Doença de Sjogren e vírus SARS-CoV-2: aspectos moleculares e imunogênicos relacionados ao SARS-CoV-2 em glândulas salivares

Abstract

With the emergence of COVID-19, the salivary glands were identified as a reservoir for SARS-CoV-2. Sjögren’s disease (SjD), an autoimmune epithelitis affecting exocrine glands, including the salivary glands, has implications that are not yet well understood regarding SARS-CoV-2 infection. This study, therefore, aimed to investigate three objectives, namely: (i) to evaluate the presence of SARS-CoV-2 in salivary gland biopsies from patients with SjD; (ii) to compare the tissue gene expression levels of three factors related to SARS-CoV-2 entry into cells, namely ACE2, TMPRSS2, and FURIN, in minor salivary glands of patients with primary SjD (pSjD) in relation to normal salivary glands and pleomorphic adenoma; (iii) to analyze the effects of local infusion of SARS-CoV-2 Spike (S) and Envelope (E) proteins in the submandibular glands (SMGs) of C57BL/6 mice. To address these objectives, using total RNA extracted from SjDp and mucocele samples, the presence of SARS-CoV-2 nucleocapsid genes (N1 and N2) was investigated by RT-qPCR in 30 SjD cases and 98 mucocele cases. The tissue expression levels of ACE2, TMPRSS2, and FURIN genes in the salivary glands of pSjD patients compared to normal salivary glands and pleomorphic adenoma were also evaluated by RT-qPCR. Finally, the S (S1+S2), E proteins or a control buffer solution were injected directly into the SMGs of C57BL/6 mice. The effects of the infusion were assessed through histological analyses, flow cytometry, immunohistochemistry, and ELISA. (i) The virus was detected in 5/128 of the analyzed samples, with 4/30 (13.3%) in the SjDp group and 1/98 (1.0%) in the mucocele group. (ii) ACE2 levels were upregulated in SjDp compared to normal salivary gland and pleomorphic adenoma (p = 0.002 and p < 0.001, respectively). TMPRSS2 and FURIN mRNA levels were also elevated in SjDp compared to pleomorphic adenoma (p < 0.05). There was an association between ACE2 levels and the presence of anti SSA/SSB in SjDp. Neither ACE2, TMPRSS2, nor FURIN were associated with N1 or N2 SARS-CoV-2 positivity in SjDp cases. (iii) Intra-SMG infusion of S protein led to a significant reduction in salivary flow rate (p = 0.003) and immune cell infiltration in the SMGs in two-thirds (4/6) of the mice, which was not observed with control or E protein treatment. Consequently, the focus score of the S protein-treated group was significantly elevated compared to the control (p = 0.029). S protein induced changes in the activation of CD4+ T cells and B cells in the SMGs, accompanied by an increase in pSTAT3 compared to control (p < 0.05). Finally, S protein led to the production of antinuclear antibodies in circulation. In summary, this study indicates that there are changes in the microenvironment of normal or SjD-affected salivary glands when aspects related to SARS-CoV-2 are evaluated. Additionally, SARS-CoV-2 S protein triggered significant inflammatory changes in the SMGs of C57BL/6 mice.