p38γ and p38δ modulate innate immune response by regulating MEF2D activation.

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 Mapk12D171A/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.

p38δ controls Mitogen- and Stress-activated Kinase-1 (MSK1) function in response to toll-like receptor activation in macrophages.

Mitogen- and Stress-activated Kinase (MSK) 1 is a nuclear protein, activated by p38α Mitogen-Activated Kinase (MAPK) and extracellular signal-regulated kinase (ERK1/2), that modulate the production of certain cytokines in macrophages. Using knockout cells and specific kinase inhibitors, we show that, besides p38α and ERK1/2, another p38MAPK, p38δ, mediates MSK phosphorylation and activation, in LPS-stimulated macrophages. Additionally, recombinant MSK1 was phosphorylated and activated by recombinant p38δ, to the same extent than by p38α, in in vitro experiments. Moreover, the phosphorylation of the transcription factors CREB and ATF1, that are MSK physiological substrates, and the expression of the CREB-dependent gene encoding DUSP1, were impaired in p38δ-deficient macrophages. Also, the transcription of IL-1Ra mRNA, that is MSK-dependent, was reduced. Our results indicate that MSK activation can be one possible mechanism by which p38δ regulates the production of a variety of inflammatory molecules involved in immune innate response.

TPL2 kinase expression is regulated by the p38γ/p38δ-dependent association of aconitase-1 with TPL2 mRNA.

p38γ and p38δ (p38γ/p38δ) regulate inflammation, in part by controlling tumor progression locus 2 (TPL2) expression in myeloid cells. Here, we demonstrate that TPL2 protein levels are dramatically reduced in p38γ/p38δ-deficient (p38γ/δ-/-) cells and tissues without affecting TPL2 messenger ribonucleic acid (mRNA) expression. We show that p38γ/p38δ posttranscriptionally regulates the TPL2 amount at two different levels. p38γ/p38δ interacts with the TPL2/A20 Binding Inhibitor of NF-κB2 (ABIN2)/Nuclear Factor κB1p105 (NF-κB1p105) complex, increasing TPL2 protein stability. Additionally, p38γ/p38δ regulates TPL2 mRNA translation by modulating the repressor function of TPL2 3' Untranslated region (UTR) mediated by its association with aconitase-1 (ACO1). ACO1 overexpression in wild-type cells increases the translational repression induced by TPL2 3'UTR and severely decreases TPL2 protein levels. p38δ binds to ACO1, and p38δ expression in p38γ/δ-/- cells fully restores TPL2 protein to wild-type levels by reducing the translational repression of TPL2 mRNA. This study reveals a unique mechanism of posttranscriptional regulation of TPL2 expression, which given its central role in innate immune response, likely has great relevance in physiopathology.

The 3F Library: Fluorinated Fsp3 -Rich Fragments for Expeditious 19 F†NMR Based Screening.

Fragment-based drug discovery (FBDD) is a popular method in academia and the pharmaceutical industry for the discovery of early lead candidates. Despite its wide-spread use, the approach still suffers from laborious screening workflows and a limited diversity in the fragments applied. Presented here is the design, synthesis, and biological evaluation of the first fragment library specifically tailored to tackle both these challenges. The 3F library of 115 fluorinated, Fsp3 -rich fragments is shape diverse and natural-product-like with desirable physicochemical properties. The library is perfectly suited for rapid and efficient screening by NMR spectroscopy in a two-stage workflow of 19 F NMR and subsequent 1 H NMR methods. Hits against four diverse protein targets are widely distributed among the fragment scaffolds in the 3F library and a 67 % validation rate was achieved using secondary assays. This collection is the first synthetic fragment library tailor-made for 19 F NMR screening and the results demonstrate that the approach should find broad application in the FBDD community.

Myeloid cell deficiency of p38γ/p38δ protects against candidiasis and regulates antifungal immunity.

Candida albicans is a frequent aetiologic agent of sepsis associated with high mortality in immunocompromised patients. Developing new antifungal therapies is a medical need due to the low efficiency and resistance to current antifungal drugs. Here, we show that p38γ and p38δ regulate the innate immune response to C. albicans We describe a new TAK1-TPL2-MKK1-ERK1/2 pathway in macrophages, which is activated by Dectin-1 engagement and positively regulated by p38γ/p38δ. In mice, p38γ/p38δ deficiency protects against C. albicans infection by increasing ROS and iNOS production and thus the antifungal capacity of neutrophils and macrophages, and by decreasing the hyper-inflammation that leads to severe host damage. Leucocyte recruitment to infected kidneys and production of inflammatory mediators are decreased in p38γ/δ-null mice, reducing septic shock. p38γ/p38δ in myeloid cells are critical for this effect. Moreover, pharmacological inhibition of p38γ/p38δ in mice reduces fungal burden, revealing that these p38MAPKs may be therapeutic targets for treating C. albicans infection in humans.