Myeloid NCOA4 sequesters KEAP1 to reduce ferroptosis for protection against salmonellosis in mice

Abstract

Salmonellosis, caused by Salmonella enterica serovar Typhimurium, is a significant global threat. Host immunity limits bacterial replication by inducing hepcidin, which degrades ferroportin, reducing iron transfer. However, this boosts macrophage iron storage, aiding intracellular pathogens like Salmonella. Mice lacking ferritin heavy chain (FTH1) in myeloid cells suffer worsened Salmonella infection. Nuclear receptor co-activator 4 (NCOA4) regulates iron release via FTH1 degradation during low iron, but its role in salmonellosis is unclear. Here, we reveal that myeloid NCOA4 deficiency augments spleen iron levels and increases cellular iron accumulation, oxidative stress, and ferroptosis in bone marrow-derived macrophages. This deficiency also increases susceptibility to Salmonella-induced colitis in mice. Mechanistically, NCOA4 suppresses oxidative stress by directly binding to the E3 ubiquitin ligase Kelch-like ECH-associated protein 1 (KEAP1) and stabilizing the antioxidant transcription factor nuclear factor-erythroid 2-related factor 2 (NRF2). Activation of NRF2 protects myeloid NCOA4 knockout mice from Salmonella-induced colitis. Antioxidant Tempol and myeloid cell-targeted curcumin offer protection against colitis in myeloid NCOA4-deficient mice. A low iron diet and ferroptosis inhibition also mitigate the heightened colitis in these mice. Overexpression of myeloid cell-specific NCOA4 confers protection against Salmonella-induced colitis via upregulating NRF2 signaling. Serum iron was reduced in myeloid NCOA4-overexpressing mice, but not in NCOA4-deficient mice. Targeted serum metabolomics analysis revealed that many lipids were decreased in myeloid NCOA4-deficient mice, while several of them were increased in myeloid NCOA4-overexpressing mice. Together, this study not only advances our understanding of NCOA4/KEAP1/NRF2/ferroptosis axis but also paves the way for novel myeloid cell-targeted therapies to combat salmonellosis.

Keywords: Salmonella; iron metabolism; macrophages; oxidative stress.

Fig. 1:. Myeloid NCOA4 depletion increases cellular iron, mitochondrial iron, cell death, and lipid peroxidation levels in BMDM cells.

(A) qPCR analysis and (B) Immunoblot analysis, (C) Quantification of FerroOrange staining for cellular iron, (D) Representative imaging flow cytometry single cell images, (E) Quantification of mitoFerroGreen positive cells, (F) Quantification of DAPI positive dead cells, (G) Representative single cell images, (H) Histogram of imaging flow cytometry analysis of BODIPY C11 staining, (I) Positive BODIPY C11 staining cell percentage and (J) relative intensity quantification in BMDM from myeloid NCOA4 KO and WT mice (n=3–4). Values above blots represent mean ± S.D. Statistical significance was denoted as *p<0.05, ***p<0.001, ****p<0.0001 using Student’s t-test.

Fig. 2:. Myeloid NCOA4 knockout mice display shortened colon length, impaired Salmonella clearance, heightened inflammation, and reduced NRF2 signaling antioxidant proteins in Salmonella-treated mouse colon tissues.

(A) Body weight, (B) representative images of dissected colon, (C) colon length, (D) Colony Formation Unit (CFU) of Salmonella, qPCR analysis of the proinflammatory cytokines (E) Il1β, (F) Tnfα, (G) Il6, and (H) Cxcl1, (I) immunoblot analysis, were conducted on colon tissues from control or Salmonella-treated KO mice (n=3–8) and WT mice (n=3–6). ns, not significant. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 Vs WT. #p<0.05, ##p<0.01, ###p<0.001 Vs Salmonella treated WT mice. Two-way ANOVA followed by Tukey’s multiple comparisons test for (C and I). Student’s t-test for (D-H).

Fig. 3:. The IVR and Kelch domains of KEAP1 are essential for NCOA4 binding and its degradation through lysosome but not proteasome.

(A) Predicted (13 high confidence) interaction of E3 (including KEAP1) and substrate (NCOA4) in Ubibrowser database. Co-immunoprecipitation (IP) analysis using (B) anti-HA or (C) anti-Myc antibody in HEK293 cells co-transfected with Myc-KEAP1 and HA-NCOA4 plasmids. Immunoblot analysis in HEK293T cells transiently transfected with NCOA4 and KEAP1 plasmids with or without (D) a proteasomal inhibitor 10 μM MG132 treatment for 4h, (E) an autolysosomal inhibitor 150 μM Chloroquine (CQ) for 24h. (F) Diagram of truncated constructs of KEAP1. Each domain (NTR, BTB, IVR, Kelch and CTR) of the KEAP1 protein (indicated by boxes), its putative molecular function, and number of amino acids are shown. (G) Co-IP analysis in HEK293 cells co-transfected with different KEAP1 and HA-NCOA4 plasmids. To detect different KEAP1 mutant constructs, we used two KEAP1 antibodies with different immunogens: Left, Santa Cruz, sc-514914, 39–65aa. Right, Proteintech, 10503–2-AP, 325–624aa.

Fig. 4:. Activation of NRF2 protects myeloid NCOA4 KO mice from Salmonella-induced colitis.

(A) Body weights and (B) colon lengths, qPCR analysis of the proinflammatory cytokines (C) Il1β and (D) Tnfα, (E) Immunoblot analysis of key antioxidant proteins in the NRF2 signaling pathway and NCOA4 were conducted on colon tissues from control or Salmonella-treated KO mice (n=3–8) and WT mice (n=3–7) treated with either vehicle control or NRF2 activator Oltipraz. Statistical significance was denoted as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 vs WT control or #p<0.05, ##p<0.01 vs KO control. Two-way ANOVA followed by Tukey’s multiple comparisons test was applied.

Fig. 5:. Antioxidants Tempol and Myeloid Cell Targeting Nanoparticles Conjugated Curcumin Protect Against Salmonella-Induced Colitis in Myeloid NCOA4-Deficient Mice.

(A) Colon length, qPCR analysis of the proinflammatory cytokines (B) Il6, (C) Tnfα, and (D) Il1β, (E) immunoblot analysis of key antioxidant proteins in the NRF2 signaling pathway in colon tissues from Salmonella-treated KO mice (n=3–4) and WT mice (n=3–4) that received vehicle or 0.064% Tempol in drinking water. (F) Colon length, qPCR analysis of the proinflammatory cytokines (G) Il6, (H) Tnfα, and (I) Il1β, (J) immunoblot analysis of key antioxidant proteins in the NRF2 signaling pathway in colon tissues from Salmonella-treated KO mice (n=3–4) and WT mice (n=3–4) that received curcumin (Cur) or myeloid cell targeting nanoparticles conjugated curcumin (nano-Cur) treatment. Statistical significance was denoted as *p<0.05, **p<0.01, ***p<0.001 or #p<0.05, ###p<0.001 vs KO control. Two-way ANOVA followed by Tukey’s multiple comparisons test was applied.

Fig. 6:. A Low Iron Diet and ferroptosis inhibition abolishes the susceptibility of myeloid NCOA4 knockout mice to Salmonella-induced colitis.

(A) colon length, qPCR analysis of the proinflammatory cytokines (B) Tnfα and (C) Il1β, (D)Nos2 and (E) Sic11a2 in colon tissues from Salmonella-treated KO mice (n=3–4) and WT mice (n=3–4) that received either a control (40 ppm iron) or a low iron (3.5 ppm iron) diet. (F) Colon length, qPCR analysis of proinflammatory cytokines (G) Tnfα and (H) Il6, (I) iron uptake transporter Tfrc in colon tissues from myeloid NCOA4 KO and their WT control mice treated with vehicle control (n=3–5) or a daily 1 mg/kg dose of Ferrostatin-1 (n=3–4) via i.p. injection starting 2 days before the Salmonella infection. Statistical significance was denoted as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Two-way ANOVA followed by Tukey’s multiple comparisons test was applied.

Fig. 7:. Myeloid cell-specific NCOA4 overexpression protects mice from Salmonella-induced colitis via upregulation of NRF2 signaling.

(A) qPCR analysis and (B) immunoblot analysis of NCOA4 in BMDM cells from WT (n=3–4) and myeloid cell-specific NCOA4 OE (n=3–4) mice. (C) colon lengths and (D) representative image of colons, and qPCR analysis of (E) Tfrc, (F) Slc11a2, (G) Nos2 and (H) Tnfα, (I) Cxcl1, and (J) immunoblot analysis in colons from OE (n=4) and WT mice treated with Salmonella (n=4–5). *p<0.05, **p<0.01, ***p<0.001, Student’s t-test.