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. 2015 Feb 28;1(1):5.
doi: 10.23937/2469-5726/1510005.

Suppression of experimental arthritis through AMP-activated protein kinase activation and autophagy modulation

Huimin Yan  1 , Hui-Fang Zhou, Ying Hu, Christine T N Pham
Affiliations

Suppression of experimental arthritis through AMP-activated protein kinase activation and autophagy modulation

Huimin Yan et al. J Rheum Dis Treat. .

Abstract

Autophagy plays a central role in various disease processes. However, its contribution to inflammatory arthritides such as rheumatoid arthritis (RA) is unclear. We observed that autophagy is engaged in the K/BxN serum transfer model of RA but autophagic flux is severely impaired. Metformin is an anti-diabetic drug that has been shown to stimulate autophagy. Induction of autophagic flux, through metformin-mediated AMP-activated protein kinase (AMPK) activation and interruption of mammalian target of rapamycin (mTOR) signaling mitigated the inflammation in experimental arthritis. Further investigation into the effects of metformin suggest that the drug directly activates AMPK and dose-dependently suppressed the release of TNF-α, IL-6, and MCP-1 by macrophages while enhancing the release of IL-10 in vitro. In vivo, metformin treatment significantly suppressed clinical arthritis and inflammatory cytokine production. Mechanistic studies suggest that metformin exerts its anti-inflammatory effects by correcting the impaired autophagic flux observed in the K/BxN arthritis model and suppressing NF-κB-mediated signaling through selective degradation of IκB kinase (IKK). These findings establish a central role for autophagy in inflammatory arthritis and argue that autophagy modulators such as metformin may represent potential therapeutic agents for the treatment of RA.

Keywords: AMP-activated protein kinase (AMPK); Autophagy; inflammatory arthritis; mammalian target of rapamycin (mTOR); metformin.

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Conflict of interest statement

Conflict: The authors declare no conflict of interest

Figures

Figure 1
Figure 1. Metformin suppresses KRN arthritis
(A) Cohorts of mice were injected i.p daily with saline or metformin (150 mg/kg of body weight) starting one day prior (day −1) to KRN serum transfer and continued daily for the duration of the experiment. On day 0, they were injected i.p. with 175 μl of KRN serum. Changes in arthritis score and ankle thickness were assessed daily. (B) Representative micrographs of day 9 joint sections stained with H&E and toluidine blue. Scale bars = 200 μm (H&E), 50 μm (toluidine blue) (C) Graphical representations of inflammatory cellular infiltrates, bone erosions and cartilage degradation. (D) Day 9 paws were homogenized and cleared lysates were assayed for inflammatory cytokines. Levels are expressed as picograms (pg) per mg of total protein extracts. (E) Arthritis was induced with KRN serum transfer on day 0 and metformin started on day 0 or day 2 after disease is established. Values are presented as mean ± SEM, n = 15 mice in the saline group and 5 mice for each metformin treatment group. *P < 0.05, **P < 0.01, ***P < 0.001
Figure 2
Figure 2. Metformin directly suppresses macrophage inflammatory activity in vitro
(A) Day 5 thioglycollate-elicited peritoneal macrophages were cultured with the indicated metformin concentrations. At 48 h, supernatants were collected and assayed for cytokines by cytometric bead arrays. Values represent mean ± SEM of triplicate samples derived from 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (B) Cultured macrophages were lysed, cleared, fractionated by SDS-PAGE, and probed for phospho-AMPK. Actin served as control for protein loading.
Figure 3
Figure 3. Metformin suppresses inflammatory macrophage phenotype in KRN arthritis
Day 9 paws were stained for (A) macrophages (Mac-3, green) and TNF-α (red) and (B) Mac-3 (green) and phospho-STAT1 (red). In saline control animals there was significant colocalization (yellow) of TNF-α and phospho-STAT1 with Mac-3+ cells (arrowheads) while metformin treatment suppressed both TNF-α and STAT1 activation in macrophages (arrows). DAPI (blue) stained nuclei. Scale bar = 25 μm.
Figure 4
Figure 4. Metformin activates AMPK and suppresses mTORC1 activity in KRN arthritis
(A) Protein lysates from day 5, 7, or 9 paws obtained from KRN arthritic mice were probed for p-AMPK and total AMPK. Actin served as control for protein loading. (B) Day 9 paws were stained for Mac-3 (arrows, green) and p-AMPK (red). Colocalization (arrowheads) appeared orange/yellow. (C) Day 9 paws were stained for phospho (p)-mTOR and p-S6 (red). DAPI (blue) stained nuclei. Scale bar = 25 μm. (D) Intracellular level of p-mTOR and p-S6 was analyzed using ImageJ program as detailed in the Materials and Methods section and presented as integrated optical density (IntDen). Values represent mean ± SEM, n = 4–5 mice per treatment group. *P < 0.05 compared with saline.
Figure 5
Figure 5. Metformin enhances autophagic flux
(A) Paw lysates from normal and day 9 KRN serum-induced arthritic mice were probed for LC3-I, the lipidated form LC3-II, and p62. Actin served as control for protein loading. (B) Paw sections from untreated arthritic mice or arthritic mice treated with saline or metformin were stained for LC3 (green) and the lysosomal marker LAMP-1 (red). Note the punctate LC3 staining in untreated or saline control arthritic paws. Colocalization of LC3 and LAMP-1 (arrow) indicates autolysosome. Scale bar = 10 μm. (C) Treatment with metformin led to degradation of the lipidated LC3-II form and p62, indicating enhanced autophagic flux. (D) Day 9 paws were stained for phospho (p)-ULK1Ser555 (red). DAPI (blue) stained nuclei. Scale bar = 25 μm. (E) Intracellular level of p-ULK1was analyzed using ImageJ program as detailed in the Materials and Methods section and presented as integrated optical density (IntDen). Values represent mean ± SEM, n = 4–5 mice per treatment group. *P < 0.05 compared with saline.
Figure 6
Figure 6. Metformin promotes selective degradation of NF-κB protein Iκ kinase (IKK) through autophagy
(A) Day 9 paw lysates from saline controls and metformin-treated animals were probed for NF-κB proteins (IKKα, IκB-β, p65, and phospho-p65). Actin served as protein loading control. (B) Day 9 paw sections were probed for ubiquitin (green) and p62 (red). Colocalization (arrowheads, yellow) suggests ubiquinated-p62 aggregates. (C) Equivalent amounts of protein were immunoprecipitated with anti-ubiquitin antibody and probed with anti-IKKα antibody. High molecular weight protein complexes likely represent poly-ubiquinated IKKα. (D) Paw sections were also probed for IKKα and LAMP-1. Accumulation of IKKα was evident in the saline controls while IKKα level was significantly lower in metformin-treated animals. Colocalization of IKKα and LAMP-1 (arrows) in metformin treatment suggests IKKα targeted to the lysosomes.
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References

    1. McInnes IB, Schett G. Cytokines in the pathogenesis of rheumatoid arthritis. Nat Rev Immunol. 2007;7:429–42. - PubMed
    1. Zhang K, Shen X, Wu J, Sakaki K, Saunders T, Rutkowski DT, et al. Endoplasmic reticulum stress activates cleavage of CREBH to induce a systemic inflammatory response. Cell. 2006;124:587–99. - PubMed
    1. Zhang K, Kaufman RJ. From endoplasmic-reticulum stress to the inflammatory response. Nature. 2008;454:455–62. - PMC - PubMed
    1. Schmid D, Munz C. Innate and adaptive immunity through autophagy. Immunity. 2007;27:11–21. - PMC - PubMed
    1. Munz C. Enhancing immunity through autophagy. Annu Rev Immunol. 2009;27:423–49. - PubMed

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