Sijian Wang

Objective: To determine the critical roles of PU.1/cathepsin S activation in regulating inflammatory responses of macrophages during periodontitis.Background: Cathepsin S (CatS) is a cysteine protease and exerts important roles in the immune response. Elevated CatS has been found in the gingival tissues of periodontitis patients and is involved in alveolar bone destruction. However, the underlying mechanism of CatS‐driven IL‐6 production in periodontitis remains unclear.Methods: Western blot was applied to measure mature cathepsin S(mCatS) and IL‐6 expression in gingival tissues from periodontitis patients and RAW264.7 cells exposed to lipopolysaccharide from Porphyromonas gingivalis (P.g. LPS). Immunofluorescence was applied to confirm the localization of PU.1, and CatS in the gingival tissues of periodontitis patients. ELISA was performed to determine IL‐6 production by the P.g. LPS‐exposed RAW264.7 cells. Knockdown by shRNA was used to determine the effects of PU.1 on p38/ nuclear factor (NF)‐κB activation, mCatS expression and IL‐6 production in RAW264.7 cells.Results: The expressions mCatS and IL‐6 were significantly upregulated in gingival macrophages. In cultured RAW264.7 cells, increased mCatS and IL‐6 protein paralleled the activation of p38 and NF‐κB after exposure to P.g. LPS. CatS knockdown by shRNA significantly decreased P.g. LPS‐induced IL‐6 expression and p38/NF‐κB activation. PU.1 was significantly increased in P.g. LPS‐exposed RAW264.7 cells, and PU.1 knockdown dramatically abolished the P.g. LPS‐induced upregulation of mCatS and IL‐6 and the activation of p38 and NF‐κB. Furthermore, PU.1 and CatS colocalized in macrophages within the gingival tissues of periodontitis patients.Conclusion: PU.1‐dependent CatS drives IL‐6 production in macrophages by activating p38 and NF‐κB in periodontitis.

Despite a complex cascade of cellular events to reconstruct the damaged extracellular matrix, ligament healing results in a mechanically inferior scarred ligament. During normal healing, granulation tissue expands into any residual normal ligamentous tissue (creeping substitution), resulting in a larger region of healing, greater mechanical compromise and an inefficient repair process. To control creeping substitution and possibly enhance the repair process, the antiinflammatory cytokine, interleukin‐4 (IL‐4), was administered to rats before and after rupture of their medial collateral ligaments. In vitro experiments showed a time‐dependent effect on fibroblast proliferation after IL‐4 treatment. In vivo treatments with IL‐4 (100 ng/mL IV) for 5 days resulted in decreased wound size and type III collagen and increased type I procollagen, indicating a more regenerative early healing in response to the IL‐4 treatment. However, continued treatment of IL‐4 to day 11 antagonized this early benefit and slowed healing. Together, these results suggest that IL‐4 not only influences the macrophages and T lymphocytes but also stimulates fibroblasts associated with the proliferative phase of healing in a dose‐, cell‐, and time‐dependent manner. Although treatment significantly influenced healing in the first week after injury, IL‐4 alone was unable to maintain this early regenerative response.