(514) Anti-Inflammatory Effects of MEK1/2 Inhibitors on Cystic Fibrosis Macrophages

Background: Chronic and recurrent infections and inflammation are a significant driver of lung pathology in people with CF (PwCF). Although CFTR modulator therapies are improving the lives of PwCF, chronic infections have not been eliminated. Therefore, there is still a critical need for new therapeutic strategies to eliminate bacterial infections and reduce detrimental inflammatory responses. Our laboratory is investigating the role of the MEK1/2-ERK1/2 signaling pathway as a regulator of macrophage mediated inflammation. We previously demonstrated that MEK1/2 inhibitor administration could reduce pulmonary inflammation in murine models of LPS-induced acute lung injury or P. aeruginosa infection. Cellular models of CF have indicated that overactivation of the MEK1/2-ERK1/2 pathway may exacerbate detrimental pro-inflammatory responses. Pharmacologic compounds targeting the MEK1/2 pathway for inhibition have the potential to modulate the inflammatory response to reduce inflammation. However, as with all potential anti-inflammatory therapies, there is a risk of immunosuppression or impairing host defense mechanisms. In this study, we determined if pharmacologic MEK1/2 inhibitor compounds could reduce pro-inflammatory responses of human CF macrophages without impairing macrophage host defenses.

Methods: Peripheral blood mononuclear cells were isolated from peripheral blood of PwCF and cultured with 20 ng/ml recombinant human M-CSF for 7-10 days to generate human monocyte-derived macrophages. To determine the effect of MEK1/2 inhibitor compounds on CF macrophage pro-inflammatory responses, macrophages were stimulated with 50 ng/ml P. aeruginosa LPS with addition of either vehicle (DMSO), 0.5 mM PD0325901, 10 mM CI-1040, or 0.5 mM GSK1120212 for 4 hours. Protein lysates were used to measure pro-IL-1b and supernatants were used to measure the secretion of IL-8. In separate experiments, flow cytometry was used to evaluate if MEK1/2 inhibitors impaired macrophage or neutrophil phagocytosis and phagosome acidification of serum-opsonized pHrodo Red S. aureus or E. coli bioparticles.

Results: The addition of MEK1/2 inhibitor compounds to macrophages significantly reduced LPS-induced activation of ERK1/2, production of the inflammatory cytokine pro-IL1b, and secretion of IL-8. Treatment of macrophages from PwCF with either opsonized Staphylococcus aureus or Escherichia coli pHrodo particles in the presence of MEK1/2 inhibitors did not significantly reduce phagocytosis or phagosome acidification compared to vehicle-treated controls; while cytochalasin-D treated controls demonstrated robust inhibition of phagocytosis. Preliminary results also indicated that treatment of neutrophils with MEK1/2 inhibitor compounds did not reduce phagocytosis or phagosome acidification.

Conclusions: MEK1/2 inhibitor compounds significantly decrease pro-inflammatory responses of human CF macrophages following stimulation with LPS. However, treatment of macrophages with MEK1/2 inhibitor compounds did not alter phagocytosis or phagosome acidification of pHrodo Red labeled bioparticles. Together, these data support the hypothesis that MEK1/2 inhibitor compounds can be utilized to reduce detrimental inflammation without impairing host defense mechanisms. Future studies will evaluate the effects of MEK1/2 inhibitor compounds on the effector functions of CF neutrophils and on the in vivo response to infection.

Acknowledgements: Supported by the Cystic Fibrosis Foundation awards LONG19F5-CI, LONG21R3, and MCCOY19R0.

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