Transferrin receptor 2 deficiency promotes macrophage polarization and inflammatory arthritis.

OBJECTIVE: Rheumatoid arthritis is an inflammatory joint disease in which synovial iron deposition has been described. Transferrin receptor 2 (Tfr2) represents a critical regulator of systemic iron levels. Loss of Tfr2 function in humans and mice results in iron overload. As iron contributes to inflammatory processes, we investigated whether Tfr2-deletion affects the pathogenesis of inflammatory arthritis in an iron-dependent manner. METHODS: Using a global and conditional genetic disruption of Tfr2, we assessed the relevance of Tfr2 in K/BxN serum-transfer arthritis (STA) and macrophage polarization. RESULTS: Male Tfr2-/- mice subjected to STA developed pronounced joint swelling, and bone erosion as compared to Tfr2+/+ littermate-controls (P < 0.01). Furthermore, an increase of neutrophils and macrophages/monocytes was observed in the inflammatory infiltrate within the paws of Tfr2-/- mice. To elucidate whether Tfr2 in myeloid cells has a direct role in the pathogenesis of arthritis or whether the effects were mediated via the systemic iron overload, we induced STA in Tfr2fl/fl-LysMCre + mice, which showed normal iron-loading. Cre + female mice displayed increased disease development compared to Cre-controls. As macrophages regulate iron availability and innate immunity, we hypothesized that Tfr2-deficiency would polarize macrophages toward a pro-inflammatory state (M1) that contributes to arthritis progression. In response to IFN-γ stimulation, Tfr2-/- macrophages showed increased expression of M1-like cytokines, IFN-γ-target genes, nitric-oxide production, and prolonged STAT1 activation compared to Tfr2+/+ macrophages (P < 0.01), while pre-treatment with ruxolitinib abolished Tfr2-driven M1-like polarization. CONCLUSION: Taken together, these findings suggest a protective role of Tfr2 in macrophages on the progression of arthritis via suppression of M1-like polarization.

Prolactin blocks the expression of receptor activator of nuclear factor κB ligand and reduces osteoclastogenesis and bone loss in murine inflammatory arthritis.

BACKGROUND: Prolactin (PRL) reduces joint inflammation, pannus formation, and bone destruction in rats with polyarticular adjuvant-induced arthritis (AIA). Here, we investigate the mechanism of PRL protection against bone loss in AIA and in monoarticular AIA (MAIA). METHODS: Joint inflammation, trabecular bone loss, and osteoclastogenesis were evaluated in rats with AIA treated with PRL (via osmotic minipumps) and in mice with MAIA that were null (Prlr-/-) or not (Prlr+/+) for the PRL receptor. To help define target cells, synovial fibroblasts from Prlr+/+ mice were treated or not with proinflammatory cytokines ((Cyt), including TNFα, IL-1ß, and interferon (IFN)γ) with or without PRL, and these synovial cells were co-cultured or not with bone marrow osteoclast progenitors from Prlr+/+ or Prlr-/- mice. RESULTS: In AIA, PRL treatment reduced joint swelling, increased trabecular bone area, lowered osteoclast density, and reduced mRNA levels of osteoclast-associated genes (tartrate-resistant acid phosphatase (Trap)), cathepsin K (Ctsk), matrix metalloproteinase 9 (Mmp9), and receptor activator of nuclear factor κB or RANK (Tnfrsf11a)), of genes encoding cytokines with osteoclastogenic activity (Tnfa, Il1b, Il6, and receptor activator of nuclear factor κB ligand or RANKL (Tnfrsf11)), and of genes encoding for transcription factors and cytokines related to T helper (Th)17 cells (Rora, Rorc, Il17a, Il21, Il22) and to regulatory T cells (Foxp3, Ebi3, Il12a, Tgfb1, Il10). Prlr-/- mice with MAIA showed enhanced joint swelling, reduced trabecular bone area, increased osteoclast density, and elevated expression of Tnfa, Il1b, Il6, Trap, Tnfrsf11a, Tnfrsf11, Il17a, Il21, Il22, 1 l23, Foxp3, and Il10. The expression of the long PRL receptor form increased in arthritic joints, and in synovial membranes and cultured synovial fibroblasts treated with Cyt. PRL induced the phosphorylation/activation of signal transducer and activator of transcription-3 (STAT3) and inhibited the Cyt-induced expression of Il1b, Il6, and Tnfrsf11 in synovial fibroblast cultures. The STAT3 inhibitor S31-201 blocked inhibition of Tnfrsf11 by PRL. Finally, PRL acted on both synovial fibroblasts and osteoclast precursor cells to downregulate Cyt-induced osteoclast differentiation. CONCLUSION: PRL protects against osteoclastogenesis and bone loss in inflammatory arthritis by inhibiting cytokine-induced expression of RANKL in joints and synovial fibroblasts via its canonical STAT3 signaling pathway.

Prolactin-derived vasoinhibins increase anxiety- and depression-related behaviors.

The hormone prolactin (PRL) regulates neuroendocrine and emotional stress responses. It is found in the hypothalamus, where the protein is partially cleaved to vasoinhibins, a family of N-terminal antiangiogenic PRL fragments ranging from 14 to 18kDa molecular masses, with unknown effects on the stress response. Here, we show that the intracerebroventricular administration of a recombinant vasoinhibin, containing the first 123 amino acids of human PRL that correspond to a 14kDa PRL, exerts anxiogenic and depressive-like effects detected in the elevated plus-maze, the open field, and the forced swimming tests. To investigate whether stressor exposure affects the generation of vasoinhibins in the hypothalamus, the concentrations of PRL mRNA, PRL, and vasoinhibins were evaluated in hypothalamic extracts of virgin female rats immobilized for 30min at different time points after stress onset. The hypothalamic levels of PRL mRNA and protein were higher at 60min but declined at 360min to levels seen in non-stressed animals. The elevation of hypothalamic PRL did not correlate with the stress-induced increase in circulating PRL levels, nor was it modified by blocking adenohypophyseal PRL secretion with bromocriptine. A vasoinhibin having an electrophoretic migration rate corresponding to 17kDa was detected in the hypothalamus. Despite the elevation in hypothalamic PRL, the levels of this hypothalamic vasoinhibin were similar in stressed and non-stressed rats. Stress reduced the rate of cleavage of PRL to this vasoinhibin as shown by the incubation of recombinant PRL with hypothalamic extracts from stressed rats. These results suggest that vasoinhibins are potent anxiogenic and depressive factors and that stress increases PRL levels in the hypothalamus partly by reducing its conversion to vasoinhibins. The reciprocal interplay between PRL and vasoinhibins may represent an effective mechanism to regulate anxiety and depression.