MEK1/2 inhibition decreases pro-inflammatory responses in macrophages from people with cystic fibrosis and mitigates severity of illness in experimental murine methicillin-resistant Staphylococcus aureus infection

Abstract

Chronic pulmonary bacterial infections and associated inflammation remain a cause of morbidity and mortality in people with cystic fibrosis (PwCF) despite new modulator therapies. Therapies targeting host factors that dampen detrimental inflammation without suppressing immune responses critical for controlling infections remain limited, while the development of lung infections caused by antimicrobial resistant bacteria is an increasing global problem, and a significant challenge in CF. Pharmacological compounds targeting the mammalian MAPK proteins MEK1 and MEK2, referred to as MEK1/2 inhibitor compounds, have potential combined anti-microbial and anti-inflammatory effects. Here we examined the immunomodulatory properties of MEK1/2 inhibitor compounds PD0325901, trametinib, and CI-1040 on CF innate immune cells. Human CF macrophage and neutrophil phagocytic functions were assessed by quantifying phagocytosis of serum opsonized pHrodo red E. coli, Staphylococcus aureus, and zymosan bioparticles. MEK1/2 inhibitor compounds reduced CF macrophage pro-inflammatory cytokine production without impairing CF macrophage or neutrophil phagocytic abilities. Wild-type C57BL6/J and Cftr ^tm1kth (F508del homozygous) mice were used to evaluate the in vivo therapeutic potential of PD0325901 compared to vehicle treatment in an intranasal methicillin-resistant Staphylococcus aureus (MRSA) infection with the community-acquired MRSA strain USA300. In both wild-type and CF mice, PD0325901 reduced inflammation associated body mass loss. Wild-type mice treated with PD0325901 had significant reduction in neutrophil-mediated inflammation compared to vehicle treatment groups, with preserved clearance of bacteria in lung, liver, or spleen 1 day after infection in either wild-type or CF mouse models. In summary, this study provides the first data evaluating the therapeutic potential of MEK1/2 inhibitor to modulate CF immune cells and demonstrates that MEK1/2 inhibitors diminish pro-inflammatory responses without impairing host defense mechanisms required for acute pathogen clearance.

Keywords: S. aureus; cystic fibrosis; macrophage; neutrophil; therapeutic.

Figure 1

MEK1/2 inhibitors reduce CF macrophage TLR4 and TLR2 pro-inflammatory responses. Human CF macrophages were stimulated with 50 ng/mL of P. aeruginosa LPS (A-D), 1 μg/mL Pam3CSK4 (E, F) or 100 ng/mL FSL1 (E, G) for 4 hours with the addition of vehicle, PD0325901, Trametinib, or CI-1040 to media at the initiation of stimulation. (A) Protein lysates from a representative experiment from one donor, (B) densitometry quantitation of the ratio of pro-IL-1 to GAPDH from n=6 donors. (C) Measurement of the levels of IL-8 or (D) TNF from supernatants collected at 4 hours from n=5-6 donors. (E) Protein lysates from one representative experiment of three; (F, G) densitometry quantitation from n=3 donors. (B, C, D, F, G) Data are the mean ± SEM, each point represents one individual donor. Statistical analyses were performed by One-Way ANOVA with Tukey multiple comparisons, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001, ns is not significant.

Figure 2

MEK1/2 inhibitors do not impair CF macrophage phagocytosis. Representative flow cytometry gating used for phagocytosis of (A) pHrodo Red E coli bioparticles, (B) pHrodo Red S. aureus bioparticles, or (C) pHrodo Red Zymosan bioparticles. Quantitation of the percent of macrophages positive for pHrodo red fluorescence following incubation with serum opsonized (D) pHrodo Red E coli bioparticles, (E) pHrodo Red S. aureus bioparticles, or (F) pHrodo Red Zymosan bioparticles. Cells were exposed to vehicle, MEK1/2 inhibitor compounds, or cytochalasin D during the 1 hour phagocytosis incubation period with pHrodo red bioparticles. Data are the mean ± SEM and each point represents data from one individual donor. Statistical analyses were performed by One-Way ANOVA with Tukey multiple comparisons, * P<0.05, ** P<0.01, **** P<0.0001.

Figure 3

MEK1/2 inhibitors do not impair CF neutrophil phagocytosis. Representative flow cytometry gating used for phagocytosis of (A) opsonized pHrodo red E. coli bioparticles or (B) opsonized pHrodo red S. aureus bioparticles. Quantitation of the percent of neutrophils positive for pHrodo red fluorescence following incubation with opsonized (C) opsonized pHrodo red E. coli bioparticles or (D) opsonized pHrodo S. aureus bioparticles. Cells were exposed to vehicle, MEK1/2 inhibitor compounds, or cytochalasin D during the 1 hour phagocytosis incubation period with pHrodo red bioparticles. Data are the mean ± SEM and each point represents one individual donor. Statistical analyses were performed by One-Way ANOVA with Tukey multiple comparisons, ** P<0.01.

Figure 4

MEK1/2 inhibitor administration does not impair host defense of mice during S. aureus infection. Anesthetized mice were provided i.p. treatment with 20 mg/kg PD0325901 (black filled circles) or vehicle control (open circles) immediately prior to intranasal inoculation with 1 x 10⁷ CFU of S. aureus USA300; mock-infected (open square) animals received sterile PBS by intranasal instillation. (A) Body mass of C57BL6/J mice was measured following infection or mock infection for six days. Data are the mean SEM from n=5-10 males and n=5-10 females for each infected treatment group, and n=2-7 males and n=2-7 females for mock infected groups across the six day period. On day 1 after infection, (B) total BAL cells, (C) total BAL neutrophils, and (D) total BAL macrophages were quantified, (E) neutrophil elastase (NE) in BAL fluid (BALF) was quantified by ELISA, and (F) CFU burdens in lung homogenates were enumerated and normalized to gram of tissue collected; data points represent an individual mouse, n=9-10 mice per group combined from two independent experiments. In separate experiments (G) body mass of Cftr ^tm1kth F508del homozygous mice was measured on day 1 after infection, and (H) CFU burdens in lung, liver, and spleen homogenates were enumerated. Data points represent an individual mouse and are combined from three independent experiments; data points were omitted from the graphs if the CFU recovery was below the limit of detection (99 CFU/mL) prior to normalization to gram of tissue. (I-L) IHC staining for CD45 on representative lungs from a (I-J) vehicle-treated or (K-L) PD0325901-treated CF mice. (M) Percent of total lung area positive for CD45 staining quantified by ImageJ Software. (N) NE in BALF of CF mice 1 day after infection quantified, n=4 per group. Statistical analyses were performed with (A) Two-Way ANOVA with multiple comparisons with results shown comparing S. aureus + vehicle to S. aureus + PD0325901 groups, (B-E) One-Way ANOVA with Tukey’s multiple comparisons, (F, G, M, N) Unpaired t-test, and (H) multiple unpaired t-tests.