Plasmacytoid dendritic cells promote the pathogenesis of Sjögren's syndrome

Abstract

Plasmacytoid dendritic cells (pDCs) produce type I interferons (IFNs) and promote pathogenesis of multiple autoimmune diseases. Autoimmune Sjögren's syndrome (SS) primarily affects salivary and lacrimal glands, causing their inflammation, destruction and dysfunction. pDCs and type I IFN activity are elevated in salivary glands of SS patients, and this study seeks to elucidate the in vivo actions of pDCs in SS pathogenesis using the non-obese diabetic (NOD) mouse model. We confirmed the type I IFN-dependency of SS development in female NOD mice and elevation of pDC-type I IFN in their submandibular glands (SMGs). We administered a pDC-depleting anti-BST2/CD317 antibody to female NOD mice from 4 to 7 weeks of age at the early stage of SS, and assessed SS pathologies at age 10 weeks, the time of disease onset. Depletion of pDCs impeded the development of SMG inflammation and secretory dysfunction. It drastically reduced the amount of type I IFN mRNA and the number of total leukocytes, and T- and B lymphocytes in SMGs. Gene expression analyses showed that pDC depletion markedly diminished SMG expression of IL-7, BAFF, TNF-α, IFN-γ, CXCL9, CXCL11, CD40, CD40L, Lt-α, Lt-β and NOS2. Hence, pDCs critically contribute to the development and onset of SS-like salivary gland exocrinopathy.

Keywords: Autoimmune inflammation; Hyposalivation; Innate immune cells; Salivary gland; Sialadenitis; Type I interferons.

Figure 1.. Blockade of IFNAR1 impedes the development of SS-like salivary gland pathologies in NOD mice.

Anti-IFNAR1 antibody or IgG was i.p. administered to 4-week-old female NOD mice, once every 5 days for a total of 4 times. All the analyses were performed when these mice were 10 weeks of age. (A) Tile images of H&E stained areas of one representative SMG sample each treatment group (scale Bar=100 μm). Bar graph shows the average number of leukocyte foci per SMG section. (B) Stimulated saliva flow rate normalized to body weight. (C) Real-time PCR analysis of IL-7 and BAFF mRNA levels in SMGs. Gene expression was presented relative to that of β-actin. (D) Relative level of anti-M3R in the sera as determined by ELISA. (E) Detection of serum autoantibodies (scale bar=50 μm). Bar graphs show the fluorescence intensity of autoantibody staining of the 1:40-diluted sera, left, and the autoantibody titers, right. All data are the representative or average of analyses of 6 mice for each group. Error bars represent the standard error of mean (SEM). *P< 0.05, ** P<0.01, ***P<0.001.

Figure 2.. pDCs are present in the SMGs of female NOD mice, accompanied by local upregulation of type I IFN-responsive genes.

(A) Flow cytometric analysis of the percentage of pDCs (CD11b⁻CD11c^midB220⁺Siglec-H⁺BST2⁺) in total SMG cells of BALB/c mice and NOD mice aged 13 weeks. The top panels show the negative control staining profile of the cells (a mixture of BALB/c and NOD SMG cells at the 1:1 ratio) under each gating. Bar graph shows the mean percentage of pDCs in total SMG cells, calculated as: Percentage of pDCs in the total SMG cells (%) = % mononuclear cells in SMG cells × % CD11b⁻CD11c^mid cells in mononuclear cells × % B220⁺Siglec-H⁺ cells among CD11b⁻CD11c^mid cells × % B220⁺BST2⁺ cells among B220⁺Siglec-H⁺ cells × 100. (B) Immunohistochemical staining with anti-BST2 antibody of SMG sections from female NOD mice aged 4, 7, and 10 weeks (scale bar = 50 μm). Red arrows indicate the representative areas stained positive for BST2. Negative staining control: SMG sections from female NOD mice aged 10 weeks stained without the without the primary antibody. (C) Real-time PCR analysis of IRF-1 and IRF-7 levels in the SMGs of female NOD mice. The results are presented relative to that of β-actin. All data are representative or the average of analyses of 4–7 mice for each group.

Figure 3.. Administration of anti-BST2 antibody efficiently depletes pDCs and reduces expression of type I IFNs, type I IFN-responsive genes and SS-promoting cytokines in SMGs.

Anti-BST2 or isotype rat IgG was i.p. administered into 9-week-old female NOD mice on day 0 and day 2. The analyses were performed 24 hours after the last injection. (A) Percentages of pDCs (defined as CD11b⁻CD11c^midB220⁺Siglec-H⁺BST2⁺) among total SMG cells, SMG-draining lymph node, and spleen cells based on flow cytometric analysis. (B) Real-time PCR analysis of the mRNA levels of type I IFNs, type I IFN-responsive genes and SS-promoting cytokines in the SMGs. The results are presented relative to that of β-actin. All data are representative or the average of analyses of 3 mice for each group.

Figure 4.. Administration of anti-BST2 antibody over a 3-week period reduces leukocyte infiltration of SMGs and improves salivary secretion in female NOD mice.

Anti-BST2 or isotype rat IgG was i.p. administered into 4-week-old female NOD mice, 3 times weekly for 3 weeks. All the analyses were performed when mice were 10 weeks of age. (A) Stimulated saliva flow rate normalized to body weight. (B) Images of H&E staining of SMG sections (scale Bar=100 μm). Bar graph shows the average number of leukocyte foci per SMG section. (C) Flow cytometric analysis of lymphocyte populations in the SMGs. (D) The clustergram of RT² Profiler PCR Array results displaying gene expression levels with statistically significant changes upon anti-BST2 treatment. All data are representative or the average of analyses of 6 mice for each group.

Figure 5.. Administration of anti-BST2 antibody does not significantly affect serum autoantibody levels.

(A) Anti-BST2 or the isotype rat IgG was i.p. administered into 4-week-old female NOD mice, 3 times weekly for 3 weeks. Immunofluorescence staining images show the detection of serum autoantibodies, measured by the HEp-2 human epithelial cell substrate slides, in mice aged 10 weeks (scale bar = 50 μm). The two images shown for the anti-BST2 treatment group represent serum samples from two of the NOD mice in this group. Bar graphs show the fluorescence intensity of autoantibody staining of the 1:40-diluted sera, left, and the autoantibody titers, right. (B) Relative level of anti-M3R in the sera as determined by ELISA. All data are representative or the average of analyses of 6 mice for each group.