Tracking early autoimmune disease by bioluminescent imaging of NF-kappaB activation reveals pathology in multiple organ systems

Abstract

It is desirable to have an early and sensitive detection marker of autoimmune disease in intact animals. Nuclear factor (NF)-kappaB is a transcription factor that is associated with inflammatory responses and immune disorders. Previously, we demonstrated that so-called idiotypic-driven T-B cell collaboration in mice doubly transgenic for paired immunoglobulin and T cell receptor transgenes resulted in a systemic autoimmune disease with systemic lupus erythematosus-like features. Here, we investigated NF-kappaB activation by including an NF-kappaB-responsive luciferase reporter transgene in this animal model. Triply transgenic mice developed bioluminescence signals from diseased organs before onset of clinical symptoms and autoantibody production, and light emissions correlated with disease progression. Signals were obtained from secondary lymphoid organs, inflamed intestines, skin lesions, and arthritic joints. Moreover, bioluminescence imaging and immunohistochemistry demonstrated that a minority of mice suffered from an autoimmune disease of the small intestine, in which light emissions correlated with antibodies against tissue transglutaminase and gliadin. Detection of luciferase by immunohistochemistry revealed NF-kappaB activation in collaborating B and T cells, as well as in macrophages. These results demonstrate that bioluminescent in vivo imaging of NF-kappaB activation can be used for early and sensitive detection of autoimmune disease in an experimental mouse model, offering new possibilities for the evaluation of anti-inflammatory drugs.

Figure 1

Weight, survival, and clinical signs in triply transgenic mice. A cohort of 13 TCR⁺Id⁺Luc⁺ triply transgenic mice and 8 Id⁺Luc⁺ transgenic control littermates were inspected for disease and weighed weekly. A: Weight (left) and survival (right) of mice. B: Cumulative incidences of skin, joint and inflammatory bowel disease.

Figure 2

NF-κB bioluminescence precedes and correlates with disease. A: Photons emitted from ventral aspects of mice with increasing age. Left: a representative TCR⁺Id⁺Luc⁺ mouse investigated at the indicated time-points. Right: an Id⁺Luc⁺ control. B–D: Focal disease. A common light intensity bar for all panels in B–D is shown (right). B: Early detection of skin disease on the dorsal aspect of head and upper thorax. Ears and eyes are traced in some frames with a black line. C: A representative 20-week-old triply transgenic mouse with severe skin disease including scarring is shown. Focal areas detected by bioluminescence (left) and photo (right), have been numbered. D: Early signals from the hind paw of a mouse that later developed arthritis. Clinical arthritis was only evident starting from week 14 (asterisks). E: Photons from ventral organs after sacrifice and skin removal. The ventricle and kidney are abbreviated as Vent and Kidn. The surface trace of the colon in the TCR⁺Id⁺Luc⁺ mouse is indicated with black lines. F: Signals from isolated organs postdissection. G: a) A whole spleen from a triple transgenic mouse displaying a focus of high bioluminescence signals (detected by the IVIS100) is shown. b) Light emissions from cross sections of the same spleen with focus were detected by a microscope with an ultrasensitive CCD camera (see Materials and Methods). Light emissions can be seen from the white pulp (W, traced with a black line). c) Immunofluorescence of an area with intense signals (see arrow from b) stained for IgG (red), peanut agglutinin [PNA] (green), and nuclei (blue). An active immune response with PNA⁺ IgG⁺ germinal centers (yellow), PNA⁺ IgG⁻ germinal centers (green), and IgG⁺ B cells (red) can been seen. H: NF-κB-dependent bioluminescence in triple transgenic treated with L-012; note the colocalization of NF-κB activity and inflammatory (L-012) signals from area of the distal colon. The inset shows NF-κB-dependent light emission postdissection from the distal third of the colon.

Figure 3

Statistical evaluation of NF-κB bioluminescence signals in triply transgenic mice. A: Ventral surfaces of TCR⁺Id⁺Luc⁺ and Id⁺Luc⁺ mice (n = 13, 10 respectively) were visualized and the photons emitted from the indicated areas were measured. Note the different scales on the y axis. *P < 0.05, Mann Whitney. B: Quantification of signals from organs of mice, P values (Mann Whitney) are indicated.

Figure 4

NF-κB bioluminescence correlates with autoantibody levels. A: IgG ANA titers correlated with emitted photons from the ventral sides of 10-week-old triply transgenic mice. A linear regression analysis is shown. B: The presence of IgG skin autoantibodies correlated with bioluminescence from ears measured at the indicated time points. Triply transgenic mice were divided into those positive or negative for serum autoantibodies against skin antigens at any of the indicated time points. The number of autoantibody-positive mice (out of 13) at each time-point is indicated. *P < 0.05, Mann Whitney.

Figure 5

Luciferase protein expression was detected in T cells, B cells, and macrophages, and in inflamed tissues. A–D: Representative examples of immunofluorescence analyses of 15 to 19-week-old mice are shown. A: Lymph node B cell follicles of Id⁺Luc⁺ (control) and TCR⁺Id⁺Luc⁺ mice were stained for luciferase protein (green), Id (anti-λ2/3 2B6 mAb, red), and nuclei (DAPI, blue). NF-κB-activated Id⁺ B cells are yellow. B: Left: Interfollicular area showing Id-specific T cells (TCR-clonotype specific mAb GB113, red), and luciferase (green), and nuclei (DAPI, blue). An NF-κB activated Id-specific T cell (yellow), two non-activated Id-specific cells (red), as well as two NF-κB activated non-Id-specific T cells (green) are seen (T cells expressing exclusively endogenous TCRα-chains are not Id-specific and do not stain with GB11314). Right: interfollicular areas from Id⁺Luc⁺ (control) and TCR⁺Id⁺Luc⁺ mice showing CD11b (red) and luciferase (green) and nuclei (DAPI, blue). NF-κB⁺CD11b⁺ macrophages are yellow. C: Triple staining of lymph node follicle showing Id⁺ B cells (anti-λ2/3 2B6 mAb, blue), Id-specific T cells (TCR-clonotype specific mAb GB113, red), and luciferase (green). The inset shows magnification and splitting of channels (right). Several Id⁺ B cells and Id-specific T cells cluster together, and both cell types are luciferase protein positive. D: Sections of an inflamed colon from a TCR⁺Id⁺Luc⁺ mouse. Left top: High magnification (×400) of area near erosion stained for Id (red), luciferase (green), and nuclei (blue). Id⁺NF-κB⁺ B cells are yellow. Left, bottom: Co-localization of IgG (red) and C3 (green) at sites of erosion (asterisks). A cellular infiltrate is seen in the middle left of the panel. Right, low magnification (×100): luciferase (red), and complement C3 (green), nuclei (blue). The colon architecture is effaced by cellular infiltrates and erosions (asterisks). Areas where C3 and luciferase expression co-localize are yellow. E–F: Small intestines of mice with inflammation. E: H&E stain. Left, mononuclear cells infiltrating a villus (arrow). Right, shortening of villi. F: Staining of luciferase (red) and C3 (green) in triply transgenic (left) and doubly transgenic control mice (right).

Figure 6

Antibodies related to intestinal pathology correlate with signals from inflamed small intestines. Sera were screened for antibodies associated with small intestinal pathology. A: Levels of serum anti-transglutaminase 2 (TG2) autoantibodies (left) and anti-Gliadin (right) correlated with NF-κB bioluminescence from small intestines isolated from TCR⁺Id⁺Luc⁺ mice. B: Serum from a 15-week-old triply transgenic mouse with small intestinal light emissions gave an anti-endomycium staining pattern, [IgG (red), and smooth muscle cell nuclei (blue)]. C: Autoantibodies directed toward the small intestine of a BALB/c RAG2^−/− mouse. Left: serum from triply transgenic mouse (15 weeks old) with small intestinal signal and disease. Right: negative stain, serum from triply transgenic littermate (15 weeks) without small intestinal disease.