The Predictive Value of MAP2K1/2 Mutations on Efficiency of Immunotherapy in Melanoma.

Background: MAP2K1/2 genes are mutated in approximately 8% of melanoma patients; however, the impact of MAP2K1/2 gene alterations on the efficiency of immunotherapy has not been clarified. This study focused on the correlation between MAP2K1/2 gene mutations and the treatment response. Methods: Six metastatic melanoma clinical cohorts treated with immune checkpoint inhibitors [anti-cytotoxic T lymphocyte antigen-4 (CTLA-4) or anti-programmed cell death-1 (PD-1)] were recruited in this study. RNA expression profiling results from each of these six cohorts and the Cancer Genome Atlas (TCGA) melanoma cohort were analysed to explore the mechanism related to immune activation. Results: Compared to patients with wild-type MAP2K1/2, those with MAP2K1/2 mutations in an independent anti-CTLA-4-treated cohort had higher objective response rates, longer progression-free survival, and longer overall survival (OS). These findings were further validated in a pooled anti-CTLA-4-treated cohort in terms of the OS. However, there was no correlation between MAP2K1/2 mutations and OS in the anti-PD-1-treated cohort. Subgroup Cox regression analysis suggested that patients with MAP2K1/2 mutations received fewer benefits from anti-PD-1 monotherapy than from anti-CTLA-4 treatment. Furthermore, transcriptome profiling analysis revealed that melanoma tumours with MAP2K mutation was enriched in CD8+ T cells, B cells, and neutrophil cells, also expressed high levels of CD33 and IL10, implying a potential mechanism underlying the benefit of melanoma patients with MAP2K1/2 mutations from anti-CTLA-4 treatment. Conclusions: MAP2K1/2 mutations were identified as an independent predictive factor for anti-CTLA-4 therapy in melanoma patients. Anti-CTLA-4 treatment might be more effective than anti-PD-1 therapy for patients with MAP2K1/2-mutated melanoma.

MEK2 Negatively Regulates Lipopolysaccharide-Mediated IL-1ß Production through HIF-1α Expression.

LPS-activated macrophages require metabolic reprogramming and glucose uptake mediated by hypoxia-inducible factor (HIF)-1 α and glucose transporter 1 (Glut1) expression for proinflammatory cytokine production, especially IL-1ß. This process is tightly regulated through activation of MAPK kinases, including the MEK/ERK pathway as well as several transcription factors including HIF-1α. Although MAPK kinase (MEK) 2 deficiency had no significant effect on NO, TNF-α, or IL-12 production in response to LPS challenge, MEK2-deficient murine bone marrow-derived macrophages (BMDMs) exhibited lower IL-10 production. Importantly, MEK2-deficient BMDMs exhibited a preserved ERK1/2 phosphorylation, higher HIF-1α and Glut1 levels, and substantially increased IL-1ß as well as IL-6 production in response to LPS stimulation. Knockdown of HIF-1α expression via short interference RNA decreased the level of HIF-1α expression in MEK2-deficient BMDMs and decreased IL-1ß production in response to LPS treatment. Furthermore, we performed gain of function experiments by overexpressing MEK2 protein in RAW264.7 cells. LPS stimulation of MEK2 overexpressed in RAW264.7 cells led to a marked decreased IL-1ß production. Finally, we investigated the role of Mek1 and Mek2 double and triple mutation on ERK phosphorylation, HIF-1α expression, and IL-1ß production. We found that MEK2 is the major kinase, which inversely proportionally regulates HIF-1α and IL-1ß expression independent of ERK activation. Our findings demonstrate a novel regulatory function for MEK2 in response to TLR4 activation in IL-1ß production through modulating HIF-1α expression.

MEK1/2 Inhibition Promotes Macrophage Reparative Properties.

Macrophages have important functional roles in regulating the timely promotion and resolution of inflammation. Although many of the intracellular signaling pathways involved in the proinflammatory responses of macrophages are well characterized, the components that regulate macrophage reparative properties are less well understood. We identified the MEK1/2 pathway as a key regulator of macrophage reparative properties. Pharmacological inhibition of the MEK1/2 pathway by a MEK1/2 inhibitor (MEKi) significantly increased expression of IL-4/IL-13 (M2)-responsive genes in murine bone marrow-derived and alveolar macrophages. Deletion of the MEK1 gene using LysMCre+/+Mek1fl/fl macrophages as an alternate approach yielded similar results. MEKi enhanced STAT6 phosphorylation, and MEKi-induced changes in M2 polarization were dependent on STAT6. In addition, MEKi treatment significantly increased murine and human macrophage efferocytosis of apoptotic cells, independent of macrophage polarization and STAT6. These phenotypes were associated with increased gene and protein expression of Mertk, Tyro3, and Abca1, three proteins that promote macrophage efferocytosis. We also studied the effects of MEKi on in vivo macrophage efferocytosis and polarization. MEKi-treated mice had increased efferocytosis of apoptotic polymorphonuclear leukocytes instilled into the peritoneum. Furthermore, administration of MEKi after LPS-induced lung injury led to improved recovery of weight, fewer neutrophils in the alveolar compartment, and greater macrophage M2 polarization. Collectively, these results show that MEK1/2 inhibition is capable of promoting the reparative properties of murine and human macrophages. These studies suggest that the MEK1/2 pathway may be a therapeutic target to promote the resolution of inflammation via modulation of macrophage functions.

Rhinovirus-induced MMP-9 expression is dependent on Fra-1, which is modulated by formoterol and dexamethasone.

Matrix metalloproteinase-9 is implicated in airway inflammation and airway remodeling in asthma. We have previously confirmed that human rhinovirus-16 (HRV-16) infection increases MMP-9 expression both in vivo and in vitro. However, the role of the AP-1 sites within the MMP-9 promoter and the effect of commonly used asthma pharmacotherapies in modulating human rhinovirus (HRV)-induced MMP-9 production have not yet been elucidated. Experiments were performed in vitro in the human bronchial epithelial (HBE) cell line BEAS-2B and in primary HBE cells obtained from non-transplanted lungs. Using site-directed mutagenesis approaches, AP-1 sites were found to be necessary for HRV-induced MMP-9 promoter drive. EMSAs and supershift assays identified complexes consisting of Fos-related Ag-1 (Fra-1) in addition to other AP-1 subunits. Small interfering RNA approaches indicated that Fra-1 was induced upon HRV-16 infection in BEAS-2B cells and was necessary for MMP-9 expression in both BEAS-2B and primary HBE cells. Inhibition of MEK1/2 activity using PD98059 and U0126 reduced Fra-1 expression, DNA binding, MMP-9 promoter drive, and MMP-9 protein production. The long-acting ß(2)-agonist formoterol and the glucocorticoid dexamethasone significantly reduced HRV-induced ERK phosphorylation, Fra-1, and MMP-9 expression in BEAS-2B cells. These data indicate that HRV-induced activation of the MEK/ERK MAPK pathway and Fra-1 expression are necessary for the upregulation of MMP-9 and can be modulated by two distinct but commonly used asthma pharmacotherapies. Together, these results offer insights into the mechanisms by which long-acting ß(2)-agonists and glucocorticoids might reduce HRV-related asthma exacerbations.

Cell intrinsic roles of apoptosis-associated speck-like protein in regulating innate and adaptive immune responses.

The role of apoptosis-associated speck-Like protein (ASC) in the assembly of the inflammasome complex within macrophages has been elucidated in several studies. In this particular role, ASC functions as an adaptor protein by linking nod-like receptors (NLRs) and procaspase-1, thereby leading to the activation of caspase-1 to cleave inflammatory cytokines IL-1ß and IL-18 and inducing pyroptosis. It has been noted that ASC maintains inflammasome-independent roles, including but not limited to controlling the expression of Dock2 and mitogen-activated protein kinases (MAPK/ERK2) and regulating the NF-κB pathway. This paper will emphasize the major roles of ASC during pathogen infection, the mechanisms by which it mediates inflammation, and discuss its more recently discovered functions.

Pathophysiological role of Toll-like receptor 5 engagement by bacterial flagellin in colonic inflammation.

Commensal and enteroinvasive microbes in the human gut release bacterial flagellin, a specific microbial ligand of Toll-like receptor 5 (TLR5). However, the pathophysiological role of bacterial flagellin in gastrointestinal inflammation has not been determined. Here we evaluated the role of bacterial flagellin using native human colonic mucosa and the mouse colitis model of dextran sulfate sodium (DSS). We demonstrate that, in intact human colonic mucosa, the flagellin/TLR5 response occurs only after exposure to the basolateral, not the apical, surface, implying a basolaterally polarized TLR5 response in human colonic mucosa. In this context, flagellin exposure to injured colonic mucosa due to DSS administration in mice resulted in a TLR5-associated response evaluated by in vivo activation of mitogen-activated protein kinase/extracellular signal-related kinase 1/2 (MEK1/2) and elevated IL-6, TNF-alpha, and keratinocyte-derived chemokine production, whereas intact colonic mucosa did not respond to flagellin. Moreover, flagellin exposure to injured mouse colon in vivo, but not to intact colon, also significantly aggravated colonic inflammation, increased mouse mortality, and enhanced histopathological damage in the colonic mucosa. However, the TLR2-specific agonist, peptidoglycan or lipoteichoic acid, did not cause an inflammatory response in intact or DSS-injured mouse colon. Furthermore, intracolonic flagellin administration in mice causes severe apoptosis in colonic epithelium disrupted by DSS administration. These data suggest that intracolonic flagellin via TLR5 engagement is able to elicit inflammatory responses in disrupted colon, whereas the normal colon is not responsive to bacterial flagellin. These results demonstrate that bacterial flagellin plays an important role in the development and progress of colitis.