Iron overload promotes hemochromatosis-associated osteoarthritis via the mTORC1-p70S6K/4E-BP1 pathway

Joint iron overload in hemochromatosis induces M1 polarization in synovial macrophages, releasing pro-inflammatory factors and leading to osteoarthritis development. However, the mechanism by which iron overload regulates M1 polarization remains unclear. This study aims to elucidate the mechanism by which synovial iron overload promotes macrophage M1 polarization.

In vitro, RAW264.7 macrophages were treated with iron and divided into five groups based on the concentration of the iron chelator, desferrioxamine (DFO): Ctrl, Fe, DFO1, DFO2, and DFO3. In vivo, rats were categorized into five groups based on iron overload and intra-articular DFO injection: A-Ctrl, A-Fe, A-DFO1, A-DFO2, and A-DFO3. Osteoarthritis was induced by transecting the left knee anterior cruciate ligament. Macrophage morphology was observed; Prussian Blue staining quantified iron deposition in macrophages, synovium, and liver; serum iron concentration was measured using the ferrozine method; cartilage damage was assessed using H&E and Safranin O-Fast Green staining; qPCR detected iNOS and Arg-1 expression; Western Blot analyzed the protein expression of iNOS, Arg-1, 4E-BP1, phosphorylated 4E-BP1, p70S6K, and phosphorylated p70S6K; ELISA measured TNF-α and IL-6 concentrations in supernatants; and immunohistochemistry examined the protein expression of F4/80, iNOS, Arg-1, 4E-BP1, phosphorylated 4E-BP1, p70S6K, and phosphorylated p70S6K in the synovium.

In vitro, iron-treated macrophages exhibited Prussian Blue staining indicative of iron overload and morphological changes towards M1 polarization. qPCR and Western Blot revealed increased expression of the M1 polarization markers iNOS and its protein. ELISA showed elevated TNF-α and IL-6 levels in supernatants. In vivo, ferrozine assay indicated significantly increased serum iron concentrations in all groups except A-Ctrl; Prussian Blue staining showed increased liver iron deposition in all groups except A-Ctrl. Iron deposition in rat synovium decreased in a DFO concentration-dependent manner; immunohistochemistry showed a corresponding decrease in iNOS and phosphorylated 4E-BP1 expression, and an increase in Arg-1 expression.

Intracellular iron overload may exacerbate joint cartilage damage by promoting synovial macrophage M1 polarization through phosphorylation of 4E-BP1 in the mTORC1-p70S6K/4E-BP1 pathway.