p38γ and p38δ modulate innate immune response by regulating MEF2D activation

  1. Alejandra Escós
  2. Ester Diaz-Mora
  3. Michael Pattison
  4. Pilar Fajardo
  5. Diego González-Romero
  6. Ana Risco
  7. José Martín-Gómez
  8. Éric Bonneil
  9. Nahum Sonenberg
  10. Seyed Mehdi Jafarnejad
  11. Juan José Sanz-Ezquerro
  12. Steven C Ley
  13. Ana Cuenda

  • Curated by eLife

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    eLife assessment

    The authors have established a model for studying p38g/d signaling, which is an important accomplishment given that previous models have been compromised by changes in the TPL2/ERK pathway. Compelling evidence is presented to support the conclusions.

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Abstract

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.

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  1. Version published to 10.7554/elife.86200 on eLife
  2. eLife

    eLife assessment

    The authors have established a model for studying p38g/d signaling, which is an important accomplishment given that previous models have been compromised by changes in the TPL2/ERK pathway. Compelling evidence is presented to support the conclusions.

  3. eLife

    Reviewer #1 (Public Review):

    Studies of the p38g/d MAP kinase signaling pathways using loss-of-function approaches are compromised the finding that the expression of the ERK family MAP3K Tpl2 is down-regulated. Dissection of the specific roles of p38g/d is therefore difficult. Here the authors report that compound mutant mice with a kinase-inactive p38y MAPK mutation and p38d deficiency show no defects in Tpl2 expression. The importance of this study is therefore that they describe a mouse model that can be used to examine p38g/d MAP kinase function. The data presented are solid and convincing. The authors show that p38g/d MAP kinase signaling contributes to macrophage responses to endotoxin. Moreover, the authors identify Ser44 as an inhibitory site of MEF2D phosphorylation by p38d.

  4. eLife

    Reviewer #2 (Public Review):

    This paper addresses the specific function of p38γ/p38δ isoforms in inflammation. This was achieved by developing a novel mouse model in which p38γ was replaced by a kinase-inactive mutant (D171A mutation in a p38δ knock out background (p38γ/δKIKO). The results demonstrate that the p38γ/p38δ MAPKs are required for regulating the expression of inflammatory mediators implicated in the innate immune response. The phosphorylation of the transcription factor MEF2D at Ser444 constitutes one potential mechanism by which p38γ/p38δ suppresses iNOS and IL-1β mRNA expression.

    The strength of this paper resides in the novelty of the mouse model that permitted to assess the specific requirement of p38γ/p38δ isoforms independently of the loss of TPL2 expression caused by compound deletion of the p38γ/p38δ alleles. The finding that p38γ/p38δ MAPKs inhibit MEF2D activity by phosphorylation at Ser444 is also novel.

    One weakness lies in the lack of consistency between the expression profiles performed by RNA-seq/qPCR/cytokine arrays to identify inflammatory mediators whose expression is dependent on p38γ/δ in the two in vivo models of septic shock (i.e. fungal infection and induced by LPS) and in LPS activated macrophages in vitro.

    The other issue is that gene expression analyses are performed using bone marrow-derived macrophages (BMDM) (Figs. 3 and 5A), whereas the proteomic analysis employs peritoneal macrophages given that "p38γ and p38δ are expressed at much higher levels in these macrophages than in BMDM (p11)" (Fig. 4). Although the authors state on p11 "Additionally, the LPS-induced cytokine production in peritoneal macrophages was comparable to that of BMDM", only two cytokines were measured, i.e. IL1b and IFNg (SI Appendix Fig. S4B). This really emphasises the importance of verifying that i) MEF2D is indeed a substrate of p38δ in macrophages and ii) p38γ/δ-mediated phosphorylation of MEF2D at Ser444 negatively regulates the expression of iNOS and IL-1β transcripts in macrophages.

    Finally, no experiment was performed to demonstrate that the lower fungal burden or increased survival rate following LPS-induced sepsis in p38γ/δKIKO mice (Fig. 1) is a consequence of impaired production of inflammatory mediators by p38γ/δKIKO macrophages. This important issue should be addressed.

  5. eLife

    Reviewer #3 (Public Review):

    This paper investigates the role of the p38g and p38d kinases in the immune response using genetically modified mice that are deficient in p38d and express a kinase-inactive form of p38g. This model avoids the possible confounding effect of the downregulation of the ERK1/2 activator Tpl2, which is observed in mice that are deficient for both p38g and p38d, making it more straightforward to determine the contribution of p38g/p38d to specific phenotypes. The mice that express kinase-inactive p38g and lack p38d show reduced susceptibility to both C. albicans infection and LPS-induced septic shock. Macrophages derived from these mice show dysregulated expression of a number of genes involved in innate immunity. Phospho-proteomics analysis identifies the transcription factor MEF2D as one of the targets of p38g/p38d in macrophages, and in vitro assays show that p38d can phosphorylate several residues of MEF2D including Ser444. Reporter assays provide evidence that a MEF2D-S444A mutant has enhanced transcriptional activity compared with the WT MEF2D, and this is also supported by analyzing the mRNA levels of MEF2D targets in fibroblasts overexpressing both proteins. Taken together, these results support that S444A phosphorylation negatively regulates MEF2D activity.

    The manuscript contains a number of interesting observations supporting a role for p38g/p38d in the control of the innate immune response independently of the regulation of the Tpl2-ERK1/2 pathway. It also provides evidence that p38d but not p38g can phosphorylate MEF2D, which inhibits its transcriptional activity, and it is therefore a candidate target for some of the gene expression changes observed. Altogether, the manuscript adds new and exciting information on the functions performed by p38 MAPKs in macrophages and introduces a new mouse model that will be useful for further studies.

  6. Version published to 10.1101/2022.12.09.519777v1 on bioRxiv