Neutrophil extracellular traps mediate bone erosion in rheumatoid arthritis by enhancing RANKL-induced osteoclastogenesis.

BACKGROUND AND PURPOSE: Rheumatoid arthritis (RA) is a chronic autoimmune disease that can cause bone erosion due to increased osteoclastogenesis. Neutrophils involvement in osteoclastogenesis remains uncertain. Given that neutrophil extracellular traps (NETs) can act as inflammatory mediators in rheumatoid arthritis, we investigated the role of NETs in stimulating bone loss by potentiating osteoclastogenesis during arthritis. EXPERIMENTAL APPROACH: The level of NETs in synovial fluid from arthritis patients was assessed. Bone loss was evaluated by histology and micro-CT in antigen-induced arthritis (AIA)-induced WT mice treated with DNase or in Padi4-deficient mice (Padi4flox/flox LysMCRE ). The size and function of osteoclasts and the levels of RANKL and osteoprotegerin (OPG) released by osteoblasts that were incubated with NETs were measured. The expression of osteoclastogenic marker genes and protein levels were evaluated by qPCR and western blotting. To assess the participation of TLR4 and TLR9 in osteoclastogenesis, cells from Tlr4-/- and Tlr9-/- mice were cultured with NETs. KEY RESULTS: Rheumatoid arthritis patients had higher levels of NETs in synovial fluid than osteoarthritis patients, which correlated with increased levels of RANKL/OPG. Moreover, patients with bone erosion had higher levels of NETs. Inhibiting NETs with DNase or Padi4 deletion alleviated bone loss in arthritic mice. Consistently, NETs enhanced RANKL-induced osteoclastogenesis that was dependent on TLR4 and TLR9 and increased osteoclast resorptive functions in vitro. In addition, NETs stimulated the release of RANKL and inhibited osteoprotegerin in osteoblasts, favouring osteoclastogenesis. CONCLUSIONS AND IMPLICATIONS: Inhibiting NETs could be an alternative strategy to reduce bone erosion in arthritis patients.

The metabolic function of pyruvate kinase M2 regulates reactive oxygen species production and microbial killing by neutrophils.

Neutrophils rely predominantly on glycolytic metabolism for their biological functions, including reactive oxygen species (ROS) production. Although pyruvate kinase M2 (PKM2) is a glycolytic enzyme known to be involved in metabolic reprogramming and gene transcription in many immune cell types, its role in neutrophils remains poorly understood. Here, we report that PKM2 regulates ROS production and microbial killing by neutrophils. Zymosan-activated neutrophils showed increased cytoplasmic expression of PKM2. Pharmacological inhibition or genetic deficiency of PKM2 in neutrophils reduced ROS production and Staphylococcus aureus killing in vitro. In addition, this also resulted in phosphoenolpyruvate (PEP) accumulation and decreased dihydroxyacetone phosphate (DHAP) production, which is required for de novo synthesis of diacylglycerol (DAG) from glycolysis. In vivo, PKM2 deficiency in myeloid cells impaired the control of infection with Staphylococcus aureus. Our results fill the gap in the current knowledge of the importance of lower glycolysis for ROS production in neutrophils, highlighting the role of PKM2 in regulating the DHAP and DAG synthesis to promote ROS production in neutrophils.

STING is an intrinsic checkpoint inhibitor that restrains the TH17 cell pathogenic program.

External and intrinsic factors regulate the transcriptional profile of T helper 17 (TH17) cells, thereby affecting their pathogenic potential and revealing their context-dependent plasticity. The stimulator of interferon genes (STING), a component of the intracellular DNA-sensing pathway, triggers immune responses but remains largely unexplored in T cells. Here, we describe an intrinsic role of STING in limiting the TH17 cell pathogenic program. We demonstrate that non-pathogenic TH17 cells express higher levels of STING than those activated under pathogenic conditions. Activation of STING induces interleukin-10 (IL-10) production in TH17 cells, decreasing IL-17A and IL-23R expression in a type I interferon (IFN)-independent manner. Mechanistically, STING-induced IL-10 production partially requires aryl hydrocarbon receptor (AhR) signaling, while the decrease of IL-17A expression occurs due to a reduction of Rorγt transcriptional activity. Our findings reveal a regulatory function of STING in the TH17 cell activation program, proposing it as a valuable target to limit TH17-cell-mediated inflammation.

Kallikrein 5 Inhibition by the Lympho-Epithelial Kazal-Type Related Inhibitor Hinders Matriptase-Dependent Carcinogenesis.

Head and neck squamous cell carcinoma remains challenging to treat with no improvement in survival rates over the past 50 years. Thus, there is an urgent need to discover more reliable therapeutic targets and biomarkers for HNSCC. Matriptase, a type-II transmembrane serine protease, induces malignant transformation in epithelial stem cells through proteolytic activation of pro-HGF and PAR-2, triggering PI3K-AKT-mTOR and NFKB signaling. The serine protease inhibitor lympho-epithelial Kazal-type-related inhibitor (LEKTI) inhibits the matriptase-driven proteolytic pathway, directly blocking kallikreins in epithelial differentiation. Hence, we hypothesized LEKTI could inhibit matriptase-dependent squamous cell carcinogenesis, thus implicating kallikreins in this process. Double-transgenic mice with simultaneous expression of matriptase and LEKTI under the keratin-5 promoter showed a prominent rescue of K5-Matriptase+/0 premalignant phenotype. Notably, in DMBA-induced SCC, heterotopic co-expression of LEKTI and matriptase delayed matriptase-driven tumor incidence and progression. Co-expression of LEKTI reverted altered Kallikrein-5 expression observed in the skin of K5-Matriptase+/0 mice, indicating that matriptase-dependent proteolytic pathway inhibition by LEKTI occurs through kallikreins. Moreover, we showed that Kallikrein-5 is necessary for PAR-2-mediated IL-8 release, YAP1-TAZ/TEAD activation, and matriptase-mediated oral squamous cell carcinoma migration. Collectively, our data identify a third signaling pathway for matriptase-dependent carcinogenesis in vivo. These findings are critical for the identification of more reliable biomarkers and effective therapeutic targets in Head and Neck cancer.

PKM2 promotes Th17 cell differentiation and autoimmune inflammation by fine-tuning STAT3 activation.

Th17 cell differentiation and pathogenicity depend on metabolic reprogramming inducing shifts toward glycolysis. Here, we show that the pyruvate kinase M2 (PKM2), a glycolytic enzyme required for cancer cell proliferation and tumor progression, is a key factor mediating Th17 cell differentiation and autoimmune inflammation. We found that PKM2 is highly expressed throughout the differentiation of Th17 cells in vitro and during experimental autoimmune encephalomyelitis (EAE) development. Strikingly, PKM2 is not required for the metabolic reprogramming and proliferative capacity of Th17 cells. However, T cell-specific PKM2 deletion impairs Th17 cell differentiation and ameliorates symptoms of EAE by decreasing Th17 cell-mediated inflammation and demyelination. Mechanistically, PKM2 translocates into the nucleus and interacts with STAT3, enhancing its activation and thereby increasing Th17 cell differentiation. Thus, PKM2 acts as a critical nonmetabolic regulator that fine-tunes Th17 cell differentiation and function in autoimmune-mediated inflammation.