T cell specific STAT3 deficiency abrogates lupus nephritis

Abstract

Signal Transducer and Activator of Transcription (STAT) 3 is a regulator of T cell responses to external stimuli, such as pro-inflammatory cytokines and chemokines. We have previously shown that STAT3 is activated (phosphorylated) at high levels in SLE T cells and mediates chemokine induced migration and T:B cell interactions. Stattic, a small molecular STAT3 inhibitor can partially ameliorate lupus nephritis in mice. To better understand the role of STAT3 in T cell pathophysiology in lupus nephritis and its potential as a treatment target, we silenced its expression in T cells using a cd4-driven CRE-Flox model. We found that lupus prone mice that do not express STAT3 in T cells, did not develop lymphadenopathy, splenomegaly or glomerulonephritis. Moreover, the production of anti-dsDNA antibodies was decreased in these mice compared to controls. To dissect the mechanism, we also used a nephrotoxic serum model of nephritis. In this model, T cell specific silencing of STAT3 resulted in amelioration of nephrotoxic serum induced kidney damage. Taken together our results suggest that in mouse models of autoimmune nephritis, T cell specific silencing of STAT3 can hamper their ability to help B cells to produce autoantibodies and induce cell tissue infiltration. We propose that STAT3 inhibition in T cells represents a novel approach in the treatment of SLE and lupus nephritis in particular.

Introduction

Although the pathophysiology of systemic lupus erythematosus (SLE) is complex and not solely dependent on a single population of cells, T cells do play a central role in propagating the autoimmune response ¹. SLE T cells provide help to (auto)antibody producing B cells, infiltrate the kidneys, the skin and other tissues, and produce pro-inflammatory cytokines ². In addition the regulatory Treg cells are underperforming in SLE ^{3, 4} partly because of deficient production of interleukin (IL)-2 ⁵.

We have shown in previous work that, at least in part, the phenotype of SLE T cells depends on the aberrant activation (phosphorylation) of the transcription factor STAT3 (signal transducer and activator of transcription-3). Phosphorylated (p)-STAT3 enables SLE T cells to respond to chemokines and migrate more efficiently in vitro ⁶. Moreover, inhibition of p-STAT3 using the small molecule Stattic resulted in decreased production of most pro-inflammatory cytokines by SLE and murine lupus T cells in vitro ⁷. When we used the same small molecule Stattic to treat lupus prone mice, we observed that although STAT3 inhibition delayed by few weeks the development of nephritis, the mice did eventually develop proteinuria, at even higher levels than the control-treated mice.

Non-cell specific inhibition of STAT3 using a small molecule, while providing some preliminary evidence of the importance of this pathway, left several questions unanswered. STAT3 is widely expressed in a variety of immune and non-immune cells, such as B cells and monocytes/macrophages which also play significant role in the pathophysiology of SLE.

Therefore, Stattic may have delayed the onset of nephritis due to inhibition of STAT3 in these cells. More importantly, inhibition of STAT3 may have paradoxical pro-inflammatory effects ⁸. Specifically, STAT3 activation is known to change the balance of M1/M2 macrophages towards the regulatory M2 macrophages. Therefore, Stattic by non-specifically inhibiting STAT3 may have increased the M1/M2 ratio and promoted tissue inflammation.

To address the role of STAT3 in SLE T cell pathophysiology and its potential as a treatment target, we generated mice that lacked STAT3 expression only in T cells. Herein we present evidence that this T cell specific STAT3 deletion results in amelioration of nephritis in a mouse model that develops spontaneously lupus and in an experimental nephritis model.

Materials and Methods

Mice

B6.Stat3^fl/fl mice (Jackson Laboratories, Bar Harbor, ME) were crossed with B6.Cd4^cre (Jackson Laboratories, Bar Harbor, ME) to generate B6.cd4^cre− Stat3^fl.fl (wild type B6) and B6.cd4^cre+ Stat3^fl.fl (STAT3 T cell conditional knock out B6) mice. Similarly, we used lupus prone B6.MRL Fas^lpr mice (Jackson Laboratories, Bar Harbor, ME) to generate B6.MRL Fas^lpr cd4^cre− Stat3^fl.fl (wild type B6.lpr) and B6. MRL Fas^lpr cd4^cre+ Stat3^fl.fl (STAT3 T cell conditional knock out B6.lpr). All mice were housed at the Beth Israel Deaconess Medical Center pathogen-free animal facility (Boston, MA). Our protocol was approved by the BIDMC IACUC.

2.2. Experimental Nephrotoxic serum-induced nephritis (NSN)

Nephrotoxic rabbit serum was kindly given to us by Professor Tanya Mayadas (Brigham and Women’s Hospital and Harvard Medical School, Boston, MA). B6.cd4^cre− Stat3^fl.fl (B6 WT) and B6.cd4^cre+ Stat3^fl.fl (B6 STAT3 CKO) were injected with 0.2 mg rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) in Freund complete adjuvant (Sigma-Aldrich, Deisenhofen, Germany) as previously described ⁹. After 72 hours (see Fig 3A), the mice were injected intravenously with 50 mg heat inactivated nephrotoxic rabbit serum. The mice were followed for 21 days; urine was collected weekly. At the end of the experiment the mice were sacrificed and the kidneys harvested.

Figure 3. T cell specific STAT3 deficiency alleviates experimental anti-glomerular basement membrane antibody-induced glomerulonephritis (AIGN).

B6 Cd4^CRE−Stat3^/fl (B6) and B6 Cd4^CRE+Stat3^fl/fl (B6 STAT3 CKO) were injected with nephrotoxic serum (see Methods) and observed for 3 weeks. (A) Timeline of AIGN experimental setup. (B) The albumin/creatine ratio in the urine of mice from day 0 until the end of the experiment (day 21) is shown here (n=5/group). (C) The mice were sacrificed at the end of the experiment and their kidneys were harvested and stained with PAS. A representative kidney section is shown here. *= p<0.05. Error bar represents SEM.

Urine and serum analyses

Urine and serum were collected at various time points from individual mice. Levels of albumin and creatine were assessed in urine using commercially available kits: Albuwell M (Exocell) and Creatinine Parameter Assay Kit (R & D Systems) respectively. IgG specific to dsDNA was quantified by ELISA (Alpha Diagnostics).

Histopathology

Kidneys were harvested from exsanguinated mice and immediately fixed in formalin. Tissues were subsequently paraffin embedded, sectioned and stained with H and E. For each animal, 20 glomeruli and 20 tubulo-interstitial areas were scored in a blinded fashion ⁷. For immunofluorescence, tissues were snap frozen, sectioned and fixed in acetone. Slides were blocked with 10% BSA and stained with FITC labelled goat anti-mouse IgG or appropriate isotype controls (Jackson ImmunoResearch).

Statistical analysis

Statistical analyses were performed in GraphPad Prism version 5.0 software. Statistical significance was determined by t-tests (two-tailed). Statistical significance was defined as p<0.05. 3–5 mice were used for each experiment as indicated.

Results

T cell specific STAT3 deficiency alleviates the autoimmune response in lupus prone mice.

We used the Cre-Flox method to eliminate the Stat3 gene in cells that express CD4 in B6.lpr lupus prone mice. Since all thymocytes express CD4 at the double positive stage, all mature T cells and not just CD4⁺, did not express STAT3. We then followed these mice up to 40 weeks of age. We observed a significant decrease in the titers of anti-dsDNA antibody in the sera of the STAT3 deficient (B6.lpr STAT3 CKO) vs. wild type mice (B6.lpr WT) (Fig 1A, p<0.05). At 40 weeks we sacrificed these mice and harvested their spleens and lymph nodes. We observed a significant decrease in the size of the spleen and lymph nodes in B6.lpr STAT3 CKO vs. B6.lpr WT mice (Fig 2A, p<0.05). These data suggested that eliminating STAT3 expression only in T cells was sufficient to decrease the (auto) immune reaction in lupus prone mice.

Figure 1. T cell specific STAT3 deficiency abrogates the development of autoimmunity in lupus prone mice.

B6.lpr Cd4^CRE−Stat3^/fl (B6.lpr WT) and B6.lpr Cd4^CRE+Stat3^fl/fl (B6.lpr STAT3 CKO) were observed for 40 weeks. Serum was collected at various time points. (A) The serum level of anti-dsDNA antibodies is shown here (n=5). (B) The mice were sacrificed at 40 weeks and their spleens and lymph nodes were harvested and weighed. A representative pair of spleens and lymph nodes (left panel) and cumulative weight results from 5 WT and CKO B6.lpr mice (right panel) are shown here. *= p<0.05. Error bar represents SEM.

Figure 2. T cell specific STAT3 deficiency abrogates the development of nephritis in lupus prone mice.

B6.lpr Cd4^CRE−Stat3^/fl (B6.lpr WT) and B6.lpr Cd4^CRE+Stat3^fl/fl (B6.lpr STAT3 CKO) were observed for 40 weeks. Urine was collected every 4 weeks starting at 24 weeks. (A) The albumin/creatine ratio in the urine of mice is shown here (n=5). (B) The mice were sacrificed at 40 weeks and their kidneys were harvested and stained with PAS (left panel). The percent of glomeruli with crescents from 20 randomly chosen glomeruli was assessed in each mouse. Cumulative results from 5 mice per group are shown on the right panel. *= p<0.05. Error bar represents SEM.

T cell specific STAT3 deficiency prevents the development of nephritis in lupus prone mice.

We collected urine from both B6.lpr STAT3 CKO and B6.lpr WT mice every 4 weeks, starting at 28 weeks. We observed that B6.lpr WT mice had significantly higher levels of proteinuria compared to B6.lpr STAT3 CKO (Fig 2A) overtime. We then harvested the kidneys and performed histopathologic analysis. This showed a decrease by more than 50% on average of crescent formation in the B6.lpr STAT3 CKO vs. B6.lpr WT mice (Fig 2B, p<0.05). In addition, there was a significant decrease in IgG deposition as measured by immunofluorescence (Fig 2B). We then asked whether cell infiltration is decreased in the absence of STAT3 and found that not only glomerular but also interstitial pathology is alleviated in B6.lpr STAT3 CKO vs. B6.lpr WT mice (Fig 2C, p<0.05). We concluded that T cell specific STAT3 inhibition is associated with a clinically meaningful improvement in both glomerular and interstitial kidney pathology in lupus prone mice.

T cell specific STAT3 deficiency prevents experimental anti-glomerular basement membrane antibody induced glomerulonephritis (AIGN).

Arguably, STAT3 inhibition results in profound changes in how T cells get activated, differentiate, and migrate. The results from the lupus nephritis model suggested that the decrease in autoantibody production and deposition was the main reason that the Cd4-conditional STAT3-deficient mice had significantly milder disease. To further understand the role of STAT3 in T cell induced autoimmunity, we used the AIGN model of nephritis (Fig 3A). In this model, nephrotoxic serum is injected in the mice, which promptly develop glomerulonephritis. After injecting the mice with serum, we observed that Cd4-conditional STAT3-deficient mice developed significantly milder renal disease than wild type mice (Fig 2B, p<0.05 at day 21). The histopathology of the kidneys showed milder glomerular pathology in the STAT3-deficient mice (Fig 3C). The results from this model affirmed that STAT3 is not only important in T cell mediated-germinal center responses but also in the T cell response to deposition of immune globulins in the glomerular basement membrane of the kidney.

Discussion

We present data that genetically deleting the signaling molecule STAT3 exclusively in T cells, both murine lupus and experimental murine nephritis are significantly alleviated. STAT3 deficiency resulted in a profound decrease in B cell activation, autoantibody production, and cellular infiltration of the kidneys. Moreover, as opposed to non-specific STAT3 inhibition using Stattic, which may have affected B cells and monocytes, this approach proves the importance of T cell activation, migration and differentiation in the development of two different antibody-mediated nephritis models.

The exact reasons that STAT3 is aberrantly activated in SLE T cells are still under investigation. Activated STAT3 is regarded crucial for the development of pro-inflammatory Th cells such as Tfh and Th17 cells, especially in response to such cytokines as IL-6 and IL-23 ¹⁰. Moreover, STAT3 may play a role in downregulating Treg, a phenomenon well described in SLE ⁴. We have shown that IL-23/IL-23R signaling is upregulated in SLE and murine lupus ¹¹ and proposed that the enhanced IL-23 expression is mainly responsible for the STAT3 upregulation in SLE. Another potential mechanism for STAT3 activation is the exposure of SLE T cells to circulating chemokines such as SDF-1 ¹², which may help explain the decreased in T cell infiltration in the renal interstitial area in the absence of STAT3.

Our results suggest that T cell specific targeting of STAT3 may be a better therapeutic strategy than non-specific inhibition in SLE. Targeting upstream of STAT3 such as IL-23 or IL-6 inhibition may also prove efficacious; given though multiple pathways that converge at STAT3, this may not be as efficacious. Currently, Ustekinumab, an antibody that blocks the common p40 subunit of IL-23 and IL-12 is being evaluated in a phase III trial in SLE after showing positive phase II results ¹³. As it targets both IL-23 and IL-12 though, this antibody inhibits not only pro-inflammatory Th1 and Th17 cytokines but also the regulatory cytokine IL-2 ¹⁴, which could compromise its effectiveness. Baricitinib, a Jak1/2 inhibitor ¹⁵, that may inhibit among other mechanisms IL-6 dependent STAT3 activation, was modestly efficacious in SLE. The dose used to achieve effectiveness though has been shown to increase incidence of thrombosis in patients with rheumatoid arthritis, making this approach potentially toxic.

In conclusion, we present evidence that inhibition of STAT3 in T cells can ameliorate significantly lupus and experimental nephritis in mice. In contrast to non-specific STAT3 inhibition that may be suboptimal, T cell specific STAT3 targeting represents a novel approach to the treatment of SLE and lupus nephritis in particular.