Receptor activator of NF-kB ligand (RANKL) generates intracellular reactive oxygen species (ROS), which increase RANKL-mediated signaling in osteoclast (OC) precursor bone marrow macrophages (BMMs). Here we show that a ROS scavenging protein DJ-1 negatively regulates RANKL-driven OC differentiation, also called osteoclastogenesis. DJ-1 ablation in mice leads to a decreased bone volume and an increase in OC numbers. In vitro, the activation of RANK-dependent signals is enhanced in DJ-1-deficient BMMs as compared to wild-type BMMs. DJ-1 suppresses the activation of both RANK-TRAF6 and RANK-FcRγ/Syk signaling pathways because of activation of Src homology region 2 domain-containing phosphatase-1, which is inhibited by ROS. Ablation of DJ-1 in mouse models of arthritis and RANKL-induced bone disease leads to an increase in the number of OCs, and exacerbation of bone damage. Overall, our results suggest that DJ-1 plays a role in bone homeostasis in normal physiology and in bone-associated pathology by negatively regulating osteoclastogenesis.

Objective: Interleukin‐32 (IL‐32) is a recently discovered cytokine that appears to play a critical role in human rheumatoid arthritis (RA). It is highly expressed in synovium and fibroblast‐like synoviocytes (FLS) from RA patients, but not in patients with osteoarthritis (OA). This study was undertaken to assess IL‐32 levels in RA synovial fluid (SF) and to investigate the secretion and regulation of IL‐32 in RA FLS.Methods: FLS and SF were obtained from the joints of RA patients. The secretion and expression of IL‐32 and activation of signaling molecules were examined by enzyme‐linked immunosorbent assay, immunoblotting, immunoprecipitation, reverse transcriptase–polymerase chain reaction, and small interfering RNA (siRNA) transfection.Results: IL‐32 levels were high in RA SF compared with OA SF. Furthermore, RA FLS expressed and secreted IL‐32 when stimulated with tumor necrosis factor α (TNFα). TNFα‐induced expression of IL‐32 was significantly suppressed, in a dose‐dependent manner, by inhibitors of Syk, protein kinase Cδ (PKCδ), and JNK and by knockdown of these kinases and c‐Jun with siRNA. We also observed that PKCδ mediated the activation of JNK and c‐Jun, and experiments using specific inhibitors and siRNA demonstrated that Syk was the upstream kinase for the activation of PKCδ.Conclusion: The present findings suggest that IL‐32 may be a newly identified prognostic biomarker in RA, thereby adding valuable knowledge to the understanding of this disease. The results also demonstrate that the production of IL‐32 in RA FLS is regulated by Syk/PKCδ‐mediated signaling events.

Food allergy is a hypersensitive immune reaction to food proteins. We have previously demonstrated the presence of IL-10-producing CD5⁺ B cells and suggested their potential role in regulating cow’s milk casein allergy in humans and IgE-mediated anaphylaxis in mice. In this study, we determined whether IL-10-producing CD5⁺ regulatory B cells control casein-induced food allergic responses in mice and, if so, the underlying mechanisms. The induction of oral tolerance (OT) by casein suppressed casein-induced allergic responses including the decrease of body temperature, symptom score, diarrhea, recruitment of mast cells and eosinophils into jejunum and other biological parameters in mice. Notably, the population of IL-10-producing CD5⁺ B cells was increased in mesenteric lymph node (MLN), but not in spleen or peritoneal cavity (PeC) in OT mice. The adoptive transfer of CD5⁺ B cells from MLN, but not those from spleen and PeC, suppressed the casein-induced allergic responses in an allergen-specific and IL-10-dependent manner. The inhibitory effect of IL-10-producing CD5⁺ B cells on casein-induced allergic response was dependent on Foxp3⁺ regulatory T cells. Taken together, mesenteric IL-10-producing regulatory B cells control food allergy via Foxp3⁺ regulatory T cells and could potentially act as a therapeutic regulator for food allergy.

Mast cells are critical for various allergic disorders. Mast cells express Src family kinases, which relay positive and negative regulatory signals by Ag. Lyn, for example, initiates activating signaling events, but it also induces inhibitory signals. Fyn and Hck are reported to be positive regulators, but little is known about the roles of other Src kinases, including Fgr, in mast cells. In this study, we define the role of Fgr. Endogenous Fgr associates with FcεRI and promotes phosphorylation of Syk, Syk substrates, which include linkers for activation of T cells, SLP76, and Gab2, and downstream targets such as Akt and the MAPKs in Ag-stimulated mast cells. As a consequence, Fgr positively regulates degranulation, production of eicosanoids, and cytokines. Fgr and Fyn appeared to act in concert, as phosphorylation of Syk and degranulation are enhanced by overexpression of Fgr and further augmented by overexpression of Fyn but are suppressed by overexpression of Lyn. Moreover, knockdown of Fgr by small interfering RNAs (siRNAs) further suppressed degranulation in Fyn-deficient bone marrow-derived mast cells. Overexpression of Fyn or Fgr restored phosphorylation of Syk and partially restored degranulation in Fyn-deficient cells. Additionally, knockdown of Fgr by siRNAs inhibited association of Syk with FcεRIγ as well as the tyrosine phosphorylation of FcεRIγ. Of note, the injection of Fgr siRNAs diminished the protein level of Fgr in mice and simultaneously inhibited IgE-mediated anaphylaxis. In conclusion, Fgr positively regulates mast cell through activation of Syk. These findings help clarify the interplay among Src family kinases and identify Fgr as a potential therapeutic target for allergic diseases.