Background Evidence from this laboratory had indicated that the epithelia of primary Sjögren's syndrome (SS) patients, by virtue of an intrinsically activated cell status, may have a crucial pathogenetic role in this disorder. The degradation of necrotic cell debris by exonucleases, such as DNase1, is thought protective against the inflammagenic action of necrotic cell-derived substances;
Objectives therefore, we comparatively investigated various tissues from SS patients and non-SS controls for the handling of and response to necrotic cell debris (secondarily necrotic cells; SNEC), including the activation of inflammasome.
Methods The mRNA and protein expression of DNase1 was investigated in salivary gland (SG) tissues, peripheral blood mononuclear cells (PBMC) and long-term cultured non-neoplastic salivary gland epithelial cell (SGEC) lines by real-time PCR, immunohistochemistry and immunoblotting. DNase1 activity was measured in cytosolic extracts of cultured SGEC and in SGEC culture supernatants by single radial enzyme diffusion (SRED assay). The mRNA and protein expression of various inflammasome-related genes were compared in SG tissues from SS patients and non-SS controls. The constitutive, as well as the SNEC-induced expression of inflammasome-related and cell activation genes were also comparatively studied in cultured SGEC. The presence of extracellular DNA (exDNA) in SG tissues and cultured SGEC was examined by confocal microscopy, using monoclonal anti-dsDNA antibody, DAPI and Ribogreen staining.
Results The SG tissues of SS patients with intense lymphocytic infiltrates (≥2 infiltrations/4mm2) manifested significantly reduced DNase1 mRNA levels, compared to non-SS controls (p=0.023). Significantly decreased DNase1 mRNA and protein expression (both for p<0.01), as well as impaired cytosolic DNase1 activity (p=0.019) were also observed in SGEC lines (but not the PBMC) of SS patients, compared to controls. In line to these findings, significant amounts of exDNA (DNA strands) were observed in SG tissues of SS patients (particularly around epithelial ductal cells), as well as in the extracellular space of SS-SGEC (but not nonSS-SGEC) cultures. High expression of the inflammasome-related molecules PYCARD/ASC (1.6-fold increase) and AIM2 (5.8-fold increase), which are involved in inflammasome activation though DNA-sensing, was detected in the epithelial cells of SG tissues and constitutively in cultured SGEC lines from SS patients. SGEC treatment with SNEC was found to induce the expression of PYCARD/ASC, AIM2, several cell activation markers (ICAM-1, MHC-I, CD86, Fas, IFN-β) and DNase1, as well as the activity of DNase1 in SGEC culture supernatants. The SNEC-induced activation of inflammasome in SGEC was also demonstrated by detection of caspase-1 activation (intracellular p20 expression; 4.8-fold increase) and of IL-1β secretion in culture supernatants (2.5-fold increase).
Conclusions These findings provide first evidence that the salivary epithelia of SS patients manifest impaired endogenous DNase1 activity, as well as constitutive activation of inflammasome that likely owes to the exposure of cells to necrotic debris. Such aberrations may hold a key pathogenetic role in the tissue inflammatory reactions of SS and may also explain the “intrinsic activation status” that characterizes the epithelia of these patients.
Disclosure of Interest : None declared
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