BACKGROUND: Macrophages are involved in the pathogenesis of pulmonary arterial hypertension (PAH). Caspase-8 (FLICE), an apical component of cell death pathways, is significantly upregulated in macrophages of PAH animal models. However, its role in PAH remains unclear. Caspase-8 plays a critical role in regulating inflammatory responses via inflammasome activation, cell death, and cytokine induction. This study investigated the mechanism of regulation of IL-1β (interleukin 1β) activation in macrophages by caspase-8. METHODS: A hypoxia + SU5416-induced PAH mouse model and monocrotaline-induced rat model of PAH were constructed and the role of caspase-8 was analyzed. RESULTS: Caspase-8 and cleaved-caspase-8 were significantly upregulated in the lung tissues of SU5416 and hypoxia-treated PAH mice and monocrotaline-treated rats. Pharmacological inhibition of caspase-8 alleviated PAH compared with wild-type mice, observed as a significant reduction in right ventricular systolic pressure, ratio of right ventricular wall to left ventricular wall plus ventricular septum, pulmonary vascular media thickness, and pulmonary vascular muscularization; caspase-8 ablated mice also showed significant remission. Mechanistically, increased proliferation of pulmonary arterial smooth muscle cellss is closely associated with activation of the NLRP3 (NOD [nucleotide oligomerization domain]-, LRR [leucine-rich repeat]-, and PYD [pyrin domain]-containing protein 3) inflammasome and the IL-1β signaling pathway. Although caspase-8 did not affect extracellular matrix synthesis, it promoted inflammatory cell infiltration and pulmonary arterial smooth muscle cell proliferation via NLRP3/IL-1β activation during the development stage of PAH. CONCLUSIONS: Taken together, our study suggests that macrophage-derived IL-1β via caspase-8-dependent canonical inflammasome is required for macrophages to play a pathogenic role in pulmonary perivascular inflammation.

Caspase-8 is an initiator of death receptor-induced apoptosis and an inhibitor of RIPK3-MLKL-dependent necroptosis. In addition, caspase-8 has been implicated in diseases such as lymphoproliferation, immunodeficiency, and autoimmunity in humans. Although auto-cleavage is indispensable for caspase-8 activation, its physiological functions remain poorly understood. Here, we generated a caspase-8 mutant lacking E385 in auto-cleavage site knock-in mouse (Casp8ΔE385/ΔE385). Casp8ΔE385/ΔE385 cells were expectedly resistant to Fas-induced apoptosis, however, Casp8ΔE385/ΔE385 cells could switch TNF-α-induced apoptosis to necroptosis by attenuating RIPK1 cleavage. More importantly, CASP8(ΔE385) sensitized cells to RIPK3-MLKL-dependent necroptosis through promoting complex II formation and RIPK1-RIPK3 activation. Notably, Casp8ΔE385/ΔE385Ripk3−/− mice partially rescued the perinatal death of Ripk1−/− mice by blocking apoptosis and necroptosis. In contrast to the Casp8−/−Ripk3−/− and Casp8−/−Mlkl−/− mice appearing autoimmune lymphoproliferative syndrome (ALPS), both Casp8ΔE385/ΔE385Ripk3−/− and Casp8ΔE385/ΔE385Mlkl−/− mice developed transplantable lymphopenia that could be significantly reversed by RIPK1 heterozygosity, but not by RIPK1 kinase dead mutation. Collectively, these results demonstrate previously unappreciated roles for caspase-8 auto-cleavage in regulating necroptosis and maintaining lymphocytes homeostasis.

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