical significance was indicated by **<i>P</i> < 0.01, ***<i>P</i> < 0.001, ****<i>P</i> < 0.0001.</p

Gut microbiota and MyD88-mediated signaling promote the onset of colitis in LTAC mice.

<p>(A) Fasted mice were rectally administered with FITC-labeled dextran (2 mg / 10 g body weight) and after 3 hours blood was collected. Concentration of the FITC-dextran in the sera from WT (n = 5, filled bar) and LTAC mice (n = 7, open bar) are depicted. Data are shown as mean ± s.e.m.. (B) Bacterial titre in liver from WT (n = 5, filled bar) and LTAC mice (n = 5, open bar). Data are shown as mean ± s.e.m.. CFU, colony forming units. nd, not detected. (C and D) Six- to eight-week-old LTAC mice were subjected to intake of drinking water containing antibiotics cocktail (Abx) for 8 weeks. (C) Picture of the cecum and colon after Abx-treated (+) or left untreated (-) LTAC mice. (D) HE staining with proximal colons harvested from Abx-treated (+) or left untreated (-) LTAC mice. Data are representative of 4 to 6 individual mice. Scale bar, 200 μm. (E) HE staining with proximal colon harvested from each genotype of 12- to 16-week-old mice. Data are representative of 4 to 6 individual mice. Scale bar, 200 μm. (F) Histological score determined based upon criteria for colitis is depicted in each mice. WT (n = 4, filled bar), LTAC (equal to Abx(–)) (n = 6, open bar), LTAC Abx (+) (n = 6, hatched bar), LTAC x MyD88^{<b>–/–</b>}(n = 4, vertical striped bar). Data are shown as mean ± s.e.m.. In (A), Mann-Whitney test was performed. In (B), unpaired <i>t</i> test was performed. In (F), one-way ANOVA Bonferroni’s multiple comparison test was performed. Statistical significance is indicated by *<i>P</i> < 0.05, ****<i>P</i> < 0.0001.</p

Extracellular Signal-Regulated Protein Kinase 2 Is Required for Efficient Generation of B Cells Bearing Antigen-Specific Immunoglobulin G

Activation of extracellular signal-regulated protein kinase (ERK) has been implicated in proliferation as well as differentiation in a wide variety of cell types. Using B-cell-specific gene-targeted mice, we report here that in T-cell-dependent immune responses, ERK2 is required to generate efficient immunoglobulin G (IgG) production. In its absence, the proportion of antigen-specific surface IgG1-bearing cells and the subsequent number of IgG1 antibody-secreting cells were decreased, despite apparently unimpaired class switch recombination. Notably, this defect was countered by overexpression of the antiapoptotic factor Bcl-2. Together, our results suggest that ERK2 plays a key role in efficient generation of antigen-specific IgG-bearing B cells by promoting their survival