ILF2 and ILF3 are autoantigens in canine systemic autoimmune disease

Abstract

Dogs can spontaneously develop complex systemic autoimmune disorders, with similarities to human autoimmune disease. Autoantibodies directed at self-antigens are a key feature of these autoimmune diseases. Here we report the identification of interleukin enhancer-binding factors 2 and 3 (ILF2 and ILF3) as autoantigens in canine immune-mediated rheumatic disease. The ILF2 autoantibodies were discovered in a small, selected canine cohort through the use of human protein arrays; a method not previously described in dogs. Subsequently, ILF3 autoantibodies were also identified in the same cohort. The results were validated with an independent method in a larger cohort of dogs. ILF2 and ILF3 autoantibodies were found exclusively, and at a high frequency, in dogs that showed a speckled pattern of antinuclear antibodies on immunofluorescence. ILF2 and ILF3 autoantibodies were also found at low frequency in human patients with SLE and Sjögren's syndrome. These autoantibodies have the potential to be used as diagnostic biomarkers for canine, and possibly also human, autoimmune disease.

Figure 1

Identification of ILF2 as a potential autoantigen in canine IMRD. ILF2 was identified as a potential autoantigen in canine immune-mediated rheumatic disease (IMRD) by a protein array screen of 12 IMRD patients and 9 healthy controls. All of the dogs were of the breed Nova Scotia duck tolling retriever (NSDTR). Log(ln)-transformed fluorescence signal intensities are the mean of protein duplicates. The cutoff value was calculated from healthy controls as the mean + 3 SD (ln-transformed data). IMRD ANA^S, IMRD patients with speckled antinuclear antibodies (ANA) pattern. IMRD ANA^H, IMRD patients with homogenous ANA pattern.

Figure 2

Validation of ILF2 with an independent method. Radio-ligand binding assay was used to validate ILF2 as an autoantigen in the discovery cohort (a) and in an extended cohort. (b) represent discovery and extended cohort together. All of the patients with immune-mediated rheumatic disease (IMRD) were of the breed Nova Scotia duck tolling retriever (NSDTR). The cutoff value was calculated from healthy controls as the mean + 5 SD. All samples were analysed in duplicate. Autoantibody index = (sample value mean − negative control)/(positive control − negative control) * 100. IMRD ANA^S, IMRD patients with speckled antinuclear antibodies (ANA) pattern IMRD. ANA^H, IMRD patients with homogenous ANA pattern IMRD. ANA^neg, IMRD patients without ANA. SRMA, steroid-responsive meningitis-arteritis.

Figure 3

Screening the canine cohort for ILF3 and RBMX autoantibodies. A radio-ligand binding assay was used to screen for ILF3 (a) and RBMX (b) autoantibodies in dogs. The cutoff value for ILF3 was calculated from healthy controls as the mean + 5 SD. For RBMX, the mean + 7 SD was used as cutoff value because of a clear separation of negative and positive samples. All samples were analysed in duplicate. Autoantibody index = (sample value mean − negative control)/(positive control − negative control) * 100. NSDTR, Nova Scotia duck tolling retriever. IMRD ANA^S, IMRD patients with speckled antinuclear antibodies (ANA) pattern. IMRD ANA^H, IMRD patients with homogenous ANA pattern. IMRD ANA^neg, IMRD patients without antinuclear antibodies. SRMA, steroid-responsive meningitis-arteritis.

Figure 4

Reactivity to ILF2, ILF3 and RBMX. The number of positive samples to ILF2, ILF3 and RBMX is presented in each field.

Figure 5

Sera from ILF2- and ILF3-positive patients and commercial ILF2 and ILF3 antibodies display a speckled ANA pattern. Indirect immunofluorescence microscopy images of HEp-2 cells incubated with sera from an ILF2- and ILF3-positive IMRD patient (a), a commercial polyclonal ILF2 antibody (b); and a commercial polyclonal ILF3 antibody (c). (a) In the nucleoplasm, fine tiny speckles can be observed and mitotic cells have unstained chromatin mass (⇨). The nucleoli are not stained (→). (b,c) In the nucleoplasm, fine tiny speckles can be observed and mitotic cells have unstained chromatin mass (⇨). Nucleoli are stained in many of the cells (→) but not all.