Comunicación

FUNCTIONAL STUDY OF A LATE-ONSET AUTOINFLAMMATORY DISEASE DUE TO SOMATIC NLRC4 MOSAICISM

Autores:

ALEJANDRO ELEAZAR PEÑIN FRANCH1, LAURA HURTADO NAVARRO1, MARÍA CRISTINA MOLINA LÓPEZ1, Daniela Ionescu2, Luis Miguel Fernández Pereira3, Anna Mensa Vilaró4, Juan Ignacio Arostegui4, PABLO PELEGRIN VIVANCOS1

Afiliaciones:

(1) CIRUGÍA DIGESTIVA, ENDOCRINA Y TRASPLANTE DE ÓRGANOS ABDOMINALES, IMIB-Arrixaca, España
(2) Departamento de Inmunología y genética molecular, Hospital San Pedro de Alcántara, Cáceres, España (Extremadura)
(3) Departamento de inmunología y genética molecular, Hospital San Pedro de Alcántara, Cáceres, España (Extremadura)
(4) Departamento de inmunología, Hospital Clinic, Barcelona, España (Cataluña)

Comunicación:

Antecedentes:

Autoinflammatory diseases (AIDs) are inherited disorders of innate immunity that usually start during childhood. However, several studies have recently reported an increasing number of patients with AID starting in adulthood. Inflammasomes are multiprotein complexes that are present in some cells of innate immunity. Inflammasomes activation produce an inflammatory response mediated by cytokines of the interleukin (IL)-1 family, such as IL-1β and IL-18. This study was undertaken to characterize the cause underlying a patient with a somatic mosaicism due to the post-zygotic p.Ser171Phe NLRC4 variant.

Métodos:

Human peripheral blood mononuclear cells (PBMCs) were collected from a patient with p.Ser171Phe NLRC4 mosaicism, four patients with Cryopyrin-Associated Periodic Syndromes (CAPS) with germline p.Ala439Thr NLRP3 variant and control individuals (n=4-6), cultured and stimulated to activate NLRP3 and NLRC4 inflammasomes. NLRP3 activation was carried out using bacterial lipopolysaccharide (LPS) at 100ng/ml during 2h and ATP at 3mM during 30 min. NLRC4 activation was carried out using LPS, at the same conditions as for NLRP3 activation, and protective antigen (PA)+LFn-Fla mix (Flatox) at 4μg and 2μg respectively during 5h. Cell-free supernatants were collected to quantify secreted IL-1β, IL-18, IL-6 and TNF-α by ELISA. Intracellular ASC-speck formation was evaluated in monocytes by flow cytometry. Wild-type and mutant NLRC4 expression vectors were generated by overlapping PCR and expressed in HEK293T cells to quantify NLRC4 oligomerization with or without the expression of ASC.

Resultados:

In vitro studies were performed expressing NLRC4 constructs in HEK293T cells. Expression of wild-type and mutant NLRC4 were similar after transfection. Both wild-type and mutant NLRC4 showed spontaneous oligomerization, and this oligomerization co-localized with ASC when it was also transfected. Oligomerization analysis showed a higher percentage of cells with oligomers of p.Ser171Phe NLRC4 than wild-type, both in presence and absence of ASC. Ex vivo analyses stimulating blood samples of the patient with the somatic NLRC4 mosaicism with LPS showed a higher percentage of monocytes with ASC specks, with a subsequent increase in IL-18 but failed to release IL-1β when compared to controls, suggesting a higher activation of NLRC4-inflammasome that controlled IL-18, but not IL-1β release. These results were similar than when NLRC4 stimulation was performed on the sample from the NLRC4 mosaicism. TNF-α and IL-6 release did not change between control and the NLRC4 mosaicism patient.

Conclusiones:

We have identified that the post-zygotic p.Ser171Phe NLRC4 variant could be a plausible cause of the late-onset AID in the studied patient, since functional studies support the gain-of-function behaviour of the mutation similarly that in previously reported NLRC4 pathogenic variants.


Dirección

Campus de Ciencias de la Salud
Carretera Buenavista s/n, 30120 El Palmar
Murcia, España

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