Potential Tamoxifen Repurposing to Combat Infections by Multidrug-Resistant Gram-Negative Bacilli

Abstract

The development of new strategic therapies for multidrug-resistant bacteria, like the use of non-antimicrobial approaches and/or drugs repurposed to be used as monotherapies or in combination with clinically relevant antibiotics, has become urgent. A therapeutic alternative for infections by multidrug-resistant Gram-negative bacilli (MDR-GNB) is immune system modulation to improve the infection clearance. We showed that immunocompetent mice pretreated with tamoxifen at 80 mg/kg/d for three days and infected with Acinetobacter baumannii, Pseudomonas aeruginosa, or Escherichia coli in peritoneal sepsis models showed reduced release of the monocyte chemotactic protein-1 (MCP-1) and its signaling pathway interleukin-18 (IL-18), and phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2). This reduction of MCP-1 induced the reduction of migration of inflammatory monocytes and neutrophils from the bone marrow to the blood. Indeed, pretreatment with tamoxifen in murine peritoneal sepsis models reduced the bacterial load in tissues and blood, and increased mice survival from 0% to 60-100%. Together, these data show that tamoxifen presents therapeutic efficacy against MDR A. baumannii, P. aeruginosa, and E. coli in experimental models of infection and may be a new candidate to be repurposed as a treatment for GNB infections.

Keywords: animal model; bacteria; immune system; infection; repurposing drug; tamoxifen.

Figure 1

Bone marrow immune cells’ migration to the blood in response to monocyte chemotactic protein-1 (MCP-1) and interleukin-18 (IL-18) during bacterial infection. (A) Myeloid cells, (B) inflammatory monocytes, and (C) neutrophils were identified as CD11b+, CD11b+Ly6C^hi, and CD11b+Ly6G+, respectively, by flow cytometry in the bone marrow and blood of mice infected with MLD100 of A. baumannii ATCC17978, P. aeruginosa PAO1, or E. coli ATCC25922 strains for 24 h. (D,E) Serum MCP-1 and IL-18 levels (enzyme-linked immunosorbent assays (ELISAs)), 6 and 24 h post-infection, in mice infected with minimal lethal dose 100 (MLD100) of A. baumannii ATCC17978, P. aeruginosa PAO1, or E. coli ATCC25922 strains. (F) RAW 264.7 cells were infected with A. baumannii ATCC17978, P. aeruginosa PAO1, or E. coli ATCC25922 strains for 2 h and proteins were collected for phospho-p44/42 Mitogen-Activated Protein Kinases (extracellular signal-regulated kinase 1/2 (Erk1/2)) and β-actin immunoblotting. Data are representative of six mice per group, and expressed as mean ± standard error of the mean (SEM). * p < 0.05: infected vs. control (CTL). CTL: non-infected mice, %: the percentage of myeloid cells, inflammatory monocytes and neutrophils from total cells in bone marrow or blood.

Figure 2

Role of MCP-1 in the bone marrow immune cells’ migration to the blood during bacterial infection. (A) Wild-type (WT) and MCP-1 knockout (KO) mice were infected with minimal lethal dose 100 (MLD100) of A. baumannii ATCC17978, P. aeruginosa PAO1, or E. coli ATCC25922 strains. Twenty-four hours post-infection, serum was harvested for MCP-1 ELISAs. (B) Inflammatory monocytes and (C) neutrophils were identified as CD11b+Ly6C^hi and CD11b+Ly6G+ by flow cytometry, respectively, in the bone marrow and blood of wild-type and MCP-1 KO mice infected with MLD100 of A. baumannii ATCC17978, P. aeruginosa PAO1, or E. coli ATCC25922 strains for 24 h. Data are representative of six mice per group, and expressed as mean ± SEM. * p < 0.05: WT vs. MCP-1 KO. WT: wild-type, MCP-1 KO: mice lacking MCP-1, CTL: non-infected mice, %: the percentage of inflammatory monocytes and neutrophils from total cells in bone marrow or blood.

Figure 3

Tamoxifen reduces, after bacterial infection, the release of MCP-1 and IL-18 in vitro and in vivo, and ERK phosphorylation in vitro. (A,B) RAW 264.7 cells were treated with 2.5 mg/L of tamoxifen for 24 h and infected with A. baumannii ATCC17978, P. aeruginosa PAO1, or E. coli ATCC25922 strains for 2 h. MCP-1 levels and ERK-phosphorylation were determined by ELISA and immunoblotting assays, respectively. Data are representative of three independent experiments, and expressed as mean ± SEM. (C,D) Mice received firstly tamoxifen (80 mg/kg/d, for three days), followed by infection with a minimal lethal dose 100 (MLD100) of A. baumannii ATCC17978, P. aeruginosa PAO1, or E. coli ATCC25922 strains. At 6 and 24 h post-infection, serum was harvested for MCP-1 and IL-18 ELISA assays. Data are representative of six mice per group and are expressed as mean ± SEM. * p < 0.05: treated vs. INF, ** p < 0.05: treated vs. CTL. CTL: non-infected mice. TAM: tamoxifen. INF: infected.

Figure 4

Tamoxifen impairs, after bacterial infection, the migration of immune cells from the bone marrow to the blood. Mice received firstly tamoxifen (80 mg/kg/d, for three days), followed by infection with minimal lethal dose 100 of A. baumannii ATCC17978, P. aeruginosa PAO1, or E. coli ATCC25922 strains. Twenty-four hours post-infection, (A) myeloid cells, (B) inflammatory monocytes, (C) neutrophils and (D) Tip-DC were identified as CD11b+, CD11b+Ly6C^hi, CD11b+Ly6G+, and CD11b+CD11c+MHII+ respectively, by flow cytometry in the bone marrow and blood of mice. Data are representative of five/six mice per group and are expressed as mean ± SEM. * p < 0.05: treated vs. infected. ** p < 0.05: CTL vs. infected, ^# p < 0.05: TAM vs. infected. CTL: non-infected mice, TAM: tamoxifen, %: the percentage of myeloid cells, inflammatory monocytes, neutrophils and Tip-DC from total cells in bone marrow or blood.

Figure 5

Tamoxifen impairs slightly, after bacterial infection in MCP-1-deficient mice, the migration of immune cells from the bone marrow to the blood through MCP-1 regulation. (A) WT and MCP-1 KO mice received firstly tamoxifen (80 mg/kg/d, for three days), followed by infection with a minimal lethal dose 100 of A. baumannii ATCC17978, P. aeruginosa PAO1, or E. coli ATCC25922 strains. Twenty-four hours post-infection, (A) inflammatory monocytes and (B) neutrophils were identified as CD11b+, CD11b+Ly6C^hi, and CD11b+Ly6G+, respectively, by flow cytometry in the bone marrow and blood of mice. Data are representative of six mice per group and are expressed as mean ± SEM. CTL: non-infected mice. * p < 0.05: infected WT vs. infected MCP-1 KO, ** p < 0.05: infected MCP-1 KO vs. infected MCP-1 KO + TAM, *** p < 0.05: infected WT + TAM vs. infected MCP-1 KO + TAM. CTL: non-infected mice, TAM: tamoxifen, %: the percentage of inflammatory monocytes and neutrophils from total cells in blood.

Figure 6

Tamoxifen induces the phagocytic activity of macrophages and neutrophils. RAW 264.7 cells and HL-60 neurophils were incubated with tamoxifen for 2 or 6 h and infected for 2 h with A. baumannii ATCC17978, P. aeruginosa PAO1, or E. coli ATCC25922 strains (MOI:100) (A) The adherence and invasion of these strains are expressed as the percentage of total control strain adhered or internalized to RAW 264.7 and the killing index of HL-60 neutrophil cells is expressed as the percentage. (B) The extracellular strains in the culture medium after 6 h of tamoxifen incubation are determined as Log colony-forming units (CFU)/mL. Data are representative of three independent experiments, and expressed as mean ± SEM. * p < 0.05: treated vs. CTL. CTL: infected cells without tamoxifen treatment. TAM: tamoxifen.

Figure 7

Tamoxifen increases the survival of mice infected with non-MDR or MDR GNB. Mice survival was monitored during 7 days for 6 mice infected with MLD100 of non-MDR and MDR A. baumannii (Ab9 and Ab186), P. aeruginosa (Pa39 and Pa238) or E. coli (C1-7-LE and EcMCR+) strains treated or not with 3 ip. doses of tamoxifen (80 mg/kg/d, for 3 days). * p < 0.05: treated vs. untreated, ** p < 0.05: treated vs. untreated, TAM: tamoxifen, MDR: multidrug-resistant.