Tumor Progression Locus 2 Promotes Induction of IFNλ, Interferon Stimulated Genes and Antigen-Specific CD8⁺ T Cell Responses and Protects against Influenza Virus

<div><p>Mitogen-activated protein kinase (MAP) cascades are important in antiviral immunity through their regulation of interferon (IFN) production as well as virus replication. Although the serine-threonine MAP kinase tumor progression locus 2 (Tpl2/MAP3K8) has been implicated as a key regulator of Type I (IFNα/β) and Type II (IFNγ) IFNs, remarkably little is known about how Tpl2 might contribute to host defense against viruses. Herein, we investigated the role of Tpl2 in antiviral immune responses against influenza virus. We demonstrate that Tpl2 is an integral component of multiple virus sensing pathways, differentially regulating the induction of IFNα/β and IFNλ in a cell-type specific manner. Although Tpl2 is important in the regulation of both IFNα/β and IFNλ, only IFNλ required Tpl2 for its induction during influenza virus infection both <i>in vitro</i> and <i>in vivo</i>. Further studies revealed an unanticipated function for Tpl2 in transducing Type I IFN signals and promoting expression of interferon-stimulated genes (ISGs). Importantly, Tpl2 signaling in nonhematopoietic cells is necessary to limit early virus replication. In addition to early innate alterations, impaired expansion of virus-specific CD8⁺ T cells accompanied delayed viral clearance in <i>Tpl2^-/-</i> mice at late time points. Consistent with its critical role in facilitating both innate and adaptive antiviral responses, Tpl2 is required for restricting morbidity and mortality associated with influenza virus infection. Collectively, these findings establish an essential role for Tpl2 in antiviral host defense mechanisms.</p></div

Tpl2 ablation limits antigen-specific CD8⁺ T cell responses and enhances susceptibility to influenza infection.

<p><b>(A-B)</b> WT and <i>Tpl2</i>^<i>-/-</i> mice were infected with 10⁴ pfu of X31 virus, and the proportion of NP_366–374 tetramer positive CD8⁺ T cells in BAL were assessed. <b>(C)</b> BAL cells were stimulated with a cocktail of influenza immunodominant peptides for 24 hr, and secretion of IFNγ was measured by ELISA; n = 5. <b>(D-E)</b> WT and <i>Tpl2</i>^<i>-/-</i> mice were infected with 10⁴ pfu of X31 virus, body weights were recorded daily for 14 days, and mice exhibiting severe signs of disease, including more than 30% weight loss were euthanized. Data are representative of 3 independent experiments; n = 5. * indicates <i>p</i><0.05, ** indicates <i>p</i><0.01, *** indicates <i>p</i><0.005.</p

Tpl2 ablation enhances virus replication and inflammatory responses during influenza infection.

<p><b>(A)</b> WT and <i>Tpl2</i>^<i>-/-</i> mice were intranasally infected with 10⁴ pfu of X31 virus, and lung viral titers were enumerated by plaque assays; n = 8 (D3 and D5) or n = 5 (D7). WT and <i>Tpl2</i>^<i>-/-</i> mice were infected with 10⁴ pfu of X31 virus, and the cytokine levels <b>(B)</b> and number of cells recovered <b>(C)</b> in BALF were measured on D7 pi; n = 6 uninfected and 10 (WT) and 8 (<i>Tpl2</i>^<i>-/-</i>) infected. <b>(D-E)</b> Chimeric mice were intranasally infected with 10⁴ pfu of X31 virus, and lung viral titers were enumerated by plaque assays D3 pi. * indicates <i>p</i><0.05, ** indicates <i>p</i><0.01.</p

Tpl2 mediates IFN signaling and induction of ISGs.

<p><b>(A)</b> BMDMs from WT and <i>Tpl2</i>^<i>-/-</i> mice were stimulated with IFNα or IFNβ for 1 hr, and STAT1 and ERK phosphorylation were assessed by immunoblotting. Data are representative of 3 independent experiments. Average intensities of pSTAT1^Tyr701<b>(B)</b> and pSTAT1^Ser727<b>(C)</b> bands normalized to actin bands by densitometric analysis. Data collected on the same day are connected by lines. <b>(D)</b> WT and <i>Tpl2</i>^<i>-/-</i> mice were infected with 10⁶ pfu of X31 virus, and the expression of <i>Ifitm3</i>, <i>Isg15</i> and <i>Oasl2</i> in lung tissue D1 pi was measured by RT-PCR with normalization to actin mRNA and WT uninfected sample (n = 7). <b>(E)</b> WT, <i>Ifnar1</i>^<i>-/-</i> and <i>Ifnar1</i>^<i>-/-</i><i>Tpl2</i>^<i>-/-</i> mice were intranasally infected with 10⁴ pfu of X31 virus, and lung viral titers on D3 pi were enumerated by plaque assays; n = 4 WT, 5 <i>Ifnar1</i>^<i>-/-</i> and 9 <i>Ifnar1</i>^<i>-/-</i><i>Tpl2</i>^<i>-/-</i> mice. * indicates <i>p</i><0.05, ** indicates <i>p</i><0.01.</p

Tpl2 differentially regulates IFN production in response to model viral ligands in a cell type-specific manner.

<p>MEFs <b>(A)</b> or BMDMs <b>(B-C)</b> from WT and <i>Tpl2</i>^<i>-/-</i> mice were transfected with 5’ppp-RNA or stimulated R848 or LPS for 24 hr, and IFNβ levels were measured by ELISA. <b>(D)</b> Plasmacytoid DCs were stimulated with R848 for 24 hr, and IFNα, β and λ levels were measured. Data are representative of 3–4 independent experiments. Graphs show means±SD. * indicates <i>p</i><0.05, ** indicates <i>p</i><0.01.</p

Tpl2 is required for optimal IFNλ production during influenza virus infection <i>in vitro</i> and <i>in vivo</i>.

<p>WT and <i>Tpl2</i>^<i>-/-</i> mice were infected with 10⁶ pfu of X31 virus, and IFNα, β <b>(A)</b>, and λ <b>(B)</b> levels in D1 lung homogenates or D3 BALF were measured by ELISA or bead based assay. For IFNα, n = 4 uninfected and 7 infected mice per group; for IFNβ, n = 2 uninfected and 3 infected per group; for D1 IFNλ n = 5 (WT) and 6 (<i>Tpl2</i>^<i>-/-</i>) uninfected and 10 (WT) and 12 (<i>Tpl2</i>^<i>-/-</i>) infected; for D3 IFNλ n = 5 uninfected and 14 infected (WT) and 13 (<i>Tpl2</i>^<i>-/-</i>) infected. <b>(C)</b> pDCs from WT and <i>Tpl2</i>^<i>-/-</i> mice were infected with WSN virus at an MOI of 0.2 for 24 hr, and IFNα, β and λ levels were measured. Data are representative of 3–4 independent experiments. * indicates <i>p</i><0.05, ** indicates <i>p</i><0.01.</p

Model of Tpl2 regulation of antiviral immune responses.

<p>During influenza virus infection, the viral RNA is recognized by TLR7 (in pDCs) or RIG-I (in other cell types). Virus recognition by these receptors activates various downstream signaling cascades, including Tpl2-ERK signaling, which either positively or negatively regulates secretion of IFNα/β or IFNλ in a cell-type specific manner. Specifically, Tpl2 inhibits TLR- and RLR-induced IFNα/β production in macrophages, but promotes IFNα/β and IFNλ in TLR-stimulated pDCs. Tpl2 is also involved in transducing Type I IFN signals. Moreover, Tpl2 regulates induction of ISGs, which are important in limiting virus replication. In addition to early innate responses, Tpl2 promotes expansion of virus-specific CD8⁺ T cells that facilitate viral clearance from infected lungs. Therefore, by integrating both innate and adaptive antiviral responses, Tpl2 promotes host protection during influenza virus infection.</p

IFNλ production is IFNAR-independent in influenza virus-infected lungs.

<p><b>(A)</b> Flt3 ligand-derived DCs from WT and <i>Ifnar1</i>^<i>-/-</i> mice were infected with WSN virus for 24 hr, and IFNλ secretion was measured by ELISA. <b>(B)</b> Flt3 ligand-derived DCs from WT and <i>Tpl2</i>^<i>-/-</i> mice were treated with IFNβ for 24 hr, and IFNλ secretion was quantitated by ELISA. Data are representative of 2 <b>(A)</b> or 3 <b>(B)</b> independent experiments. Graphs show means±SD. <b>(C)</b> WT and <i>Tpl2</i>^<i>-/-</i> mice were infected with 10⁴ pfu of X31 virus, and IFNλ levels in lung homogenates were measured by ELISA on D3 pi; n = 5 WT and 5 <i>Tpl2</i>^<i>-/-</i> mice. WT, <i>Ifnar1</i>^<i>-/-</i> and <i>Ifnar1</i>^<i>-/-</i><i>Tpl2</i>^<i>-/-</i> mice were infected with 10⁴ pfu of X31 virus, and IFNλ <b>(D)</b> and IFNα <b>(E)</b> levels in lung homogenates were measured by ELISA on D3 pi; n = 4 WT, 5 <i>Ifnar1</i>^<i>-/-</i> and 9 <i>Ifnar1</i>^<i>-/-</i><i>Tpl2</i>^<i>-/-</i> mice. Graphs show means±SEM. * indicates <i>p</i><0.05, ** indicates <i>p</i><0.01, *** indicates <i>p</i><0.001.</p

Tpl2 promotes Th1, but not Th17, differentiation in a CD45RB T cell transfer model of colitis.

<p>Wild type or <i>Tpl2</i>^{<i>−/−</i>} naïve T cells (CD45RB^hiCD25^-CD4⁺) were injected i.p. into female Rag-deficient mice. (A) Weight loss curves as a percentage of original body weight. (B) Measure of serum cytokine levels in colitic mice. Significance was measured by one-tailed Student’s t-test. (C) Representative histologic images are shown along with scoring for total pathology in the colon. Pathology scores were evaluated using Mann-Whitney U test. (D) Proportions of IFNγ and IL-17A in the spleen and mesenteric lymph nodes as measured by intracellular staining and flow cytometry. Cells were gated on CD4⁺TCRβ⁺ cells. Significance was measured by one-tailed Student’s t-test. (E) Total cells and CD4⁺TCRβ⁺ cells in the spleen and mesenteric lymph nodes. (F) Total CD4⁺IFNγ⁺ and CD4⁺IL-17A⁺ cells in the spleen and mesenteric lymph nodes. N≥12. Pooled from 3 independent experiments. Error bars represent means ± sem. *p<0.05.</p

Tumor Progression Locus 2 Differentially Regulates IFNγ and IL-17 Production by Effector CD4⁺ T Cells in a T Cell Transfer Model of Colitis

<div><p>Autoimmune diseases are approaching epidemic levels, estimated to affect 5–8% of the population. A number of autoimmune diseases are believed to be driven by autoreactive T cells, specifically by T helper 1 (Th1) cells and T helper 17 (Th17) cells. One molecule gaining interest as a therapeutic target is the serine-threonine kinase, Tpl2, which promotes expression of proinflammatory mediators. We previously demonstrated that Tpl2 regulates Th1 differentiation, secretion of the inflammatory cytokine IFNγ, and host defense against the intracellular parasite <i>Toxoplasma gondii</i>. The goal of this study was to determine whether Tpl2 also regulates Th1 or Th17 differentiation <i>in vivo</i> in a model of colitis associated with mixed Th1/Th17 pathology. <i>In vitro, Tpl2^−/−</i> naïve CD4 T cells were significantly impaired in IL-17A secretion under traditional Th17 inducing conditions. Reduced IL-17A secretion correlated with increased expression of FoxP3, a transcription factor known to antagonize RORγt function. In a murine T cell transfer model of colitis, transfer of <i>Tpl2^−/−</i> T cells resulted in reduced proportions of CD4 T cells expressing IFNγ, but not IL-17A, compared to that induced by wild type T cells. Further studies revealed that IL-17A differentiation induced by IL-6 and IL-23, cytokines implicated in driving Th17 differentiation <i>in vivo</i>, was unaffected by Tpl2 deficiency. Collectively, these results implicate Tpl2 in TGF-β-induced FoxP3 expression. Additionally, they underscore the contribution of Tpl2 to Th1 immunopathology specifically, which suggests that Tpl2 inhibitors may selectively target Th1-based inflammation.</p></div