Modeling Sjögren's syndrome with Id3 conditional knockout mice

Abstract

The Id3 gene has been shown to play important roles in the development and function of broad tissue types including B and T cells. Id3 deficient mice develop autoimmune disease similar to human Sjögren's syndrome. Both B and T lymphocytes have been implicated to contribute to the disease phenotype in this disease model. In order to gain a better understanding of individual cell types in this disease model, we generated an Id3 conditional allele. An LckCre transgene was used to induce Id3 deletion in developing T cells. We showed that the Id3 gene was efficiently disrupted in early thymocyte development prior to T cell receptor (TCR)-mediated positive selection. Consequently, thymocyte maturation was impaired in the conditional knockout mice. These mice developed exocrinopathy starting at two months of age and subsequently exhibited high incidence of lymphocyte infiltration to salivary glands between eight and 12 months of age. This progressive feature of disease development is very similar to those observed in Id3 germline knockout mice. This study establishes a new model for investigating the relationship between T cell development and autoimmune disease. Our observation provides an experimental case that autoimmune disease may be induced by acquired mutation in developing T cells.

Figure 1. Conditional deletion of the Id3 gene in developing thymocytes

(A) Generation of the Id3 conditional deletion allele. Open box: exon. Hatch box: selection markers. Solid arrowhead: loxP. Open arrowhead: FRT. Arrows: primers. (B) Representative genotyping results of the Id3^f allele. DNA samples were prepared from mouse toes. 4 primers were used: Id3-2 and Id3-3 for the loxP (1.25 kb) and the wild type (1.15 kb) Id3 allele, Tek-F and Tek-R for the LckCre transgene (0.48 kb). M: 1 kb plus DNA ladder. (C) Deletion of Id3 in thymocytes sorted from 7-week-old mice. Thymocytes were separated into the DN (CD4⁻CD8⁻) and DP/SP (CD4⁺CD8⁺, CD4⁺CD8⁻, and CD4⁻CD8⁺) groups by magnetic beads. For the upper panel, the floxed and deletion alleles were determined by competitive PCR using three primers, Id3-2, Id3ckoF and Id3ckoB shown in (A). It produced a 1.07-kb band for the Id3^f allele and a 1.34-kb band for the deleted allele. For the bottom panel, only two primers (Id3ckoF and Id3ckoB) were used to detect the floxed allele. (D) Real-time quantitative PCR analysis of Id3 expression. RNA was prepared from total thymocytes of 7-week old Id3^f/f (open bar, n=4) and Id3^f/f;LckCre (filled bar, n=3) mice. **P=0.006. The graphed results are means with SEM.

Figure 2. Disturbed γδT and αβT cell development in the thymus of Id3^f/f;LckCre mice

(A) Representative FACS analysis of αβ thymocyte development by CD4 and CD8 expression. Thymocytes were isolated from 4–6 weeks old Id3^f/f;LckCre (n=3) and the Id3^f/f littermate (n=4) controls. Dead cells were excluded by 7AAD staining. (B) Summary of the absolute numbers of DN (CD4⁻CD8⁻), DP (CD4⁺CD8⁺), CD4 SP (CD4⁺CD8⁻), and CD8 SP (CD4⁻CD8⁺) thymocytes. *P=0.027. ***P=0.0001. The statistical significance was assessed by unpaired student’s t test. (C) Representative FACS plots of γδ and αβ thymocyte population in Id3^f/f;LckCre mice and Id3^f/f mice. (D) Summary of the absolute numbers of mature γδ and αβ thymocytes. **P=0.009 (γδT cells) and 0.003 (αβT cells). The graphed results are means with SEM. (E) The CD69^hi cells in total thymocytes were gated for two-D plot analysis of CD4 and CD8 expression. (F) The TCRβ^hi thymocytes were gated for analysis of CD4 and CD8 expression. Results of (E) and (F) were representatives of four littermate pairs. (G) H&E staining of thymus section from Id3^f/f;LckCre mice and Id3^f/f mice. (H) Two-color immunofluorescence analysis of thymus section with CD4 and CD8 antibodies. The green and red signals are CD4 and CD8 SP thymocytes, respectively. The yellow signals are CD4⁺CD8⁺ DP thymocytes resulting from CD4 and CD8 double staining. The enlarged views of medulla are shown on the right. Scale bar equals 50 µm.

Figure 2. Disturbed γδT and αβT cell development in the thymus of Id3^f/f;LckCre mice

(A) Representative FACS analysis of αβ thymocyte development by CD4 and CD8 expression. Thymocytes were isolated from 4–6 weeks old Id3^f/f;LckCre (n=3) and the Id3^f/f littermate (n=4) controls. Dead cells were excluded by 7AAD staining. (B) Summary of the absolute numbers of DN (CD4⁻CD8⁻), DP (CD4⁺CD8⁺), CD4 SP (CD4⁺CD8⁻), and CD8 SP (CD4⁻CD8⁺) thymocytes. *P=0.027. ***P=0.0001. The statistical significance was assessed by unpaired student’s t test. (C) Representative FACS plots of γδ and αβ thymocyte population in Id3^f/f;LckCre mice and Id3^f/f mice. (D) Summary of the absolute numbers of mature γδ and αβ thymocytes. **P=0.009 (γδT cells) and 0.003 (αβT cells). The graphed results are means with SEM. (E) The CD69^hi cells in total thymocytes were gated for two-D plot analysis of CD4 and CD8 expression. (F) The TCRβ^hi thymocytes were gated for analysis of CD4 and CD8 expression. Results of (E) and (F) were representatives of four littermate pairs. (G) H&E staining of thymus section from Id3^f/f;LckCre mice and Id3^f/f mice. (H) Two-color immunofluorescence analysis of thymus section with CD4 and CD8 antibodies. The green and red signals are CD4 and CD8 SP thymocytes, respectively. The yellow signals are CD4⁺CD8⁺ DP thymocytes resulting from CD4 and CD8 double staining. The enlarged views of medulla are shown on the right. Scale bar equals 50 µm.

Figure 3. Sjögren’s syndrome-like symptoms in Id3^f/f;LckCre mice

(A) Decreased saliva secretion in Id3^f/f;LckCre mice after pilocarpine stimulation. Id3^f/f;LckCre mice were divided into young (6–18 weeks) and old (8–12 months) groups. The volume of saliva was determined after pilocarpine stimulation. Values were normalized by the body weight. *P=0.02 (young) and 0.046 (old). The graphed results are means with SEM. (B–E) H&E staining of parotid (B&C) and submandibular (D&E) salivary glands from 10-month-old mice. All the pictures were taken with the original magnification of 10×10. The scale bar equals 100µm. (F) Focus score of lymphocyte infiltrates in parotid and submandibular salivary glands of Id3^f/f;LckCre (n=13) and Id3^f/f mice (n=14) with age ranging from 8–12 month according to the method described previously [11]. **P=0.009. The significance was analyzed by two-tail unpaired student’s t test.

Figure 4. Absence of Id3-deficient T cells in gland tissues of young adult Id3^f/f;LckCre mice

Id3 deficient T cells were traced with a Cre reporter in 2 month old Id3^f/f;Rosa26^f/+;LckCre and Id3^f/+;Rosa26^f/+;LckCre control mice. Tissue section (20 µm) of gland-associated lymph node, parotid salivary gland, submandibular gland, and lachrymal gland were stained with x-gal. The slides were counterstained by eosin. The scale bar equals 100 µm.

Figure 5. Accumulation of activated/memory T cells in aged Id3^f/f;LckCre mice

(A) Numbers of CD4 and CD8 T cells in the spleen from 6-week old Id3^f/f;LckCre (n=3) and Id3^f/f mice (n=4). The graphed results were means with SEM. The significance was analyzed by two-tail unpaired student’s t test. **P=0.002. ***P=0.0003. (B) Numbers of CD4 and CD8 T cells in the spleen of 9–13 month old Id3^f/f;LckCre (n=4) and Id3^f/f mice (n=3). Analysis was performed as in (A). No significant difference was observed between the genotype groups. (C,D) FACS plots of activated/memory T cells (CD44^hiCD62L^low, the upper left rectangle gate) and naïve T cells (CD44^lowCD62L^hi, the lower right rectangle gate) among CD4 T cells (C) and CD8 T cells (D). Gated CD4 or CD8 lymphocytes are from spleen of 3-month old (upper panel of C and D) and 9-month old mice (bottom panel of C and D). Results are representative of at least three mice for each age and genotype group.