Background: Increasing evidence implicates cell death pathways linked to IL-1β are more complex [1, 2]. These cell death regulatory mechanisms have not yet been systematically explored in Familial Mediterranean Fever (FMF) [3].

Objectives: This study investigated how cell death is regulated in human primary monocytes from healthy donors and FMF patients.

Methods: Monocytes were isolated from PBMC’s with MACS using negative selection from 10 healthy donors and 11 FMF patients. Monocytes were incubated at 37⁰C, 5% CO ₂ with 1 hour of medium, RIPK1 inhibitor (necrostatin-1 (nec-1)) (20 uM), caspase-1/4 inhibitor VX-765 (20 uM), or RIPK3 inhibitor GSK872 (25 uM), followed by 16 hours with caspase-8 inhibitor Z-IETD-FMK (10 uM), LPS (& nigericin) (5 ng/mL; 6.7 uM), TNFα & IFNγ (50 ng/mL; 50 ng/mL), or staurosporine (10 uM). Cell death was visualized with live cell imaging. Supernatants were collected and ELISA for IL-1β, IL-6 and TNFα was performed.

Results: In human primary monocytes, staurosporine, and the combination of LPS/nigericin induced cell death, while LPS alone and the combination of TNFα/IFNγ did not. Z-IETD-FMK alone induced cell death, that could be inhibited by nec-1. In contrast, nec-1 increased cell death induced by LPS/nigericin and staurosporine. VX-765 did not have any effect on cell death. GSK872 did not affect staurosporine-induced cell death, but slightly induced cell death by LPS/nigericin. Similar to nec-1, GSK872 showed to inhibit lytic cell death induced by Z-IETD-FMK. We observed an increase in IL-1β production when monocytes were stimulated with LPS, LPS/nigericin, and Z-IETD-FMK. In contrast to having no effect on cell death induced by LPS/nigericin, VX-765 significantly inhibited IL-1β production, and showed a trend toward inhibition of Z-IETD-FMK-induced IL-1β. Nec-1 and GSK872, however, inhibited IL-1β production by Z-IETD-FMK, as well as LPS/nigericin. While IL-6 followed the same trend as IL-1 β, TNFα production was only induced by LPS and the combination LPS/nigericin. TNFα production was not dependent on nec-1, but showed a reduction in the presence of VX-765 and GSK872 in monocytes from healthy donors. When compared to monocytes from healthy donors, monocytes from FMF patients showed overall less cell death sensitivity. Nec-1 and GSK872 affected cell death in FMF monocytes in the same way as in healthy monocytes. However, VX-765 increased cell death induced by Z-IETD-FMK in FMF monocytes, contrary to healthy monocytes. Whereas VX-765 showed a trend towards a decrease in Z-IETD-FMK-induced IL-1β by healthy monocytes, we observed a trend towards increased IL-1β production in FMF monocytes. Another notable difference was that GSK872 enhanced IL-6 production in FMF monocytes stimulated with LPS/nigericin, whereas this had no effect on healthy monocytes. In FMF monocytes we observed that the TNFα production induced by LPS/nigericin was dependent on caspase-1/4 inhibitor VX-765.

Conclusion: We were able to show that the classically known cell death pathways pyroptosis, apoptosis and necroptosis affect each other in human primary monocytes, thereby showing that cell death in human monocytes is a more complex network than previously described. Strikingly, caspase-8 inhibition alone in the absence of any other stimulus induced cell death and production of IL-1β in human primary monocytes, which was at least dependent on RIPK1 and RIPK3. Cell death induced by caspase-8 was even further increased in the presence of caspase-1/-4 inhibition, which in parallel showed a trend towards increased IL-1β production. In FMF patients, we found dysregulated cell death regulatory mechanisms and IL-1β production, which suggest novel autoinflammatory mechanisms that could help explain pathology in in FMF.

REFERENCES: [1] Yaglikara E, Boluk O, Bayindir Y, Bilginer Y, Tasar MA, Ozen S, et al. The Potential Role of Cell-Death Mechanisms in the Pathogenesis of Familial Mediterranean Fever Attacks: Granzyme A and Beyond. Diagnostics (Basel). 2024;14(18).

[2] Tweedell RE, Hibler T, Kanneganti T-D. Defining PANoptosis: Biochemical and Mechanistic Evaluation of Innate Immune Cell Death Activation. Current Protocols. 2024;4(7):e1112.

[3] Gullett JM, Tweedell RE, Kanneganti TD. It’s All in the PAN: Crosstalk, Plasticity, Redundancies, Switches, and Interconnectedness Encompassed by PANoptosis Underlying the Totality of Cell Death-Associated Biological Effects. Cells. 2022;11(9).

Acknowledgements: NIL.

Disclosure of Interests: None declared.

© The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ). Neither EULAR nor the publisher make any representation as to the accuracy of the content. The authors are solely responsible for the content in their abstract including accuracy of the facts, statements, results, conclusion, citing resources etc.