Imidazole propionate ameliorates atopic dermatitis-like skin lesions by inhibiting mitochondrial ROS and mTORC2

Abstract

Background: Imidazole propionate (IMP) is a histidine metabolite produced by some gut microorganisms in the human colon. Increased levels of IMP are associated with intestinal inflammation and the development and progression of cardiovascular disease and diabetes. However, the anti-inflammatory activity of IMP has not been investigated. This study aimed to elucidate the role of IMP in treating atopic dermatitis (AD).

Methods: To understand how IMP mediates immunosuppression in AD, IMP was intraperitoneally injected into a Dermatophagoides farinae extract (DFE)/1-chloro-2,4 dinitrochlorobenzene (DNCB)-induced AD-like skin lesions mouse model. We also characterized the anti-inflammatory mechanism of IMP by inducing an AD response in keratinocytes through TNF-α/IFN-γ or IL-4 stimulation.

Results: Contrary to the prevailing view that IMP is an unhealthy microbial metabolite, we found that IMP-treated AD-like skin lesions mice showed significant improvement in their clinical symptoms, including ear thickness, epidermal and dermal thickness, and IgE levels. Furthermore, IMP antagonized the expansion of myeloid (neutrophils, macrophages, eosinophils, and mast cells) and Th cells (Th1, Th2, and Th17) in mouse skin and prevented mitochondrial reactive oxygen species production by inhibiting mitochondrial energy production. Interestingly, we found that IMP inhibited AD by reducing glucose uptake in cells to suppress proinflammatory cytokines and chemokines in an AD-like in vitro model, sequentially downregulating the PI3K and mTORC2 signaling pathways centered on Akt, and upregulating DDIT4 and AMPK.

Discussion: Our results suggest that IMP exerts anti-inflammatory effects through the metabolic reprogramming of skin inflammation, making it a promising therapeutic candidate for AD and related skin diseases.

Keywords: AMPK; DDIT4; atopic dermatitis; imidazole propionate; mTORC2; mitochondria ROS.

Figure 1

IMP attenuates the clinical symptoms of AD-like skin lesions in a mouse model. BALB/c mice were induced with DNCB and DFE, and treated with IMP (1 or 2 mg/mouse) or Rapa (0.6 mg/mouse). (A) Experimental design for the induction of AD-like skin lesions. The mice (n = 4–6/group) were divided into five groups. (B) Ear thickness was measured 24 h after DNCB or DFE application using a dial thickness gauge. (C) Mouse body weight was calculated as a percentage of the initial weight. (D) Images of the mouse ears from each representative group on day 28. (E) Representative photomicrographs of ear sections stained with hematoxylin and eosin (H&E) (×100 magnification; scale bar = 20 µm). (F) Epidermal and dermal thicknesses were measured using microphotographs of H&E-stained ear tissues. (G) Serum IgE levels were analyzed using ELISA. Data are presented as the mean ± standard error of mean (SEM). ****p < 0.0001, **p < 0.01, and *p < 0.05 indicate significant reduction compared to the AD group. Ctrl, control; AD, atopic dermatitis; DNCB, 2,4-dinitrochlorobenzene; DFE, Dermatophagoides farina extract; IMP, imidazole propionate; Rapa, rapamycin. ns: no significant.

Figure 2

IMP inhibits the inflammatory myeloid cells in the skin of AD mice. (A, B) Representative flow cytometry plots and bar charts show percentage of CD11b⁺Ly6G⁺ (neutrophils), F4/80⁺CD11b⁺ (macrophages), single-F⁺CD11b⁺ (eosinophils), and c-Kit⁺FcεRIA⁺ (mast cells) in the ear skin cells (n = 6/group). Data are presented as the mean ± SEM. ****p < 0.0001, ***p < 0.001, and **p < 0.01 indicate significant reduction compared to the AD group. AD, atopic dermatitis; IMP, imidazole propionate; Rapa, rapamycin. ns: no significant.

Figure 3

IMP regulates the T cell immune response in the inflamed skin of AD mice. (A, B) Representative flow cytometry plots and bar graphs showing percent CD4⁺ T cells expressing IFN-γ, IL-4 or IL-17A in the ear skin cells (n = 6/group). (C) Gene expression of Th1 (Ifnγ), Th2 (Il4, Il5, Il13, and Il31), and Th17 (Il17a) cytokines and Treg (Foxp3) transcription factor in the ears of AD and IMP or Rapa-treated AD mice. To determine the cytokine expression in mice, the ears were excised on day 28. Gene expression was analyzed using real-time PCR (n = 4/group). The gene expression levels were normalized to that of β-actin. Data are presented as the mean ± SEM. ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05 indicate significant reduction compared to the AD group. Ctrl, control; AD, atopic dermatitis; IMP, imidazole propionate; Rapa, rapamycin. ns: no significant.

Figure 4

IMP inhibits mitochondrial ROS production by altering the inflammatory metabolic profile in AD skin. (A) Real-time changes in the oxygen consumption rate (OCR) of skin cells in response to oligomycin, FCCP, and Rot/AA. The bar charts show the mitochondrial oxygen consumption, basal and maximal respiratory capacity, proton leak, ATP production rate and coupling efficiency. Representative histograms and bar graphs showing total mitochondrial mass (n = 3/group). (B) and mitochondrial ROS production (C). Mitochondrial mass and ROS were analyzed by flow cytometry in skin cells labeled with MitoTracker green or MitoSOX red (n = 6/group). Data are presented as the mean ± SEM. ***p < 0.001, **p < 0.01, and *p < 0.05 indicate significant reduction compared to the AD group. AD, atopic dermatitis; IMP, imidazole propionate; Rapa, rapamycin; OCR, oxygen consumption rate; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; Rot/AA, rotenone and antimycin A; ATP, adenosine triphosphate; ROS, reactive oxygen species. ns: no significant.

Figure 5

Induction of AMPK/DDIT4 by IMP inhibits mTORC2 signaling and reduces inflammatory cytokines and chemokines in AD in vitro. Human keratinocytes HaCaT cells were stimulated with TNF-α (10 ng/mL) and IFN-γ (10 ng/mL) in the presence or absence of IMP (10 or 20 µg/mL) for 6 h or indicated times (n = 3-4/group). (A) Gene expression of proinflammatory cytokines (Tnfα, Il1β, and Il6), and chemokines (Ccl17 and Ccl22) was analyzed using real-time PCR. (B, C) Representative histograms and bar graphs showing glucose uptake in HaCaT cells, which was determined by incubation with 2-NBDG for 2 h, followed by flow cytometry. (D) Gene expression of PI3K signaling molecules (Pik3cb and Pik3r1) was analyzed using real-time PCR. Protein expression of mTORC2 targets (E), and that of AMPK and DDIT4 (F) in HaCaT cells stimulated in the presence or absence of IMP (20 μg/mL) was determined by western blotting. Data are presented as the mean ± SEM. ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05 indicate significant reduction compared to TNF-α/IFN-γ. IMP, imidazole propionate; 2-NBDG, 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose; mTOR, mammalian target of rapamycin; mTORC2, mammalian target of rapamycin complex 2; Rictor, rapamycin-insensitive companion of mammalian target of rapamycin; AKT, protein kinase B; FOXO3a, forkhead box O3a; AMPK, AMP-activated protein kinase; DDIT4, DNA-damage-inducible transcript 4.