While interleukin (IL)-1β is a well-established driver of periodontitis, the specific mechanisms by which the emerging pathogen Filifactor alocis induces this cytokine are largely unknown. We conducted a comprehensive investigation into how F. alocis stimulates IL-1β in THP-1 macrophages, analyzing the response at the mRNA (reverse transcription-polymerase chain reaction), cellular protein (Western blot), and secreted protein (enzymelinked immunosorbent assay) levels. Our results show a clear, dose-dependent increase in all measures, confirming its potent effect. To explore the underlying mechanisms, we first tested the contribution of the NLRP3 inflammasome using the specific inhibitor MCC950. This inhibitor almost completely blocked (~90%) a control response but only partially inhibited (~50%) the F. alocis -stimulated secretion. A similar result was observed with the pan-caspase inhibitor Z-VAD-FMK, which almost completely suppressed the control but only partially inhibited (~50%) the response to F. alocis . Moreover, we found a dominant role for mitogen-activated protein kinase signaling pathways. Specific inhibitors of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) significantly attenuated both IL-1β mRNA expression and protein secretion, whereas the p38 inhibitor had no effect. These findings collectively demonstrate that F. alocis robustly induces IL-1β via ERK and JNK signaling with only partial dependence on canonical NLRP3 inflammasome and caspase-mediated pathways. This unique pattern suggests the involvement of other complementary pathways, underscoring the complex inflammatory processes involved in F. alocis -induced periodontitis.

Introduction
Materials and Methods
    1. Reagents
    2. Bacterial culture
    3. THP-1 culture
    4. Infection protocol
    5. RT-PCR
    6. ELISA
    7. Western blot analysis
    8. Statistical analysis
Results
    1. IL-1β induction by F. alocis
    2. NLRP3 inflammasome dependency
    3. Caspase dependency of IL-1β secretion
    4. Involvement of MAP kinases
Discussion
Funding
Conflicts of Interest
References

• So-Hee Kim(Department of Oral Microbiology, School of Dentistry, Chonnam National University, Gwangju 61186, Republic of Korea)
• Jin Chung(Department of Oral Microbiology, School of Dentistry, Chonnam National University, Gwangju 61186, Republic of Korea)
• In-Chol Kang(Department of Oral Microbiology, School of Dentistry, Chonnam National University, Gwangju 61186, Republic of Korea) Corresponding author