Accelerated pathological and clinical nephritis in systemic lupus erythematosus-prone New Zealand Mixed 2328 mice doubly deficient in TNF receptor 1 and TNF receptor 2 via a Th17-associated pathway

Abstract

TNF-alpha has both proinflammatory and immunoregulatory functions. Whereas a protective role for TNF administration in systemic lupus erythematosus (SLE)-prone (New Zealand Black x New Zealand White)F(1) mice has been established, it remains uncertain whether this effect segregates at the individual TNFR. We generated SLE-prone New Zealand Mixed 2328 mice genetically deficient in TNFR1, in TNFR2, or in both receptors. Doubly-deficient mice developed accelerated pathological and clinical nephritis with elevated levels of circulating IgG anti-dsDNA autoantibodies and increased numbers of CD4(+) T lymphocytes, especially activated memory (CD44(high)CD62L(low)) CD4(+) T cells. We show that these cells expressed a Th17 gene profile, were positive for IL-17 intracellular staining by FACS, and produced exogenous IL-17 in culture. In contrast, immunological, pathological, and clinical profiles of mice deficient in either TNFR alone did not differ from those in each other or from those in wild-type controls. Thus, total ablation of TNF-alpha-mediated signaling was highly deleterious to the host in the New Zealand Mixed 2328 SLE model. These observations may have profound ramifications for the use of TNF and TNFR antagonists in human SLE and related autoimmune disorders, as well as demonstrate, for the first time, the association of the Th17 pathway with an animal model of SLE.

FIGURE 1

Accelerated proteinuria (A) and mortality (B) in TNFR p55/p75DKO mice. A total of 31–37 female mice for each of the indicated experimental group was monitored up to 12 mo of age. Data are presented as the percentage of cumulative prevalence of severe (≥3⁺) proteinuria and as percentage of cumulative death at the ages shown. Development of severe proteinuria and mortality are significantly accelerated (p < 0.001 for each) in DKO mice compared with those in the other mouse lines.

FIGURE 2

IgG anti-dsDNA autoantibodies in the different lines of mice at 5–5.5 and 7.5–9 mo of age. n = 7–13 mice in the various groups. Each symbol represents an individual mouse. The composite results are plotted as box plots. The lines inside the boxes indicate the medians; the outer borders of the boxes indicate 25th and 75th percentiles; the bars extending from the boxes indicate the 10th and 90th percentiles. *, p < 0.001 vs other groups at 5–5.5 mo of age.

FIGURE 3

Representative H&E staining (original magnification ×200) and IgG1, IgG2b, and C3 immunofluorescence staining (original magnification ×40) of kidney sections from female NZM 2328 mice with the indicated genotypes and ages. Increased mesangial deposition with some cellularity in the glomeruli of 5.5-mo-old p55/p75 DKO mice and in 7.5-mo-old WT mice is marked with arrows.

FIGURE 4

Assessment of renal pathology in the different lines of mice. Data are presented as composite KBS in the NZM 2328 mice with the indicated genotypes at 5.5 mo of age and at 7.5 mo of age. Each symbol represents an individual mouse. Results are plotted as in Fig. 2. *, p < 0.001 vs all other groups at 5.5 mo of age.

FIGURE 5

Comparison of spleen follicle structure in NZM 2328 WT and the indicated TNFR-deficient mice. Spleen sections from 2-mo-old (A) and from 6-mo-old (B) female mice from the indicated genotypes were stained with anti-B220 (red) and anti-MOMA-1 (green) mAb. Original magnifications of ×10 and ×40 are shown.

FIGURE 6

Altered GC formation in TNFR-deficient mice. Spleen sections from the indicated strains at 2 and 6 mo of age were stained with peanut agglutinin (red) to detect GC and with anti-B220 (green) for B cells. Original magnification ×40.

FIGURE 7

Effects of various TNFR deficiencies on spleen B and T cell subsets in 5- to 5.5-mo-old mice. Numbers shown are millions of cells per spleen. The composite results are plotted as box plots. The lines inside the boxes indicate the medians; the outer borders of the boxes indicate 25th and 75th percentiles; the bars extending from the boxes indicate the 10th and 90th percentiles. There are four mice in each of the indicated test groups analyzed in the same experiments. Results are representative of three independent experiments. a, Total number of splenocytes; b, CD19-positive B lymphocytes; c, CD3-positive total T lymphocytes; d, CD8-positive T cells; e, CD4-positive T cells; f, CD4⁺ CD44^highCD62L^low/neg activated memory T cells; g, CD4⁺CD69⁺ recently activated T cells. Value of p < 0.05 among 5.5-mo-old mice for a–c; p < 0.01 for e–g; no statistical difference for d. Between DKO mice at 5.5 mo of age and WT at 9 mo of age, no significant difference was observed for a–c and e–g.

FIGURE 8

Flow cytometry analyses of T cell subsets in the various lines of mice. Representative FACS dot plots are shown at 5 mo of age in A–C, and at 8–9 mo of age in D. All data are representative for at least four independent experiments with similar results. A, Proportional increase of CD4⁺ and decrease of CD8⁺ T cells in p55/p75 DKO mice. The numbers shown in quadrants of each plot indicate percentage of total T cells (CD3⁺). B, Increased activated (CD69⁺) CD4⁺ T cells in p55/p75 DKO mice. The numbers shown in the upper right quadrant of each plot indicate percentage of total lymphocytes. C, Increased CD4⁺ activated memory T cells (CD44^highCD62L^low/neg) subset in p55/p75 DKO lupus mice. The numbers in the lower right quadrant of each plot indicate the percentage of CD4⁺CD25⁻ cells. D, As C, but at 8–9 mo of age. Importantly, p55/p75 DKO are absent because they are dead by this age. The numbers in the lower right quadrant of each plot indicate the percentage of CD4⁺CD25⁻ cells.

FIGURE 9

A, Selective gene mRNA expression of CD4⁺CD44^highCD62L^low/neg cells from 5-mo-old female p55/p75 DKO NZM.2328 mice purified by flow cytometry () in comparison with the same subset of T cells purified from age- and sex-matched C57BL/6 mice (black baseline). Data are presented as fold change values of quadruplicates tested in parallel, as described in Materials and Methods. B, Validation of the gene array data in A by real-time quantitative PCR of selected genes. Results are presented as fold change values of CD4⁺CD44^highCD62L^low/neg cells from 5-mo-old p55/p75 DKO NZM 2328 mice (), from WT NZM 2328 mice at 8–9 mo of age (□), and from WT NZM 2328 at 5 mo of age (■) compared in each case to the same subset of T cells purified in parallel from age- and sex-matched C57BL/6 mice (black baseline).

FIGURE 10

IL-17A levels in NZM2328 WT, p55/p75 DKO, and C57BL/6 mice. Naive and activated memory CD4⁺ spleen cells were isolated from 5- to 5.5-mo-old female p55/p75 DKO NZM 2328 mice, 7- to 9-mo-old female NZM 2328 WT mice, or 7- to 9-mo-old C57BL/6 mice by depleting B cells with magnetic beads, followed by flow cytometry sorting gated on CD4⁺CD62L^highCD44^low (for naive T cells) or CD4⁺CD44^highCD62L^low (for activated memory T cells). A, Naive and activated memory cells isolated from 5- to 5.5-mo-old p55/p75 DKO mice were treated as described in Materials and Methods, and stained for intracellular IL-17A and control IgG at days 0 and 5. B, Activated memory cells isolated from 7- to 9-mo-old female C57BL/6 and NZM 2328 WT mice were isolated, treated, and stained, as in A. C, IL-17A levels measured by ELISA in supernatants from cultures of activated memory T cells isolated and treated as above. Value of p < 0.001 between C57BL/6 mice and NZM2328 WT and DKO mice. No significant difference between DKO and 7- to 9-mo NZM WT.