Protein phosphatase 2A confers susceptibility to autoimmune disease through an IL-17-dependent mechanism

Abstract

The contribution of individual molecular aberrations to the pathogenesis of systemic lupus erythematosus (SLE), an autoimmune disease that affects multiple organs, is often difficult to evaluate because of the presence of abundant confounding factors. To assess the effect of increased expression of the phosphatase PP2A in T cells, as recorded in SLE patients, we generated a transgenic mouse that overexpresses the PP2Ac subunit in T cells. The transgenic mouse displays a heightened susceptibility to immune-mediated glomerulonephritis in the absence of other immune defects. CD4⁺ T cells produce increased amounts of IL-17 while the number of neutrophils in the peripheral blood is increased. IL-17 neutralization abrogated the development of glomerulonephritis. We conclude that increased PP2Ac expression participates in SLE pathogenesis by promoting inflammation through unchecked IL-17 production and facilitating the development of end-organ damage.

Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown etiology (1). Predisposition to its development results from the combined effect of a large number of genes, although the risk conferred by each is small and exerted through unknown mechanisms (2). The phenotype and function of T cells is abnormal in patients with SLE due to defects in signaling pathways and activity of transcription factors (1). These include increased calcium response and tyrosine phosphorylation upon TCR engagement as well as augmented secretion of pro-inflammatory cytokines including IL-17 (3,4). SLE-associated T cell abnormalities are thought to contribute to disease by facilitating immune tolerance loss and activation of autoreactive pro-inflammatory lymphocytes.

Protein phosphatase 2A (PP2A) is a ubiquitous and highly conserved serine/threonine phosphatase (5). Expression and activity of its catalytic subunit (PP2Ac) is increased in T cells from patients with SLE (6). Increased PP2Ac transcription is partially due to the presence of a lupus-associated SNP located in the intron 1 of PPP2CA, the gene that encodes PP2Ac (7). Loss of local inhibitory DNA methylation also contributes to its increased transcription in SLE (8). As in the case of other SLE-associated molecular abnormalities, it is not known if PP2Ac over-expression is of any pathogenic significance.

To determine whether abnormal levels of PP2Ac alter the immune response, and to dissect the mechanisms through which these contribute to SLE development, we generated a mouse that carries a T cell-restricted transgene that encodes PP2Ac. This allowed us to investigate the consequences of high PP2Ac in T cells in an otherwise normal organism. We demonstrate that over-expression of PP2Ac deviates the T cell cytokine production profile and confers increased susceptibility to inflammation and end-organ damage. Our results illustrate how defective regulation of a single autoimmune-associated molecule can alter the immune response and predispose an organism to develop autoimmunity.

MATERIALS AND METHODS

Mice

C57BL/6 PP2Ac transgenic mice were generated with a construct containing the Ppp2ca coding sequence placed between the human CD2 promoter and locus control region (9). Mice were housed in SPF conditions in accordance to the BIDMC IACUC and bred with C57BL/6J (The Jackson Laboratory). Non-transgenic littermates were used as controls. Mice between 8 and 12 weeks old were used in all experiments.

Antibodies and Reagents

Anti-PP2A C (clone 1D6, Millipore) Ab was used for western blot. Urinary albumin was quantified by ELISA (Bethyl Labs). IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 (p70), IL-17, GM-CSF, IFN-γ, TNF-α were measured using the Bioplex Mouse Cytokine Assay (Biorad). IL-21 was quantified with ELISA (Biolegend).

T cell isolation and stimulation

Naïve CD4 T cells were isolated by magnetic cell sorting (CD4⁺ CD62L⁺ T Cell Isolation Kit II, Miltenyi Biotec). Post-sorting cell purity was >95%. Cells were stimulated in full RPMI with plate bound goat anti-hamster Abs (MP Biomedicals) and soluble anti-CD3 (0.25 μg/mL, Clone 145-2C11, Biolegend) and anti-CD28 (0.5 μg/mL, Clone 37.51, Biolegend).

Antibody-induced glomerulonephritis (AIGN)

Experimental anti-glomerular basement membrane antibody-induced glomerulonephritis was induced as previously reported (10). Briefly, mice were immunized with rabbit IgG in CFA (day −3) and then injected i.v. with 200 μL of rabbit nephrotoxic serum (day 0).

Histology

Formalin fixed kidneys were processed for PAS (Periodic Acid-Schiff) and H&E. Kidney sections were evaluated blindly (10). Esterase reaction was performed on paraffin embedded kidney sections as described (10).

Neutrophil detection and isolation

Neutrophils were quantified in peripheral blood using a clinical hematology system (Hemavet, Drew Scientific) or flow cytometry. Bone marrow neutrophils were isolated using a three-layered Percoll gradient (Sigma). Neutrophils represented >80 % of the total cell population.

IL-17 neutralization

Rat anti-mouse IL-17A (Clone TC11-18H10.1, Biolegend) or purifed rat IgG₁κ isotype control (Clone RTK2071, Biolegend) were administered i.p. (100 μg).

RNA isolation and real-time PCR

RNA was isolated using TRIzol (Invitrogen). cDNA was produced from 500 ng of RNA (Reverse Transcription System, Promega). Real time PCR was performed using SYBR green (LightCycler 480 SYBR green I Master, Roche).

Statistical Analyses

Student two-tailed t tests and Mann-Whitney tests were used. A P<0.05 was considered significant. Results are expressed as the mean ± SEM, unless noted otherwise.

RESULTS

Increased PP2Ac levels do not alter the development of the immune system

PP2A transgenic (PP2Ac) mice had 5 copies of the transgene that resulted in a ~30% increase in PP2Ac expression at mRNA and protein levels (Supplemental Fig. 1). Their development was normal and no spontaneous disease was observed. A mild increase in the size of the spleen and lymph nodes was noted, but the architecture of lymphoid organs was normal (Supplemental Fig. 1). PP2Ac mice had a slight increase in the CD4:CD8 T cell ratio and a modest increase in the number of germinal center B cells in mesenteric lymph nodes (Supplemental Fig. 2).

PP2Ac over-expression promotes susceptibility to glomerulonephritis

PP2Ac mice did not develop any signs of autoimmunity, including autoantibodies, indicating that PP2Ac over-expression does not lead to loss of self-tolerance. To determine if PP2Ac mice are more susceptible to the development of organ damage once tolerance is breached, we induced AIGN in transgenic and wild type (WT) mice. To this end, mice were immunized with rabbit IgG in CFA and 3 days later injected with rabbit nephrotoxic serum. No differences in Ab titers or isotype were observed after immunization with rabbit IgG (Supplemental Fig. 2C).

Glomerular injury was significantly more severe in transgenic mice than in WT littermates (Fig. 1A). This was particularly apparent by increased extracapillary proliferation and glomerular deposition of PAS-positive material (Fig. 1B). Proteinuria was 5-fold higher in transgenic mice compared to WT mice at day 7 and 2-fold higher at day 10 after induction of AIGN (Fig. 1A). This indicated that PP2Ac over-expression can increase the susceptibility to immune-mediated tissue injury. Since neutrophils are centrally involved in the instigation of renal damage in AIGN (10), we investigated their presence in the kidney sections. Kidney infiltration by neutrophils was increased in transgenic mice (Fig. 1C) suggesting that PP2Ac facilitated AIGN damage by promoting local neutrophil accumulation.

Figure 1. Increased levels of PP2Ac promote organ damage and neutrophil infiltration.

Glomerulonephritis was induced in WT or PP2Ac transgenic mice. (A) Disease severity was quantified as proteinuria (urinary albumin:creatinine ratio) at the indicated time points (Mean ± SEM). Representative images of PAS-stained sections (B) or esterase staining to demonstrate neutrophils (C) are shown. Bars indicate 100 μm (upper panels) or 50 μm (lower panels). Data are representative of 3 experiments, each with ≥5 mice per group.

Increased PP2Ac expression by T cells raises neutrophil levels in the peripheral blood

We analyzed peripheral blood leukocyte subsets in WT and transgenic mice. Although the numbers of lymphocytes and monocytes were not different, neutrophil counts were significantly increased in transgenic mice (P=0.01; Fig. 2A). This was corroborated by flow cytometry (Fig. 2B). This effect was due to increased neutrophil generation since a significantly higher percentage of bone marrow cells were Gr-1⁺CD11b⁺ in transgenic mice than in WT littermates (Fig. 2C). To rule out the possibility that the transgene was expressed in neutrophils and the observed effects were driven by a non-T cell mediated mechanism, we quantified PP2Ac expression in purified neutrophils by real time PCR. As shown in Fig. 2D, there was no difference in the expression of PP2A in neutrophils from WT and transgenic mice suggesting that the high neutrophil bone marrow generation, high peripheral blood numbers, and accumulation in the kidneys were promoted by a T cell-derived factor.

Figure 2. Increased PP2Ac promotes neutrophil generation and accumulation.

(A) Peripheral blood neutrophils were quantified in unmanipulated mice using a clinical hematology analyzer (Hemavet). (B) Peripheral blood was stained with anti-Gr-1 and analyzed by flow cytometry. The gate indicates a granular Gr-1+ population that corresponds to neutrophils. The indicated frequency represents mean ± SEM. (C) Bone marrow cell suspensions were fractionated with Percoll density centrifugation. Cells corresponding to neutrophils were isolated and stained with anti-Gr-1 and anti-CD11b antibodies to determine the relative neutrophil abundance. (D) Ppp2ca mRNA levels were quantified in neutrophils by real-time PCR and normalized to Actb (expressed as Mean + SD). Data are representative of at least 3 experiments, each with ≥3 mice per group.

Increased PP2Ac induces IL-17 production

Because cytokine production is the main channel through which T cells influence the behavior of the immune system, we analyzed the effect of PP2Ac over-expression on cytokine production in naïve CD4 T cells. Naïve CD4 T cells were stimulated with anti-CD3 and anti-CD28 antibodies during 16 hours and the concentration of 12 cytokines was quantified in culture supernatants. Cytokine secretion was remarkably similar between WT and PP2Ac transgenic mice with the exception of IL-17A. Its concentration was ~9 fold-higher in supernatants from PP2Ac transgenic mice than in WT cells (Fig. 3A and data not shown). The increased cytokine protein levels were paralleled by increased mRNA detected by PCR (Fig. 3B). Quantitative PCR analyses confirmed that only transcription of Il17a and Il17f was affected; mRNA levels of Ifng, Il4, and Il21 were not increased (Fig. 3C). These results indicated that increased PP2Ac levels facilitate Il17a and Il17f transcription in a specific manner and suggested that the susceptibility to tissue injury was caused by increased T cell production of IL-17. To determine if IL-17 secretion occurred in vivo in transgenic mice during steady state conditions, we isolated lymph node cells and stained them with an anti-IL-17 antibody. As shown in Fig. 3D, spontaneous production of IL-17 was significantly more common in transgenic mice than in WT littermates (P=0.02). Increased IL-17 production was restricted to CD4⁺ T cells. Spontaneous IL-17 secretion could be responsible for the increased numbers of circulating neutrophils since IL-17 is a major determinant of their levels (11). Moreover, increased IL-17 levels could underlie the enhanced renal damage upon AIGN since IL-17 has been shown to be pathogenic in this model of glomerulonephritis (12).

Figure 3. Increased PP2Ac levels promote IL-17A and F production from naïve CD4 T cells.

(A) Cytokine concentrations in supernatants from naïve CD4 T cells stimulated 16 hours with anti-CD3 and anti-CD28 Abs were quantified using a suspension array system (IFN-γ, IL-4, IL-17) or ELISA (IL-21). Each dot represents one mouse. (B) PCR analyses of the expression of Il17a, Il17f, and Actb (β-actin) in resting and stimulated naïve CD4 T cells. Each lane corresponds to one mouse. (C) Real-time PCR analyses of the expression of the indicated genes in naïve CD4 T cells stimulated as in (A). Results were normalized to Actb (ΔCT); shown is fold expression relative to WT cells (ΔΔCT, mean + SEM). (D) Percentage of lymph node CD4⁺ T cells that spontaneously produce IL-17 in vivo quantified by flow cytometry. Data are representative of 2 to 4 experiments, each with ≥3 mice per group.

IL-17 neutralization abrogates the pathogenic effect of PP2Ac

In order to determine if heightened IL-17 secretion was responsible for the increased neutrophil counts and susceptibility to glomerulonephritis in PP2Ac transgenic mice, we treated mice with anti-IL-17 antibody or an isotype control. Two days after a single injection of the antibodies, numbers of peripheral blood neutrophils decreased significantly in mice treated with anti-IL-17 (Fig. 4A; P=0.02).

Figure 4. IL-17 neutralization abrogates PP2Ac-mediated neutrophil and kidney pathology.

(A) Peripheral blood neutrophils were quantified before and after treatment with anti-IL-17 or isotype control (IC). (B) PP2Ac transgenic mice received anti-IL-17 or IC every 3 days during the induction of glomerulonephritis. Kidney damage was assessed by proteinuria quantification at the indicated time points (mean + SEM). (C) Representative images of PAS-stained sections are shown. White bars indicate 100 μm (upper panels) or 50 μm (lower panels).

To evaluate the effect of IL-17 blockade on susceptibility to glomerulonephritis, we induced glomerulonephritis in mice treated with anti-IL-17 or isotype control every 3 days during the course of the experiment. As shown in Fig. 4B, treatment with anti-IL-17 decreased significantly the amount of proteinuria at days 7 and 10 and the extent of histological damage (Fig. 4C).

DISCUSSION

We provide evidence that increased PP2Ac levels in T cells cause increased production of IL-17A and F manifested in vivo by high neutrophil counts and susceptibility to glomerulonephritis. B6 mice are relatively resistant to AIGN (13) and therefore the new mouse can be used to study acute inflammatory nephritis including early stages of lupus nephritis where neutrophils enter the glomeruli in response to immune complex deposition or in situ formation (14). Because neutrophils are frequently activated in patients with SLE (15) this mouse may provide additional insights in exploring organ damage. Our results illustrate a mechanism by which inappropriate regulation of a gene associated with SLE in humans can alter the immune response and contribute to development of disease.

SLE is thought to develop gradually in individuals with risk factors (1). Loss of immune tolerance to self antigens, manifested as autoantibodies, develops years before the onset of clinical symptoms (16). However, overt clinical manifestations occur only when other poorly understood factors precipitate inflammation in target organs such as the kidney. Here we have shown that increased expression of PP2Ac does not induce tolerance failure, as we did not detect signs of autoimmunity in aging mice. Its pathogenic effect is exerted by facilitating a pro-inflammatory immune response, which promotes organ injury once tolerance is lost. IL-17 production has been shown repeatedly to be augmented in patients with SLE (3,4,17), but the cause has remained unknown.

Inflammation triggered by immune complex deposition is an important trigger for tissue injury in SLE (18). Neutrophils are key effector cells that instigate local inflammation in response to immune complexes during glomerulonephritis and leukocytoclastic vasculitis in patients with SLE (19). Our results illustrate one pathway that may be involved in the increased production of IL-17 in SLE, and demonstrate how unchecked secretion of IL-17 can facilitate kidney disease in an in vivo setting. Moreover, it indicates a mechanism that may underlie the granulopoiesis signature observed in patients with pediatric SLE (20).

In previous work we have shown that high levels of PP2A in total human T cells are associated with decreased production of IL-2 (6). IL-2 production was not affected in naïve CD4 T cells. This fact highlights the differences in the regulation of the IL-2 locus in naïve and antigen experienced T cells (21,22) and will be explored further in future work.

We have demonstrated a pathway that links a genetic and biochemical aberration associated to SLE in clinical studies (increased levels of PP2Ac) to the generation of a discrete pathogenic defect (heightened IL-17 production) and the susceptibility to develop severe kidney damage in the presence of a tolerance breach.