Tpl2 kinase regulates T cell interferon-γ production and host resistance to Toxoplasma gondii

Tpl2 (Tumor progression locus 2), also known as Cot/MAP3K8, is a hematopoietically expressed serine-threonine kinase. Tpl2 is known to have critical functions in innate immunity in regulating tumor necrosis factor–α, Toll-like receptor, and G protein–coupled receptor signaling; however, our understanding of its physiological role in T cells is limited. We investigated the potential roles of Tpl2 in T cells and found that it was induced by interleukin-12 in human and mouse T cells in a Stat4-dependent manner. Deficiency of Tpl2 was associated with impaired interferon (IFN)-γ production. Accordingly, Tpl2−/− mice had impaired host defense against Toxoplasma gondii with reduced parasite clearance and decreased IFN-γ production. Furthermore, reconstitution of Rag2−/− mice with Tpl2-deficient T cells followed by T. gondii infection recapitulated the IFN-γ defect seen in the Tpl2-deficient mice, confirming a T cell–intrinsic defect. CD4+ T cells isolated from Tpl2−/− mice showed poor induction of T-bet and failure to up-regulate Stat4 protein, which is associated with impaired TCR-dependent extracellular signal-regulated kinase activation. These data underscore the role of Tpl2 as a regulator of T helper cell lineage decisions and demonstrate that Tpl2 has an important functional role in the regulation of Th1 responses

Lymphoid tissue inducer–like cells are an innate source of IL-17 and IL-22

The interleukin (IL) 17 family of cytokines has emerged to be critical for host defense as well as the pathogenesis of autoimmune and autoinflammatory disorders, and serves to link adaptive and innate responses. Recent studies have identified a new subset of T cells that selectively produce IL-17 (Th17 cells; Bettelli, E., T. Korn, and V.K. Kuchroo. 2007. Curr. Opin. Immunol. 19:652–657; Kolls, J.K., and A. Linden. 2004. Immunity. 21:467–476), but the regulation of IL-17 production by innate immune cells is less well understood. We report that in vitro stimulation with IL-23 induced IL-17 production by recombination activating gene (Rag) 2−/− splenocytes but not Rag2−/− common γ chain−/− splenocytes. We found that a major source of IL-17 was CD4+CD3−NK1.1−CD11b−Gr1−CD11c−B220− cells, a phenotype that corresponds to lymphoid tissue inducer–like cells (LTi-like cells), which constitutively expressed the IL-23 receptor, aryl hydrocarbon receptor, and CCR6. In vivo challenge with the yeast cell wall product zymosan rapidly induced IL-17 production in these cells. Genetic deletion of signal transducer and activator of transcription 3 reduced but did not abrogate IL-17 production in LTi-like cells. Thus, it appears that splenic LTi-like cells are a rapid source of IL-17 and IL-22, which might contribute to dynamic organization of secondary lymphoid organ structure or host defense

De novoCIAS1 mutations, cytokine activation, and evidence for genetic heterogeneity in patients with neonatal-onset multisystem inflammatory disease (NOMID): A new member of the expanding family of pyrin-associated autoinflammatory diseases

Neonatal-onset multisystem inflammatory disease (NOMID; also known as chronic infantile neurologic, cutaneous, articular [CINCA] syndrome) is characterized by fever, chronic meningitis, uveitis, sensorineural hearing loss, urticarial skin rash, and a characteristic deforming arthropathy. We investigated whether patients with this disorder have mutations in CIAS1, the gene which causes Muckle-Wells syndrome and familial cold autoinflammatory syndrome, two dominantly inherited disorders with some similarities to NOMID/CINCA syndrome

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

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 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 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 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