Nrf2-dependent effects of CDDO-Me on bactericidal activity in macrophage infection models

Abstract

Introduction: Diabetes and chronic kidney disease (CKD) increase susceptibility to bacterial infections, particularly Staphylococcus aureus, which is associated with highmortality in CKD patients. Dysregulated macrophage activity and excessive oxidative stress exacerbate immune dysfunction and inflammation in these conditions. Nrf2 (nuclear factor erythroid 2-related factor 2) is a key regulator of antioxidant defenses and macrophage function. CDDO-Me, a synthetic triterpenoid, activates Nrf2, providing antioxidant and anti-inflammatoryeffects. However, its precise role in modulating macrophage activity, polarization, and bacterial clearance remains unclear.

Methods: The effects of CDDO-Me on macrophage function were evaluated in vitro (THP-1 and RAW 264.7 macrophages) and an in vivo Nrf2 knockout mouse model. Nrf2 activation was assessed via Western blot and luciferase reporter assays, oxidative stress was measured using CellROX reagent, and inflammatory responses were quantified by RT-qPCR. Intracellular S. aureus survival and macrophage polarization markers were analyzed to investigate the role of CDDO-Me in enhancing bactericidal activity.

Results: Our results showed that CDDO-Me activated the Nrf2 signaling pathway, reducing oxidative stress and inflammation in macrophages by downregulating pro-inflammatory cytokines (IL-1β, TNF-α). It modulated macrophage polarization, decreasing M1 and M2 marker expression, and significantly enhanced bactericidal activity against S. aureus. These effects were Nrf2-dependent, as demonstrated in knockout models.

Conclusion: The ability of CDDO-Me to regulate oxidative stress, inflammation, and bacterial clearance underscores its therapeutic potential for managing inflammatory and infectious diseases indiabetes and CKD.

Keywords: CDDO-Me; Nrf2 knockout mice; Staphylococcus aureus; bactericidal activity; bronchoalveolar lavage; inflammation; macrophages; oxidative stress.

Figure 1

CDDO-Me activated Nrf2 signaling pathway. (A) THP-1-derived macrophages were treated with 10, 25, or 50 nM CDDO-Me for 24 h before testing for cell viability using MTT assay (n=5). Positive control for cell death was obtained by incubating cells with EtOH. (B) Raw 264.7 macrophages were incubated for 24 h in presence of the indicated concentrations of CDDO-Me. Proteins from whole cell lysates were analyzed by Western blot and Nrf2 protein expression levels were detected using specific antibodies for Nrf2 (n=4), HO-1 (n=5), and GAPDH. Immunoblots are representative of 4 independent experiments, Data are presented as mean ± SEM and comparisons were done using one-way ANOVA. (C) THP-1-derived macrophages were treated with 25 nM CDDO-Me. Images are representative of 5 independent experiments, Student’s t-test. *p<0.05, **p<0.005, and ****p<0.001.

Figure 2

Activation of Nrf2 by CDDO-Me. (A) nuclear translocation of Nrf2 was observed in protein extracted 6 h after RAW 264.7 was incubated with increasing concentrations of CDDO-Me (n=5; one-way ANOVA). ns: non-specific signal. (B) Nuclear translocation of Nrf2 was also observed in THP-1-derived macrophages treated with CDDO-Me 25 nM (n=5, Student’s t-test). (C) THP-1-derived macrophages, transfected with ARE-Luciferase vector for 24 h, were treated with CDDO-Me 25 nM for an additional 24 (h) Expression of ARE-Luciferase reporter was assessed by chemiluminescent assay (n=3 independent experiments done in triplicate, Student’s t-test). *p<0.05 and **p<0.01.

Figure 3

Antioxidant effect of CDDO-Me on LPS-stimulated THP-1-derived macrophages. THP-1-derived macrophages were treated with 25 nM CDDO-Me for 3 h prior to stimulation with 5 ng/ml LPS. After 3 h, live cells were stained with CellROX reagent. Stained cells were PFA-fixed, and nuclei counterstained with DAPI. Seven images were taken by confocal microscopy and CellROX fluorescent signals were analyzed using Image J software (n=3 independent experiments; Scale bar = 20 µm). **p<0.01 and ****p<0.001 using one-way ANOVA.

Figure 4

Anti-inflammatory effect of CDDO-Me. (A) Experimental design. (B) THP-1-derived macrophages were treated with 25 nM CDDO-Me or control for 6 (h) Total RNA was extracted and genes coding for IL-1β, IL-6, and TNF-α were quantified by RT-qPCR (n=5). Data are presented as mean ± SEM. (C) THP-1-derived macrophages were pretreated with control or CDDO-Me prior to stimulation with low-dose LPS and incubation for an additional 3 (h) Genes coding for IL-1β, IL-6, and TNF-α were quantified by qPCR (n=5 independent experiments done in triplicate, one-way ANOVA). **p<0.01, ***p<0.005, and ****p<0.001. Analysis were performed by comparing two or three groups using Student’s t-test or one-way ANOVA, respectively.

Figure 5

Effect of CDDO-Me on genes coding for M1/M2 polarization markers. (A) Experimental designs for M0 macrophages and M1 macrophages. M0 macrophages were obtained by differentiation of THP-1 cells into macrophages using PMA. M1 macrophages were obtained by incubating THP-1-derived macrophages with 100 ng/ml LPS and 20 ng/ml IFNγ. Control or 25 nM CDDO-Me were added to macrophages 72 h after PMA differentiation, and incubated for an additional 48 h before RNA extraction and RT-qPCR analysis. Graphs showed on the left side of the dash line M1 marker genes (IL-23, CCR7, IL-1β, IL-6, and TNF-α), and on the right side M2 marker genes (PPARγ, CCL22, and IL-10). (B) Fold-change mRNA expression levels in control-treated M1 macrophages and control-treated M0 macrophages. (C) Fold-change mRNA expression levels in control-treated M0 macrophages and CDDO-Me-treated M0 macrophages. (D) Fold-change mRNA expression levels in control-treated M1 macrophages and CDDO-Me-treated M1 macrophages (n=3 independent experiments done in triplicate). Student’s t-test was used to compare genes from CDDO-Me-treated macrophages and control-treated macrophages *p< 0.05, **p<0.01, ***p< 0.005, ****p< 0.0001.

Figure 6

Effect of CDDO-Me on the bactericidal activity of macrophages. Bacterial viability was assessed using the BacLight bacterial viability kit. (A) S. aureus was incubated for 90 minutes in PBS containing 25 nM CDDO-Me or control. Fluorescent signals were measured at 45, 60, and 90 minutes by spectrophotometry (n=4 independent experiments done in triplicate). (B) THP-1-derived macrophages were pretreated with control or CDDO-Me for 24 h, infected for 1 h with S. aureus at MOI 10, before addition of gentamycin to the medium to eliminate extracellular bacteria. Intracellular survival of S. aureus was assessed 24 h after infection by CFU counts (n=4 independent experiments). (C, D) Macrophages isolated from BAL of Nrf2^+/- control littermates (C) and Nrf2^-/- mice (D) were submitted to the same treatment and infected with S. aureus at MOI 10. CFU counts of S. aureus was determined 24 h after infection (n=4 independent experiments). **p<0.01 and ****p<0.001.