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Key Points Mitogen-activated protein kinases (MAPKs) in innate immune cells are activated by a range of pattern recognition receptors, of which the best studied are Toll-like receptors (TLRs). TLR ligation induces the formation of a signalling complex that includes IL-1R-associated kinases (IRAKs) and TNFR-associated factor 6 (TRAF6), which is mediated by K63-linked polyubiquitylation. This complex interacts with and activates TGFβ-activated kinase 1 (TAK1), a MAPK kinase kinase (MAP3K) upstream of p38α and Jun N-terminal kinases (JNKs). TAK1 can also activate the IκB kinase (IKK) complex, leading to the activation of the transcription factor nuclear factor-κB and the MAP3K tumour progression locus 2 (TPL2), which is upstream of extracellular signal-regulated kinase 1 (ERK1) and ERK2. Recent genetic evidence, however, has shown that TAK1 is not required for TLR activation of MAPKs in primary macrophages, and the MAP3K involved remains to be identified. MAPK signalling has several of roles in innate immune responses, ranging from the induction of pro-inflammatory mediators, such as cytokines and chemokines, to the activation of anti-inflammatory feedback pathways. MAPKs can activate downstream kinases that have crucial roles in immunity; for example, p38α activates MAPK-activated protein kinase 2 (MK2), which promotes tumour necrosis factor (TNF) production. By contrast, the activation of mitogen- and stress-activated kinases (MSKs) by p38α or by ERK1 and ERK2 results in the increased transcription of the anti-inflammatory cytokines interleukin-10 (IL-10) and IL-1 receptor antagonist (IL-1RA). MAPK signalling induces the expression of dual specificity phosphatases (DUSPs). This establishes a negative feedback loop, in which the DUSPs dephosphorylate and inactivate MAPKs. Genetic studies have shown the crucial role of DUSPs in controlling innate immune responses. Bacterial pathogens have evolved ways to directly target MAPKs to downregulate the host immune response; for example, distinct bacterial proteins have been shown to inhibit MAPK signalling by inactivating MAPK kinase (MKK) enzymes and by activating DUSPs. Small-molecule inhibitors which target MAPK signalling have the potential to function as anti-inflammatory drugs. p38 inhibitors were the first MAPK inhibitors to be developed, but clinical results from using these compounds have been disappointing. As a result, focus in the pharmaceutical industry has shifted to targeting upstream MAP3Ks or downstream kinases, such as MK2. This Review summarizes our current understanding of the regulation and function of mitogen-activated protein kinases (MAPKs) in innate immunity, as well as the mechanisms by which pathogens manipulate MAPK activation and the potential of targeting MAPK pathways for the treatment of inflammatory diseases. Following pathogen infection or tissue damage, the stimulation of pattern recognition receptors on the cell surface and in the cytoplasm of innate immune cells activates members of each of the major mitogen-activated protein kinase (MAPK) subfamilies — the extracellular signal-regulated kinase (ERK), p38 and Jun N-terminal kinase (JNK) subfamilies. In conjunction with the activation of nuclear factor-κB and interferon-regulatory factor transcription factors, MAPK activation induces the expression of multiple genes that together regulate the inflammatory response. In this Review, we discuss our current knowledge about the regulation and the function of MAPKs in innate immunity, as well as the importance of negative feedback loops in limiting MAPK activity to prevent host tissue damage. We also examine how pathogens have evolved complex mechanisms to manipulate MAPK activation to increase their virulence. Finally, we consider the potential of the pharmacological targeting of MAPK pathways to treat autoimmune and inflammatory diseases.

Aberrant caspase recruitment domain family member 14 (CARD14) signaling has been strongly associated with inflammatory skin conditions. CARD14 acts as a scaffold protein, ultimately activating the transcription factor NF-KB. Although primarily studied in the context of inflammation, recent research has suggested its potential implications in tumorigenesis. In this study, we gathered The Cancer Genome Atlas (TCGA) tumor data to gauge the involvement of CARD14 in cancer, including genetic alterations, expression patterns, survival correlations, immune cell infiltration and functional interactions across diverse cancer types. We found heightened CARD14 expression in most tumors and there was a significant correlation between CARD14 expression and the prognosis of patients for certain tumors. For instance, patients with higher CARD14 expression had a better prognosis in sarcoma, lung, cervix and head and neck cancers. Moreover, CARD14 expression positively correlated with neutrophil infiltration in most of the cancer types analyzed. Finally, enrichment analysis showed that epithelial development and differentiation pathways were involved in the functional mechanism of CARD14. Our results show that CARD14 may have the potential to become a prognostic biomarker in several cancers, hence, further prospective studies will be required for its validation.

On the basis mainly of pharmacological experiments, the p38α MAP kinase isoform has been established as an important regulator of immune and inflammatory responses. However, the role of the related p38γ and p38δ kinases has remained unclear. Here, we show that deletion of p38γ and p38δ impaired the innate immune response to lipopolysaccharide (LPS), a Toll-like receptor 4 (TLR4) ligand, by blocking the extracellular signal-regulated kinase 1/2 (ERK1/2) activation in macrophages and dendritic cells. p38γ and p38δ were necessary to maintain steady-state levels of tumor progression locus 2 (TPL2), the MKK kinase that mediates ERK1/2 activation after TLR4 stimulation. TNFα, IL-1β, and IL-10 production were reduced in LPS-stimulated macrophages from p38γ/δ-null mice, whereas IL-12 and IFNβ production increased, in accordance with the known effects of TPL2/ERK1/2 signaling on the induction of these cytokines. Furthermore, p38γ/δ-deficient mice were less sensitive than controls to LPS-induced septic shock, showing lower TNFα and IL-1β levels after challenge. Together, our results establish p38γ and p38δ as key components in innate immune responses.

Interleukin (IL)-7 is critical for the maintenance of the peripheral T-cell compartment of the adaptive immune system. Our study identifies a role for the Nuclear Factor κ-B (NF-κB) signalling pathway in the control of IL-7 receptor expression by T cells. Following thymic selection, new T cells specifically up-regulate IL-7R even as they leave the thymus, and we reveal that this expression is strictly NF-κB dependent. NF-κB signaling was only required transiently, however, and once fully mature, naive T cells did not require NF-κB signaling to maintain IL-7R expression. Therefore, we reveal a developmental role for NF-κB signaling for the normal maturation and function of new T cells. Interleukin (IL)-7 is critical for the maintenance of the peripheral T-cell compartment of the adaptive immune system. IL-7 receptor α ( IL-7Rα) expression is subject to developmental regulation and new T cells induce expression as they leave the thymus, which is essential for their long-term survival. It is not understood how this expression is regulated. Here, we identify a role for the Nuclear Factor κ-B (NF-κB) signaling pathway in controlling expression of IL-7Rα in new T cells. Perturbations to NF-κB signaling, either by deletion of Inhibitor of Kappa-B Kinase-2 (IKK2) or by inhibiting Rel dimer activity, prevented normal IL-7Rα expression in new T cells. Defective IL-7Rα expression resulted in impaired survival and homeostatic cell division responses by T cells that could be attributed to their failure to express IL-7Rα normally. Surprisingly, NF-κB signaling was only required transiently in new T cells to allow their normal expression of IL-7Rα, because IKK2 deletion in mature T cells had no effect on IL-7Rα expression or their normal homeostatic responsiveness. Therefore, we identify a developmental function for NF-κB signaling in the homeostatic maturation of new T cells, by regulating IL-7Rα expression.

Evidence implicating p38γ and p38δ (p38γ/p38δ) in inflammation are mainly based on experiments using Mapk12/Mapk13 -deficient (p38γ/δKO) mice, which show low levels of TPL2, the kinase upstream of MKK1–ERK1/2 in myeloid cells. This could obscure p38γ/p38δ roles, since TPL2 is essential for regulating inflammation. Here, we generated a Mapk12 D171A/D171A / Mapk13 −/− (p38γ/δKIKO) mouse, expressing kinase-inactive p38γ and lacking p38δ. This mouse exhibited normal TPL2 levels, making it an excellent tool to elucidate specific p38γ/p38δ functions. p38γ/δKIKO mice showed a reduced inflammatory response and less susceptibility to lipopolysaccharide (LPS)-induced septic shock and Candida albicans infection than wild-type (WT) mice. Gene expression analyses in LPS-activated wild-type and p38γ/δKIKO macrophages revealed that p38γ/p38δ-regulated numerous genes implicated in innate immune response. Additionally, phospho-proteomic analyses and in vitro kinase assays showed that the transcription factor myocyte enhancer factor-2D (MEF2D) was phosphorylated at Ser444 via p38γ/p38δ. Mutation of MEF2D Ser444 to the non-phosphorylatable residue Ala increased its transcriptional activity and the expression of Nos2 and Il1b mRNA. These results suggest that p38γ/p38δ govern innate immune responses by regulating MEF2D phosphorylation and transcriptional activity.

The MEK-1/2 kinase TPL-2 is critical for Toll-like receptor activation of the ERK-1/2 MAP kinase pathway during inflammatory responses, but it can transform cells following C-terminal truncation. IκB kinase (IKK) complex phosphorylation of the TPL-2 C terminus regulates full-length TPL-2 activation of ERK-1/2 by a mechanism that has remained obscure. Here, we show that TPL-2 Ser-400 phosphorylation by IKK and TPL-2 Ser-443 autophosphorylation cooperated to trigger TPL-2 association with 14-3-3. Recruitment of 14-3-3 to the phosphorylated C terminus stimulated TPL-2 MEK-1 kinase activity, which was essential for TPL-2 activation of ERK-1/2. The binding of 14-3-3 to TPL-2 was also indispensible for lipopolysaccharide-induced production of tumor necrosis factor by macrophages, which is regulated by TPL-2 independently of ERK-1/2 activation. Our data identify a key step in the activation of TPL-2 signaling and provide a mechanistic insight into how C-terminal deletion triggers the oncogenic potential of TPL-2 by rendering its kinase activity independent of 14-3-3 binding.

Studies on hypoxia-sensitive pathways have revealed a series of Fe(ll)-dependent dioxygenases that regulate hypoxia-inducible factor (HIF) by prolyl and asparaginyl hydroxylation. The recognition of these unprecedented signaling processes has led to a search for other substrates of the HIF hydroxylases. Here we show that the human HIF asparaginyl hydroxylase, factor inhibiting HIF (FIH), also efficiently hydroxylates specific asparaginyl (Asn)-residues within proteins of the IκB family. After the identification of a series of ankyrin repeat domain (ARD)-containing proteins in a screen for proteins interacting with FIH, the ARDs of p105 (NFKB1) and IκBα were shown to be efficiently hydroxylated by FIH at specific Asn residues in the hairpin loops linking particular ankyrin repeats. The target Asn residue is highly conserved as part of the ankyrin consensus, and peptides derived from a diverse range of ARDcontaining proteins supported FIH enzyme activity. These findings demonstrate that this type of protein hydroxylation is not restricted to HIF and strongly suggest that FIH-dependent ARD hydroxylation is a common occurrence, potentially providing an oxygen-sensitive signal to a diverse range of processes.

TPL-2 (COT, MAP3K8) kinase activates the MEK1/2-ERK1/2 MAPK signaling pathway in innate immune responses following TLR, TNFR1 and IL-1R stimulation. TPL-2 contributes to type-1/Th17-mediated autoimmunity and control of intracellular pathogens. We recently demonstrated TPL-2 reduces severe airway allergy to house dust mite by negatively regulating type-2 responses. In the present study, we found that TPL-2 deficiency resulted in resistance to Heligmosomoides polygyrus infection, with accelerated worm expulsion, reduced fecal egg burden and reduced worm fitness. Using co-housing experiments, we found resistance to infection in TPL-2 deficient mice (Map3k8-/-) was independent of microbiota alterations in H. polygyrus infected WT and Map3k8-/-mice. Additionally, our data demonstrated immunity to H. polygyrus infection in TPL-2 deficient mice was not due to dysregulated type-2 immune responses. Genome-wide analysis of intestinal tissue from infected TPL-2-deficient mice identified elevated expression of genes involved in chemotaxis and homing of leukocytes and cells, including Ccl24 and alternatively activated genes. Indeed, Map3k8-/-mice had a significant influx of eosinophils, neutrophils, monocytes and Il4GFP+ T cells. Conditional knockout experiments demonstrated that specific deletion of TPL-2 in CD11c+ cells, but not Villin+ epithelial cells, LysM+ myeloid cells or CD4+ T cells, led to accelerated resistance to H. polygyrus. In line with a central role of CD11c+ cells, CD11c+ CD11b+ cells isolated from TPL-2-deficient mice had elevated Ccl24. Finally, Ccl24 neutralization in TPL-2 deficient mice significantly decreased the expression of Arg1, Retnla, Chil3 and Ear11 correlating with a loss of resistance to H. polygyrus. These observations suggest that TPL-2-regulated Ccl24 in CD11c+CD11b+ cells prevents accelerated type-2 mediated immunity to H. polygyrus. Collectively, this study identifies a previously unappreciated role for TPL-2 controlling immune responses to H. polygyrus infection by restricting Ccl24 production.

Persistent TH2 cytokine responses following chronic helminth infections can often lead to the development of tissue pathology and fibrotic scarring. Despite a good understanding of the cellular mechanisms involved in fibrogenesis, there are very few therapeutic options available, highlighting a significant medical need and gap in our understanding of the molecular mechanisms of TH2-mediated immunopathology. In this study, we found that the Map3 kinase, TPL-2 (Map3k8; Cot) regulated TH2-mediated intestinal, hepatic and pulmonary immunopathology following Schistosoma mansoni infection or S. mansoni egg injection. Elevated inflammation, TH2 cell responses and exacerbated fibrosis in Map3k8-/-mice was observed in mice with myeloid cell-specific (LysM) deletion of Map3k8, but not CD4 cell-specific deletion of Map3k8, indicating that TPL-2 regulated myeloid cell function to limit TH2-mediated immunopathology. Transcriptional and metabolic assays of Map3k8-/-M2 macrophages identified that TPL-2 was required for lipolysis, M2 macrophage activation and the expression of a variety of genes involved in immuno-regulatory and pro-fibrotic pathways. Taken together this study identified that TPL-2 regulated TH2-mediated inflammation by supporting lipolysis and M2 macrophage activation, preventing TH2 cell expansion and downstream immunopathology and fibrosis.

The IκB kinase (IKK) complex plays a well-documented role in innate and adaptive immunity. This function has been widely attributed to its role as the central activator of the NF-κB family of transcription factors. However, another important consequence of IKK activation is the regulation of TPL-2, a MEK kinase that is required for activation of ERK-1/2 MAP kinases in myeloid cells following Toll-like receptor and TNF receptor stimulation. In unstimulated cells, TPL-2 is stoichiometrically complexed with the NF-κB inhibitory protein NF-κB1 p105, which blocks TPL-2 access to its substrate MEK, and the ubiquitin-binding protein ABIN-2 (A20-binding inhibitor of NF-κB 2), both of which are required to maintain TPL-2 protein stability. Following agonist stimulation, the IKK complex phosphorylates p105, triggering its K48-linked ubiquitination and degradation by the proteasome. This releases TPL-2 from p105-mediated inhibition, facilitating activation of MEK, in addition to modulating NF-κB activation by liberating associated Rel subunits for translocation into the nucleus. IKK-induced proteolysis of p105, therefore, can directly regulate both NF-κB and ERK MAP kinase activation via NF-κB1 p105. TPL-2 is critical for production of the proinflammatory cytokine TNF during inflammatory responses. Consequently, there has been considerable interest in the pharmaceutical industry to develop selective TPL-2 inhibitors as drugs for the treatment of TNF-dependent inflammatory diseases, such as rheumatoid arthritis and inflammatory bowel disease. This review summarizes our current understanding of the regulation of TPL-2 signaling function, and also the complex positive and negative roles of TPL-2 in immune and inflammatory responses.