AFM41a: A Novel PAD2 Inhibitor for Sepsis Treatment—Efficacy and Mechanism

Figures

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  Figure 1. PAD2 inhibitors decreased susceptibility and mortality against Pseudomonas aeruginosa (PA) infection. (A-B) Structures and Kinct/KI values for PAD2 inhibitors AFM32a and AFM41a. (C) WT mice were intranasally challenged with 2.5×106 CFU PA 19660/mouse and subsequently administered a single dose of AFM32a (20 mg/kg of body weight) or AFM41a (20 mg/kg of body weight) or an equivalent dose of DMSO 0.5 hours after infection. Survival rates were monitored for 10 days (n=8/group), and analysis was conducted using Mantel-Cox test. (D) Lung tissue samples were collected 24 hours after PA infection and subjected to H&E staining. (E) Whole animal imaging of bioluminescence was obtained using IVIS XRII system 24 hours after mice challenged with 2.5×106 CFU PA Xen-41/mouse (arrows indicating PA spread regions). (F) Lung injury scores. (G) Quantitative analysis of bioluminescence. *p<0.05 vs DMSO; **p<0.01 vs DMSO; ***p<0.001 vs DMSO; #p<0.05 vs 20mg/kg AFM32a; ##p<0.01 vs PA+AFM32a; ✚ p<0.05 vs PA+AFM32a.

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  Figure 2. PAD2 inhibitors promoted THP-1 macrophage towards M2 polarization and increased phagocytosis. THP-1 cells were treated with PMA to induce differentiation for 22-24 hours prior to infection with Pseudomonas aeruginosa (PA) at a multiplicity of 100. Cells were then pre-treated with or without 1 µM AFM32a or AFM41a for 24 hours, then treated with or without PA for 1 hour. (A) The expression of classical cytokine genes (Tnfα, Il6, and Il10) and macrophage polarization signature genes (Nos2, Ccl2, and Mrc1) was quantified by qRT-PCR. (B) The concentrations of TNF-α, IL-6 and IL-10 were determined in cell culture supernatant. (C) Protein expression levels of iNOS and Arg-1 were measured using Western blot analysis. (D) Cells were then fixed and stained for iNOS (red), CD206 (green), and DAPI (blue). (E) Cells were then pre-treated with or without 1 µM AFM32a or AFM41a for 24 hours, then treated with or without PA for 1 hour. The phagocytic capacity of cells was measured using pHrodo Red E. coli BioParticles. Data are representative of three independent experiments expressed as means ± SEM. *p < 0.05 vs Control; ***p < 0.001 vs Control; #p < 0.05 vs PA; ##p < 0.01 vs PA; ###p < 0.001 vs…

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  Figure 3. Pad2 deficiency promoted Alveolar macrophages (AMs) towards M2 polarization and increased phagocytosis. The Pad2-/- and WT mice were infected with 2.5 X 106 CFU Pseudomonas aeruginosa (PA) 19660/mouse for 24 hours. (A) AMs from Pad2-/- and WT mice were lysed, and the expression of classical cytokine genes (Tnfα, Il6, and Il10) and macrophage polarization signature genes (Nos2, Ccl2, Mrc1, and Arg1) was quantified by qRT-PCR. (B) BALF was collected from both groups of mice, and the levels of inflammation cytokines (TNF-α, IL-6, and IL-10) were determined using ELISA. (C) AMs were isolated from Pad2-/- and WT mice, cell lysates from AMs were analyzed by western blotting to assess the protein levels of macrophage polarization markers, iNOS, and Arg-1. (D) AMs were subjected to immunofluorescence staining to evaluate the expression of macrophage polarization markers, iNOS, and Arg-1. (E) Isolated AMs from Pad2-/- and WT mice were analyzed by flow cytometry to quantify the distribution of M1 and M2 macrophage populations. (F) Quantification of the proportion of M1 and M2 macrophages. (G) The phagocytic capacity of AMs was measured using pHrodo Red E. coli BioParticles. Data are…

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  Figure 4. Pad2 deficiency and PAD2 specific inhibitors increased autophagy level. (A-B) THP-1 cells were treated with PMA to induce differentiation for 22-24 hours prior to infection with Pseudomonas aeruginosa (PA) at a multiplicity of 100. Cells were then pre-treated with or without 1 µM AFM32a or AFM41a for 24 hours, then treated with or without PA for 1 hour. Protein expression levels of LC3B, Beclin-1, and P62 were measured using immunofluorescence and western blot analysis. (C-D) The Pad2-/- and WT mice were infected with 2.5×106 CFU PA 19660/mouse for 24 hours. Alveolar macrophages (AMs) of WT and Pad2-/- mice were analyzed by immunofluorescence and western blotting to assess the protein levels of autophagy related proteins, LC3, Beclin-1 and P62. Data are representative of three independent experiments expressed as means ± SEM. *p < 0.05 vs Control; #p < 0.05 vs PA.

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  Figure 5. PAD2 specific inhibitors promoted THP-1 macrophage towards M2 polarization through autophagy. THP-1 cells were treated with PMA to induce differentiation for 22-24 hours prior to infection with Pseudomonas aeruginosa (PA) at a multiplicity of 100. Cells were then pre-treated with or without 1 µM AFM32a or AFM41a for 24 hours, then treated with or without PA for 1 hour. (A) Cells were treated with 1 µM AFM41a in combination with 5 µM 3MA, and the concentrations of TNF-α, IL-6, and IL-10 were determined in the cell culture supernatant. (B) Cells were treated with 1 µM AFM41a in combination with 5 µM 3MA, and the expression of iNOS and CD206 were quantified by western blotting. (C) Cells were treated with 1 µM AFM41a in combination with 5 µM 3MA. The phagocytic capacity of cells was measured using pHrodo Red E. coli BioParticles. Data are representative of three independent experiments expressed as means ± SEM. *p < 0.05 vs Control; ***p < 0.001 vs Control; #p<0.05 vs PA; #p<0.001 vs PA; NS, not significant.

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  Figure 6. Pad2 deficiency promoted Alveolar macrophages (AMs) towards M2 polarization through autophagy. (A) The Pad2-/- and WT mice were infected with 2.5×106 CFU Pseudomonas aeruginosa (PA) Xen-41/mouse, whole animal imaging of bioluminescence was obtained using IVIS XRII system 24 hours after mice challenged with PA. (B) Pad2-/- and WT mice were infected with 2.5×106 CFU PA 19660/mouse for 24 hours, and 0.5 hours after PA challenge, mice were treated with or without 15mg/kg 3MA. Survival rates were monitored for 10 days (n=8/group), and analysis was conducted using Mantel-Cox test. (C) Lung tissue samples were collected 24 hours after PA infection and subjected to H&E staining. (D) BALF was collected from both groups of mice, with or without 3MA treatment, and the levels of inflammation cytokines (TNF-α, IL-6, and IL-10) were determined using ELISA. (E) AMs from Pad2-/- and WT mice were lysed, and the expression of iNOS and CD206 were quantified by western blotting. Data are representative of three independent experiments expressed as means ± SEM. *p < 0.05; **p<0.01 vs WT; ***p<0.001 vs WT; ##p < 0.01 vs Pad2-/-.

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  Figure 7. Schematic mechanism underlying how Pad2 deficiency or inhibition mitigates Pseudomonas aeruginosa-induced lung injury. Our findings indicate that Pad2 deficiency or inhibition, particularly with the novel inhibitor AMF41a, not only enhances survival rates and diminishes bacterial proliferation but also enhances macrophage functionality through the augmentation of autophagy in mice.