Identification of Orch3, a locus controlling dominant resistance to autoimmune orchitis, as kinesin family member 1C

Abstract

Experimental autoimmune orchitis (EAO), the principal model of non-infectious testicular inflammatory disease, can be induced in susceptible mouse strains by immunization with autologous testicular homogenate and appropriate adjuvants. As previously established, the genome of DBA/2J mice encodes genes that are capable of conferring dominant resistance to EAO, while the genome of BALB/cByJ mice does not and they are therefore susceptible to EAO. In a genome scan, we previously identified Orch3 as the major quantitative trait locus controlling dominant resistance to EAO and mapped it to chromosome 11. Here, by utilizing a forward genetic approach, we identified kinesin family member 1C (Kif1c) as a positional candidate for Orch3 and, using a transgenic approach, demonstrated that Kif1c is Orch3. Mechanistically, we showed that the resistant Kif1c(D2) allele leads to a reduced antigen-specific T cell proliferative response as a consequence of decreased MHC class II expression by antigen presenting cells, and that the L(578) → P(578) and S(1027) → P(1027) polymorphisms distinguishing the BALB/cByJ and DBA/2J alleles, respectively, can play a role in transcriptional regulation. These findings may provide mechanistic insight into how polymorphism in other kinesins such as KIF21B and KIF5A influence susceptibility and resistance to human autoimmune diseases.

Figure 1. Histopathology of autoimmune orchitis.

(A, C) Cross section of normal testis histology in an immunized C.D2-3 mouse: (A) Seminiferous tubules appear normal; (C) A seminiferous tubule (ST) containing normal meiotic spermatocytes and spermatids, with intact tubular boundary (arrows). (B, D) CByJ mouse with sever and diffuse orchitis: (B) All seminiferous tubules are necrotic and have lost cell nuclear staining; (D) Sever orchitis in one seminiferous tubule (arrows) that contains numerous neutrophils and occasional multinuclear giant macrophages (double arrow); the tubular boundary (arrows) is poorly defined. (H&E; A and B, ×4; C and D, ×40).

Figure 2. Congenic mapping places Orch3 within the Kif1c/Nlrp1a/b/c interval.

For convenience, D2 alleles have been shaded. The significance of differences in severity of EAO among CByJ, CD2F1 hybrids and CD2-ISRC lines was determined using the Kruskal-Wallis test (overall p-value<0.0001) followed by Dunn's multiple comparison test. Region outlined in blue depicts the location of Orch3.

Figure 3. Identification of Orch3 as Kif1c.

(CD2-3.2×CByJ)×CByJ backcross mice were screened from recombinants using microsatellite markers spanning the Orch3 interval. Three sub-ISRC lines were identified, fixed and homozygous progeny studied for susceptibility to EAO (D = D2 allele; C = CByJ allele). The significance of differences in EAO among CByJ, CD2-3.2a, CD2-3.2b, CD2-3.2c and Tg-Kif1c^D2 transgenic mice was determined using the Kruskal-Wallis test (overall p-value<0.0001) followed by Dunn's multiple comparison test (**p<0.01). Region outlined in red reflects location of Orch3 based on high resolution congenic mapping relative to the lower resolution mapping outlined in blue.

Figure 4. Generation of BALB/cByJ-CD11B-Kif1c^D2 transgenic (Tg-Kif1c^D2) mice.

(A) Schematic representation of the Kif1c gene used to generate the transgenic mice showing the promoter (CD11B/ITGAM), and the Kif1c gene, followed by the hGH/polyA signal sequence. Arrows indicate PCR-primers for screening. (B and C) Percentage of splenic F4/80⁺ (B) and CD11b⁺ (C) cells of Tg-Kif1c^D2 and NLC. The analysis was performed on gated live cells according to their FSC vs. SSC profile. Statistical significance was determined using the Mann-Whitney U test. Data represent the mean ± SEM of at least 5 individual mice. (D) Kif1c expression in thioglycolate-induced adherent cells by Western blotting using whole-cell extracts and the anti-Kif1c mAb. Actin was used as a loading marker. (E) mRNA expression of Kif1c was measured from sorted TCRβ⁻CD19⁻CD11b⁺ myeloid cells of CByJ mice and compared with TCRβ⁻CD19⁻CD11b⁺ myeloid cells of D2 mice. β2-microglobulin and GAPDH were used as an endogenous control. Data represent the mean ± SEM of two experiments (pool of 5 animals/each).

Figure 5. Analysis of MHC II expression on splenic CD11b⁺ myeloid cells.

(A) Scatterplot of genes differentially expressed in splenic TCRβ⁻CD19⁻CD11b⁺ myeloid cells of Tg-Kif1c^D2 and NLC mice as determined by microarray. There were 164 genes differentially expressed (FDR≤0.05) and for each gene, the log₂ fold change was plotted on the ordinate against the -log₁₀ p-value, plotted on the abscissa. Each data point represents the log₂ fold change (Tg-Kif1c^D2 minus NLC) for each gene. Dark blue data points indicate H2 genes that were downregulated in splenic CD11b⁺ cells of Tg-Kif1c^D2 mice. (B) Flow cytometric analysis of the frequency of TCRβ⁻CD19⁻CD11b⁺-myeloid cells expressing MHC II in the spleen of Tg-Kif1c^D2, D2, D.2-C3.2, and NTC. Statistical significance was determined using the Kruskal-Wallis test (overall ***p-value<0.0001) followed by Dunn's multiple comparison test (**p<0.01, *p<0.05). Data represent the mean ± SEM of at least 5 individual mice.

Figure 6. Evaluation of Ag-specific T cell stimulatory capacity of APCs.

Ag-specific T cell proliferative responses were evaluated by [³H] thymidine incorporation. (A) OVA-specific CD4 T cells, and (B) PLP_180–199-specific CD4 T cells from NLC mice were co-cultured with T cell-depleted/mitomycin C-treated/OVA pulsed APCs (A) and PLP_180–199 pulsed APCs (B). Open bars are Tg-Kif1c^D2-APCs, and closed bars are NLC-APCs. Each bar represents the mean cpm ± SEM of 3 independent experiments. The significance of the differences was determined by two-way ANOVA. OVA-specific response: effect of [OVA] (p<0.0001); effect of strain (p<0.0001); interaction (p = 0.08). PLP_180–199-specific response: effect of [PLP_180–199] (p<0.0001); effect of strain (p<0.0001); interaction (p = 0.08).

Figure 7. Structural polymorphisms at amino acid residues 578 and 1027 influence KIF1c function.

Jurkat cells were co-transfected with a plasmid containing the (A) Kif1c^D2 (open bar), Kif1c^CByJ (grey bar) alleles, or control plasmid (black bar), or (B) Kif1cD2 (PPY; open bar), mutant 578 (LPY; left striped bar), or mutant 1027 (PSY; right striped bar) plasmids, and Il2 promoter luciferase reporter. Cells were stimulated for 3 hours with PMA and calcimycin, and the luciferase activity was quantified. Data are representative of two independent experiments.