Pyrin Inflammasome Activation Abrogates Interleukin-1 Receptor Antagonist, Suggesting a New Mechanism Underlying Familial Mediterranean Fever Pathogenesis.

OBJECTIVE: Aberrant pyrin inflammasome activity triggers familial Mediterranean fever (FMF) pathogenesis, but the exact mechanism remains elusive and an obstacle to efficient treatment. We undertook this study to identify pyrin inflammasome-specific mechanisms to improve FMF treatment and diagnostics in the future. METHODS: Pyrin-specific protein secretion was assessed by proteome analysis in U937-derived macrophages, and specific findings were confirmed in pyrin inflammasome-activated monocytes from healthy blood donors and patients with FMF, stratified according to MEFV genotype categories corresponding to a suspected increase in FMF disease severity. RESULTS: Proteome data revealed a differential secretion pattern of interleukin-1 receptor antagonist (IL-1Ra) from pyrin- and NLRP3-activated U937-derived macrophages, which was verified by enzyme-linked immunosorbent assay and quantitative polymerase chain reaction. Moreover, pyrin activation significantly reduced IL1RN messenger RNA expression (P < 0.001) and IL-1Ra secretion (P < 0.01) in healthy donor and FMF monocytes, respectively. Independent of MEFV genotype, unstimulated FMF monocytes from colchicine-treated patients secreted lower amounts of IL-1Ra compared to healthy donors (P < 0.05) and displayed decreased ratios of IL-1Ra:IL-1ß (P < 0.05), suggesting a reduced antiinflammatory capacity. CONCLUSION: Our data show an inherent lack of IL-1Ra expression specific to pyrin inflammasome activation, suggesting a new mechanism underlying FMF pathogenesis. The reduced IL-1Ra levels in FMF monocytes suggest a diminished antiinflammatory capacity that potentially leaves FMF patients sensitive to proinflammatory stimuli, regardless of receiving colchicine therapy. Thus, considering the potential clinical consequence of reduced monocyte IL-1Ra secretion in FMF patients, we suggest further investigation into IL-1Ra dynamics and its potential implications for FMF treatment in the future.

Evidence of non-canonical NOTCH signaling: Delta-like 1 homolog (DLK1) directly interacts with the NOTCH1 receptor in mammals.

Canonical NOTCH signaling, known to be essential for tissue development, requires the Delta-Serrate-LAG2 (DSL) domain for NOTCH to interact with its ligand. However, despite lacking DSL, Delta-like 1 homolog (DLK1), a protein that plays a significant role in mammalian development, has been suggested to interact with NOTCH1 and act as an antagonist. This non-canonical interaction is, however controversial, and evidence for a direct interaction, still lacking in mammals. In this study, we elucidated the putative DLK1-NOTCH1 interaction in a mammalian context. Taking a global approach and using Dlk1(+/+) and Dlk1(-/-) mouse tissues at E16.5, we demonstrated that several NOTCH signaling pathways indeed are affected by DLK1 during tissue development, and this was supported by a lower activation of NOTCH1 protein in Dlk1(+/+) embryos. Likewise, but using a distinct Dlk1-manipulated (siRNA) setup in a mammalian cell line, NOTCH signaling was substantially inhibited by DLK1. Using a mammalian two-hybrid system, we firmly established that the effect of DLK1 on NOTCH signaling was due to a direct interaction between DLK1 and NOTCH1. By careful dissection of this mechanism, we found this interaction to occur between EGF domains 5 and 6 of DLK1 and EGF domains 10-15 of NOTCH1. Thus, our data provide the first evidence for a direct interaction between DLK1 and NOTCH1 in mammals, and substantiate that non-canonical NOTCH ligands exist, adding to the complexity of NOTCH signaling.

Membrane-tethered delta-like 1 homolog (DLK1) restricts adipose tissue size by inhibiting preadipocyte proliferation.

Adipocyte renewal from preadipocytes has been shown to occur throughout life and to contribute to obesity, yet very little is known about the molecular circuits that control preadipocyte expansion. The soluble form of the preadipocyte factor (also known as pref-1) delta-like 1 homolog (DLK1(S)) is known to inhibit adipogenic differentiation; however, the impact of DLK1 isoforms on preadipocyte proliferation remains to be determined. We generated preadipocytes with different levels of DLK1 and examined differentially affected gene pathways, which were functionally tested in vitro and confirmed in vivo. Here, we demonstrate for the first time that only membrane-bound DLK1 (DLK1(M)) exhibits a substantial repression effect on preadipocyte proliferation. Thus, by independently manipulating DLK1 isoform levels, we established that DLK1(M) inhibits G1-to-S-phase cell cycle progression and thereby strongly inhibits preadipocyte proliferation in vitro. Adult DLK1-null mice exhibit higher fat amounts than wild-type controls, and our in vivo analysis demonstrates that this may be explained by a marked increase in preadipocyte replication. Together, these data imply a major dual inhibitory function of DLK1 on adipogenesis, which places DLK1 as a master regulator of preadipocyte homeostasis, suggesting that DLK1 manipulation may open new avenues in obesity treatment.