IFI16 filament formation in salivary epithelial cells shapes the anti-IFI16 immune response in Sjögren's syndrome

Abstract

IFN-inducible protein 16 (IFI16) is an innate immune sensor that forms filamentous oligomers when activated by double-stranded DNA (dsDNA). Anti-IFI16 autoantibodies occur in patients with Sjögren's syndrome (SS) and associate with severe phenotypic features. We undertook this study to determine whether the structural and functional properties of IFI16 play a role in its status as an SS autoantigen. IFI16 immunostaining in labial salivary glands (LSGs) yielded striking evidence of filamentous IFI16 structures in the cytoplasm of ductal epithelial cells, representing the first microscopic description of IFI16 oligomerization in human tissues, to our knowledge. Transfection of cultured epithelial cells with dsDNA triggered the formation of cytoplasmic IFI16 filaments with similar morphology to those observed in LSGs. We found that a majority of SS anti-IFI16 autoantibodies immunoprecipitate IFI16 more effectively in the oligomeric dsDNA-bound state. Epitopes in the C-terminus of IFI16 are accessible to antibodies in the DNA-bound oligomer and are preferentially targeted by SS sera. Furthermore, cytotoxic lymphocyte granule pathways (highly enriched in the SS gland) induce striking release of IFI16•dsDNA complexes from cultured cells. Our studies reveal that IFI16 is present in a filamentous state in the target tissue of SS and suggest that this property of DNA-induced filament formation contributes to its status as an autoantigen in SS. These studies highlight the role that tissue-specific modifications and immune effector pathways might play in the selection of autoantigens in rheumatic diseases.

Keywords: Autoimmune diseases; Autoimmunity; Rheumatology.

Figure 1. Cytoplasmic IFI16 filament localization in ductal epithelial cells in labial salivary gland paraffin sections obtained from patients with SS.

(A) Immunostaining of IFI16 in labial salivary gland tissue demonstrates diffuse nuclear staining in cells of the basal layers of the duct (arrows) and irregularly shaped cytoplasmic structures in some cells in the apical portion of the duct (arrowheads). Nuclear IFI16 staining is frequently absent in cells with cytoplasmic IFI16 (cells denoted with asterisk). Scale bar: 20 μM in low-power field (left), 5 μM in high-power field (right). (B) Immunoflourescence microscopy of IFI16 (green) and E-cadherin (red) in labial salivary gland identifies cells containing cytoplasmic IFI16 structures as epithelial in origin. Nuclei are stained with DAPI (blue). Scale bar: 10 μM in low-power views (left) and 5 μM in high-power views (right). Features of the patients examined are reported in Supplemental Table 1.

Figure 2. Generation of IFI16 cytoplasmic filaments in response to dsDNA transfection in epithelial cells in vitro.

(A–C) Human salivary gland (HSG) cells were treated with recombinant IFNα prior to transfection with empty plasmid DNA. Cells were then fixed, permeabilized, and stained with anti-IFI16 antibody (green) and DAPI (blue). Representative confocal images at 100× magnification are shown. Camera settings were held constant between experiments. (D) Keratinocyte cultures were transfected with Rhodamine labeled Poly(dA:dT) (red) and then stained for IFI16 (green) and counterstained with DAPI (blue). A 3-dimensional rendering of a Z-stack series is shown. (E) DNA titration was performed in HSG cultures using increasing concentrations of plasmid DNA, followed by staining with anti-IFI16 (green) and DAPI (blue). Cells with cytoplasmic IFI16 were counted in 4 fields imaged at 40×. Mean values with standard deviation are indicated. *P < 0.05; ***P < 0.0005; ****P < 0.0001 as assessed by the Mann-Whitney U test. Scale bars: 5 μM. Data are representative of results of 3 experiments.

Figure 3. DNA colocalizes with IFI16 in the cytoplasm, but is not visualized in individual filaments ex vivo or in vitro.

(A) A representative ductal epithelial cell from a SS labial salivary gland (LSG) paraffin section was stained with DAPI (blue), anti-DNA monoclonal antibody (green), and anti-IFI16 monoclonal antibody (red). No DNA staining was detected in the cytoplasmic IFI16 containing structure. (B) Primary keratinocytes transfected with Rhodamine-labeled Poly(dA:dT) were stained with DAPI (blue) and anti-IFI16 (green). IFI16 was identified in association with DNA in large structures (arrowheads), but DNA was not visualized in an isolated IFI16 filament extending from a region of IFI16-DNA interaction (arrows). Scale bars: 5 μM.

Figure 4. IFI16 protein filaments persist after nuclease treatment.

(A) Recombinant IFI16 was combined with dsDNA600 and then treated with micrococcal nuclease and imaged by negative stain electron microscopy. (B and C) Replicate samples were analyzed by agarose gel electrophoresis and stained with SYBR green to confirm effectiveness of nuclease treatment (B) and by SDS-PAGE and Western blotting for IFI16 (C). Scale bars: 100 nM.

Figure 5. Autoantibody Immunoprecipitation of IFI16 is enhanced in the presence of dsDNA.

(A and B) Serum samples from 56 Sjögren’s Syndrome patients positive for IFI16 antibodies by ELISA were used to immunoprecipitate IFI16 in the absence or presence of 150 bp dsDNA in 10:1 IFI16/dsDNA molar ratio. Immunoprecipitation products were electrophoresed and Western Blotted with a commercial IFI16 antibody. (A) Representative data from immunoprecipitations performed with 16 patients’ sera and an N-terminal mouse monoclonal antibody (MM) are shown. Sera 1 and 4 were negative for anti-IFI16 antibodies by ELISA and were included as negative controls. Input lanes contain one-fourth protein used in each serum immunoprecipitation. (B) All 56 immunoprecipitations were quantified by densitometry. For each, the ratio of immunoprecipitated IFI16 detected in the presence versus absence of dsDNA was calculated. The dotted line indicates a ratio of 1, denoting unchanged immunoprecipitation with or without dsDNA. Median with interquartile range are indicated with solid lines.

Figure 6. C-terminal specificity of SS sera with property of IFI16-DNA binding.

(A) Cytoplasmic IFI16 filaments were induced in human salivary gland (HSG) cells and stained with commercial antibodies recognizing C-terminal (green) and N-terminal (red) epitopes. Both commercial antibodies recognize nuclear IFI16 (asterisk), but only the C-terminal antibody binds the cytoplasmic filament (arrow). (B) SS sera with or without the property of DNA-enhanced IFI16 immunoprecipitation were used to immunoprecipitate full-length IFI16 (FL) or an N-terminal fragment (IFI16ΔCT) lacking residues 597–729 at the C-terminus; data from Figure 5B specific to these 10 sera are shown. Commercial antibodies specific for the N-terminus (anti-NT) and C-terminus (anti-CT) were included. (C) Western blots were performed with anti-N terminal antibody. Blots were scanned by densitometry and the ratio of immunoprecipitated IFI16ΔCT/IFI16 FL in each was quantitated (Supplemental Table 2). Representative data obtained with 2 patient sera and the commercial antibodies is shown. Scale bar: 10 μM. Data are representative of results of 2 experiments.

Figure 7. IFI16•dsDNA filaments are released from epithelial cells following exposure to cytotoxic lymphocyte granule contents.

(A and B) IFN-treated human salivary gland (HSG) cells were transfected with biotinylated DNA to induce IFI16 filament formation and were then mock treated (A) or exposed to YT cell granule contents (GC) for 3 hours (B). Cells were fixed and stained with DAPI (blue), anti-IFI16 antibody (green), and Streptavidin-DyLight 594 (red). (C) Induction of apoptosis by GC treatment was confirmed by Western blotting for caspase 3, demonstrating intact (closed arrow) and cleaved (open arrow) forms. (D) Following GC treatment, samples of cell lysate (top panel) and supernatant (middle panel) were collected and analyzed in parallel for IFI16 and DNA content by blotting with anti-IFI16 antibody and StrepTactin-HRP. Biotinylated DNA was isolated from the supernatant of treated cells using Streptavidin DynaBeads, and the presence of IFI16•dsDNA interaction in the supernatant was confirmed by blotting for each (D, bottom panel). Scale bars: 10 μM. Data are representative of results of 3 separate experiments.