Ex vivo and in vitro production of pro-inflammatory cytokines in Blau syndrome

Abstract

The objective was to study both ex vivo and in vitro secretion of pro-inflammatory cytokines in patients affected by Blau syndrome (BS) and carrying p.E383K mutation in the CARD15/NOD2 gene associated with the disease. For ex vivo studies, peripheral blood mononuclear cells (PBMCs), serum from three patients and healthy controls have been collected. PBMCs have been cultured in the presence or absence of inflammatory enhancers, such as lipopolysaccharide (LPS) and muramyl dipeptide (MDP). The levels of interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor (TNF)-α and interferon (IFN)-γ were assayed by either immunoassay or array-based system. For in vitro studies, different constructs were created cloning human wild-type and p.E383K-mutated NOD2 cDNA into the expression vector pCMV-Tag2c. HEK293 cell lines were stably transfected, cultured with or without MDP and IL-8 level was assayed in their surnatants. Statistical analysis in both studies was performed using non-parametric tests. Both ex vivo and in vitro studies have not identified a significant increase in secretion of the analyzed proinflammatory cytokines. p.E383K-mutated NOD2 transfected cells express low level of IL-8. The ex vivo basal level results from both serum and PBMCs surnatants present similar levels of IL-1β, IL-6, TNF-α and IFN-γ in patients and controls. The presence of the stimulant agents (LPS and MDP), either individual or paired, does not lead to significant increases in all cytokines concentrations in patients compared to controls. Taken together, the ex vivo and in vitro data suggest that there is not a primary mediation of IL-1β and other pro-inflammatory cytokines in BS patients carrying p.E383K.

Blau Syndrome and Early-Onset Sarcoidosis: A Six Case Series and Review of the Literature

“…Although the penetrance of BS/EOS associated with NOD2 mutations is very high, asymptomatic family members with NOD2 mutations have been defined in the literature. [21][22][23] The father of the patient 5, 6 who had a V955I mutation did not have any symptoms. It is unclear whether the type and/or location of the NOD2 mutation has an effect on the clinical features.…”

“…There is no consensus on which IL pathway predominates in the literature. Galozzi et al 21 claimed that an exaggerated NOD2 response leading to pro-inflammatory release is not a direct mechanism explaining the pathophysiology of BS.…”

Persistent Tenosynovitis, Steroid Dependency and a Hyperpigmented Scaly Macular Rash in a Child With Juvenile Idiopathic Arthritis

“…After their binding, the signaling pathway of NF-kb is activated, resulting to the transcription of proinflammatory cytokines. The believed cytokines involved are mainly the IL-1β, like in other autoinflammatory diseases [16]. In a study from Arostegui et al, plasma cytokines of patients with Blau syndrome were analyzed and an increase in IL-6, TNF and a smaller increase in IL-1β was observed in patients compared to controls, which normalized after treatment with anakinra [6].…”

A novel nucleotide oligomerisation domain 2 mutation in a family with Blau syndrome: Phenotype and function

“…10 Mutations at this site have also been previously described in patients with BS/EOS. [11][12][13][14][15] In vitro studies in cell lines transfected with NBD mutations identified in patients with BS and EOS have reported increased activity of NF-kB in the absence of stimulation, [1][2][3][4] suggesting constitutive NOD2 activation may underlie the pathogenesis of these diseases. Our results, however, do not support this theory.…”