Serine Phosphorylation of the STAT1 Transactivation Domain Promotes Autoreactive B Cell and Systemic Autoimmunity Development

Sathi Babu Chodisetti, Adam J. Fike, Phillip P. Domeier, Stephanie L. Schell, Taryn E. Mockus, Nicholas M. Choi, Chelsea Corradetti, Baidong Hou, Hannah M. Atkins, Roberto Caricchio, Thomas Decker, Aron E. Lukacher, Nancy Olsen and Ziaur S. M. Rahman
J Immunol May 15, 2020, 204 (10) 2641-2650; DOI: https://doi.org/10.4049/jimmunol.2000170

Key Points

  • STAT1-pS727 is required for SLE-associated AFC, GC, and autoantibody responses.

  • STAT1-pS727 in B cells promotes autoimmune AFC, GC, and autoantibody responses.

  • STAT1-pS727 is not required for foreign Ag– or gut microbiota–driven responses.

Abstract

Although STAT1 tyrosine-701 phosphorylation (designated STAT1-pY701) is indispensable for STAT1 function, the requirement for STAT1 serine-727 phosphorylation (designated STAT1-pS727) during systemic autoimmune and antipathogen responses remains unclear. Using autoimmune-prone B6.Sle1b mice expressing a STAT1-S727A mutant in which serine is replaced by alanine, we report in this study that STAT1-pS727 promotes autoimmune Ab-forming cell (AFC) and germinal center (GC) responses, driving autoantibody production and systemic lupus erythematosus (SLE) development. In contrast, STAT1-pS727 is not required for GC, T follicular helper cell (Tfh), and Ab responses to various foreign Ags, including pathogens. STAT1-pS727 is also not required for gut microbiota and dietary Ag–driven GC and Tfh responses in B6.Sle1b mice. By generating B cell–specific bone marrow chimeras, we demonstrate that STAT1-pS727 plays an important B cell–intrinsic role in promoting autoimmune AFC, GC, and Tfh responses, leading to SLE-associated autoantibody production. Our analysis of the TLR7-accelerated B6.Sle1b.Yaa SLE disease model expressing a STAT1-S727A mutant reveals STAT1-pS727–mediated regulation of autoimmune AFC and GC responses and lupus nephritis development. Together, we identify previously unrecognized differential regulation of systemic autoimmune and antipathogen responses by STAT1-pS727. Our data implicate STAT1-pS727 as a therapeutic target for SLE without overtly affecting STAT1-mediated protection against pathogenic infections.

This article is featured in In This Issue, p.2615

Introduction

Systemic lupus erythematosus (SLE) is a debilitating autoimmune disease characterized by the production of high-affinity antinuclear Abs (ANA). SLE is associated with the loss of peripheral B cell tolerance in the extrafollicular Ab-forming cell (AFC) and follicular germinal center (GC) pathways (1–3). However, mechanisms by which altered AFC and GC responses lead to the development of autoreactive B cells, ANAs and SLE are incompletely understood. Identifying the specific signals that are critical for SLE-associated AFC and GC responses may inform the development of targeted therapies for SLE.

In addition to the cognate interactions between B cells and Th cells, cytokines control the development of autoantibody-producing B cells. Previous studies have demonstrated the requirement of B cell–intrinsic type I and II (IFN-γ) IFN signaling in SLE-associated AFC and GC responses (4–6). Both type I IFN and IFN-γ, that contribute to SLE development, signal through the transcription factor STAT1. STAT1 was previously shown to play a critical role in the development of spontaneous GCs, T follicular helper cells (Tfh), and IgG autoantibodies (5, 7). Thus, targeting STAT1 downstream of IFN signaling could be an attractive treatment strategy for SLE. However, deficiency of STAT1 in humans and mice results in impaired immune responses and an increased susceptibility to infections (8, 9). Therefore, it is crucial to develop highly specific therapeutics for STAT1 that would eliminate autoreactive B cells without overtly affecting STAT1-mediated antipathogen immunity.

Activation of IFNR triggers STAT1 tyrosine-701 phosphorylation (STAT1-pY701) by receptor-associated JAK tyrosine kinases, causing STAT1 dimerization and nuclear translocation. However, for optimal transcriptional activity, STAT1 also needs to be phosphorylated at serine 727 (STAT1-pS727) in its C-terminal transactivation domain (TAD) (10–12). Serine phosphorylation of the STAT1 TAD, either induced by cytoplasmic or nuclear kinases (13, 14), regulates STAT1 function by allowing the recruitment of additional transcriptional coactivators to the promoters of STAT1 target genes (10–12). By expressing a STAT1-S727A mutant, in which serine is replaced by alanine, previous in vitro studies in macrophages have shown that serine phosphorylation of the STAT1 TAD (STAT1-pS727) is important for RNA polymerase II recruitment to the promoters of STAT1 target genes and the consequent regulation of 40–50% of IFN-γ–induced genes in response to IFN-γ stimulation (14). The role of STAT1-pS727 in eliciting innate immune responses has also been described previously (10). However, the requirement of STAT1-pS727 in antipathogen AFC, GC, and Ab responses is not clear. Moreover, the role of STAT1-pS727 in autoimmune AFC and GC responses, autoantibody production, and SLE pathogenesis is not known.

By crossing B6.STAT1-S727A mutant mice (10) to the autoimmune-prone B6.Sle1b mouse model that develops a moderate level of autoimmunity without significant disease manifestations, we demonstrate a crucial role of STAT1-pS727 in autoimmune AFC and GC responses and autoantibody production (15, 16). We also observe an important B cell–specific function of STAT1-pS727 in promoting these autoimmune processes. These reduced autoimmune responses in SLE-prone B6.Sle1b mice in the absence of STAT1-S727 phosphorylation are not due to a defect in primary bone marrow (BM) and splenic B cell development in STAT1-S727A mutant mice. By analyzing the TLR7-promoted SLE disease model expressing a STAT1-S727A mutant, we observe significantly reduced SLE-associated AFC, GC, and autoantibody responses and ameliorated kidney pathology. STAT1-pS727, however, is not necessary for GC and Tfh responses to foreign Ags, including 4-hydroxy-3-nitrophenol-keyhole-limpet-hemocyanin (NP-KLH), virus-like particles (VLPs), and mouse polyomavirus (muPyV) infection. Interestingly, STAT1-pS727 is also not required for GC and Tfh responses to gut microbiota and dietary Ags. These data suggest a differential regulation of autoimmune and antipathogen responses by STAT1-pS727 and identify STAT1-pS727 as a potential therapeutic target for SLE that does not overtly compromise the protective immunity to pathogens in SLE patients.

Materials and Methods

Mice

C57BL/6J, B6.SB-Yaa/J, and B6.129S2-Ighmtm1Cgn/J (μMT) mice were originally purchased from The Jackson Laboratory and bred in house. The B6.Sle1b mice (congenic for the Sle1b sublocus) were described previously (15). B6.Sle1b.yaa (Sle1bYaa) mice were generated by crossing B6.Yaa mice to B6.Sle1b mice. Previously described B6.STAT1-S727A (B6.129P2-Stat1tm1Tdec) mice (10) were crossed to B6.Sle1b and Sle1bYaa background to generate B6.Sle1b.STAT1-S727A (Sle1b.STAT1-SA) and B6.Sle1byaa.STAT1-S727A mice. All animal studies were conducted at Pennsylvania State University Hershey Medical Center in accordance with the guidelines approved by our Institutional Animal Care and Use Committee. Animals were housed in a specific pathogen-free barrier facility.

Imiquimod treatment, viral infection, and immunization

For epicutaneous imiquimod (IMQ) treatment, 5% IMQ cream (Glenmark Pharmaceuticals) was applied on the ears of mice three times weekly for 4–12 wk, based on the experimental design as previously described (17–19). To study the systemic autoimmune responses, such as AFC, GC, Tfh, and Ab responses, mice were treated for 8 wk. For viral infection, 10–12-wk-old mice were inoculated intracerebrally with 3 × 105 PFU of muPyV strain and analyzed 12 d postinfection. For immunization studies, 10–12-wk-old mice were immunized with 200 μg/mouse of NP-KLH (Biosearch Technologies) i.p. in CFA (Sigma-Aldrich) followed by immunization with 100 μg of NP-KLH in IFA on day 7. Spleen cells were prepared from these mice and analyzed on 14 d post–primary immunization. Ten to twelve weeks old mice were immunized (i.p) with 25 μg of purified Qβ-VLPs in 250 μl of PBS as previously described (20), and spleen cells were analyzed 10 d postimmunization.

Flow cytometry

Flow cytometric analysis of total mouse splenocytes or BM cells was performed using the following Abs: B220-BV605 (RA3-6B2), CD4-AF700 (RMP4-5), CD44-APC (IM7), CD62L-PECy7 (MEK-14), PD1-PE (29F.1A12), IgM-BV605 (RMM-1), IgD-BV711 (11-26c2a), CD93-PE (AA4.1), Streptavidin (SA)–PECy5, MHC class II–PECy7 (M5/114.15.2), Ly51-biotin (6C3), CD24-APC (M1/69), CD23-biotin (B3H4). GL7-FITC (GL-7), CD95-PECy7, CXCR5-biotin (2G8), CD43-FITC (S7), CD19-biotin (1D3), CD90.2-biotin (53-2.1), CD11b-AF700 (M1/70), CD11c-FITC (HL3) (BD Biosciences), and CD8α (clone 53–6.7) (eBioscience). All cells were stained with fixable viability dye eFluor780 (Invitrogen) prior to surface staining. Stained cells were analyzed using the BD LSR II Flow Cytometer (BD Biosciences). Data were acquired using FACSDiva software (BD Biosciences) and analyzed using FlowJo software (Tree Star).

Immunofluorescence and ANA staining

Mouse spleens or kidneys were embedded in OCT compound and snap frozen over liquid nitrogen. Five micrometers–thin sections were cut on a cryostat, mounted on ColorFrost Plus Microscope Slides (Thermo Fisher Scientific), and fixed in cold acetone for 20 min. The following Abs and reagents were used for immunofluorescence staining of mouse spleen sections for GCs: PE–anti-CD4 (GK1.5; BioLegend), FITC-GL7 (RA3-6B2; BD Biosciences), and APC–anti-IgD (11-26c2a; BD Biosciences). Kidney sections were stained for C3 using FITC–anti-C3 (Immunology Consultants Laboratory) or biotin–anti-IgG (Jackson ImmunoResearch) followed by SA-PE. Antinuclear Ab (ANA) reactivity was detected by indirect immunofluorescence staining of HEp-2 cell slides using sera from indicated mice at a 1:50 dilution and probed with FITC-rat anti-mouse κ (H139-52.1). The images of stained spleen and kidney sections were captured using the Leica DM4000 fluorescence microscope and analyzed using a Leica Application Suite–Advanced Fluorescence Software (Leica Microsystems). For the measurement of the GC area, randomly selected GCs (GL-7+) were measured for total area (square micrometer) using the Leica Application Suite–Advanced Fluorescence Software quantitation tool.

Kidney histopathology

Kidneys from 6-mo-old mice were fixed in 10% neutral buffered formalin and embedded in paraffin. Kidney sections were cut at 3-μm thickness for periodic acid–Schiff. All images were obtained with an Olympus BX51 microscope and DP71 digital camera using cellSens Standard 1.12 imaging software (Olympus, Center Valley, PA). Two pathologists blinded to the genotype of mice evaluated the kidney sections. One kidney section per mouse was evaluated; each glomerulus was examined at 400× magnification and scored from 0 (normal) to 4 (severe) based on glomerular size and lobulation, presence of karyorrhectic nuclear debris, capillary basement membrane thickening, and the degree of mesangial matrix expansion and mesangial cell proliferation as described (21, 22).

ELISpot assay

ELISpot assays were performed as previously described (5, 6). Briefly, splenocytes in RPMI 1640 containing 10% FBS were plated at a concentration of 1 × 106 cells/well onto salmon sperm dsDNA- (Invitrogen), nucleosome- (histone from Sigma-Aldrich plated on a layer of dsDNA coating), or Smith/ribonucleoprotein (SmRNP)- (AROTEC Diagnostics) coated multiscreen 96-well filtration plates (MilliporeSigma, Bedford, MA). Serially diluted (1:2) cells were incubated for 12 h at 37°C. dsDNA-, nucleosome-, and SmRNP-specific AFCs were detected by biotinylated anti-κ Ab (Invitrogen) and SA–alkaline phosphatase (Vector Laboratories) or alkaline phosphatase–conjugated anti-mouse IgG (Molecular Probes). Plates were developed using the Vector Blue Alkaline Phosphatase Substrate Kit III (Vector Laboratories). ELISpots were enumerated and analyzed using a computerized ELISpot plate imaging/analysis system (Cellular Technology).

ELISA

Serum autoantibodies were measured using standard ELISA protocols as described (16). Briefly, total IgG autoantibodies titers were measured in ELISA plates coated with salmon sperm dsDNA, nucleosome, or SmRNP and detected with biotinylated secondary Ab followed by SA–alkaline phosphatase (Vector Laboratories). Plates were developed using p-Nitrophenyl Phosphate (Disodium Salt) (Thermo Fisher Scientific) substrates for alkaline phosphatase and read at λ405 nm on Synergy H1 (BioTek Instruments).

Generation of mixed BM chimeric mice

Twelve-weeks-old female B6.μMT mice were lethally irradiated with two doses of 450 rad of x-rays (X-RAD 320iX Research Irradiator; Precision X-Ray) within a 4-h interval. Within a few hours of the second irradiation, each B6.μMT recipient mice received i.v. (tail vein) 10 × 106 T cell–depleted BM cells isolated from 10-wk-old female donor mice with 80% of cells from B6.μMT mice and 20% from B6.Sle1b or Sle1b.STAT1-SA mice. Recipients were analyzed for spontaneous GC B cell and Tfh cell development and ANA-specific AFC and autoantibody responses 11 wk after BM cell transfer.

Statistical analysis

The p values were calculated using unpaired, nonparametric, Mann–Whitney, Student t test, or two-way ANOVA, with a follow-up Sidak multiple comparison test (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001). GraphPad Prism 6 software was used.

Results

STAT1-pS727 promotes autoimmune AFC and GC responses

Although the role of STAT1 tyrosine 701 phosphorylation (designated STAT1-pY701) in nuclear translocation and transcriptional activity is well described, how STAT1 serine 727 phosphorylation (designated STAT1-pS727) may regulate STAT1 function in the in vivo systems has been underexplored. Especially, the involvement of STAT1-pS727 in the regulation of autoimmune AFC, GC, Tfh, and autoantibody responses is not known. To determine the role of STAT1-pS727 in autoimmune AFC, GC, and Tfh responses, we crossed SLE-prone B6.Sle1b mice to B6.STAT1-S727A mutant mice (10) to generate Sle1b.STAT1-SA in which serine 727 in STAT1 is replaced with alanine. Spontaneous autoimmune responses assessed in 5–6-mo-old Sle1b.STAT1-SA female mice showed a reduced frequency of GC B cells and Tfh cells (Fig. 1A, 1B) and reduced size and number of splenic GCs (Fig. 1C–E) compared with B6.Sle1b control mice. Sle1b.STAT1-SA mice also had a reduced number of dsDNA and nucleosome-specific splenic (Fig. 1F) and BM (Fig. 1G) AFCs that strongly correlated with reduced serum autoantibody titers and ANA seropositivity (Fig. 1H, 1I). Together, these data highlight the critical role of STAT1-pS727 in autoreactive B cell differentiation into AFCs and GC B cells and autoantibody production.

FIGURE 1.
FIGURE 1.

STAT1-pS727 regulation of autoimmune GC, AFC, and autoantibody responses. Representative flow cytometry plots (A) and the percentage (B) of splenic B220+GL7hiCD95hi GC B cells of total B220+ B cells and CD4+CD44hiPD-1hiCXCR5hi Tfh cells of total CD4+ T cells. Representative histological images show GCs (C), quantification of GC area (D), and GC frequency (E) in spleens. Quantification of splenic (F) and BM (G) dsDNA- and nucleosome-specific AFCs by ELIspot. (H) dsDNA- and nucleosome-specific IgG Ab titers were measured by ELISA. (I) Representative images of serum ANA reactivity measured by Hep-2 clinical assay. The data shown were the cumulative results of three independent experiments generated from 5- to 6-mo-old B6.Sle1b and Sle1b.STAT1-SA female mice. Three to five mice were analyzed in each experiment. Each symbol indicates an individual mouse (B, F, and G) or GC (D and E). Statistical analysis was performed by two-way ANOVA, with a follow-up Sidak multiple comparison test (H) or unpaired, nonparametric Mann–Whitney Student t test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Reduced autoimmune responses in STAT1-S727A mutant mice are not due to a defect in primary B cell development

Next, we assessed whether the reduced autoimmune GC and AFC responses in Sle1b.STAT1-SA mice were due to defects in primary B cell development in the BM and peripheral secondary lymphoid organs. We analyzed subpopulations of the early B cell progenitors, such as B220+CD43+HSA−BP-1− fraction A, B220+CD43+HSA+BP-1− fraction B, B220+CD43+HSA+BP-1+ fraction C, B220+CD43−IgM−CD93+ fraction D, B220+CD43−IgM+CD93+ fraction E, and B220+CD43−IgM+CD93− fraction F in BM and found no significant differences in fractions A–F between Sle1b.STAT1-SA and B6.Sle1b control mice (Fig. 2A–H). We also analyzed peripheral B cell developmental stages in spleens, including B220+AA4.1+CD23−IgM+ transitional type 1, B220+AA4.1+CD23+IgM+ transitional type 2, and B220+AA4.1+CD23+IgM− transitional type 3 cells, B220+AA4.1−CD93−CD23−IgM+ marginal zone B cells, and B220+AA4.1−CD93−CD23+IgM+ mature/follicular B cells (Fig. 2I–R). We observed no significant differences in splenic B cell development between Sle1b.STAT1-SA and B6.Sle1b control mice (Fig. 2I–R), indicating that STAT1-pS727 is not required for either BM or peripheral B cell development. These data suggest that reduced autoimmune responses in SLE-prone B6.Sle1b mice in the absence of STAT1-S727 phosphorylation are not due to a defect in primary B cell development in STAT1-S727A mutant mice.

FIGURE 2.
FIGURE 2.

Mice expressing STAT1-S727A mutant have normal B cell development. Flow cytometry analysis showing gating strategies (A, E, I, L, and P) and percentages of B cell developmental fraction A (B220+CD43+HSA−BP-1−) (A and B), fraction B (B220+CD43+HSA+BP-1−) (A and C), fraction C (B220+CD43+HSA+BP-1+) (A and D), fraction D (B220+CD43−IgM−CD93+) (E and F), fraction E (B220+CD43−IgM+CD93+) (E and G), and fraction F (B220+CD43−IgM+CD93−) (E and H) of live cell gated BM cells from B6.Sle1b and Sle1b.STAT1-SA mice. Splenocytes from the same mice were characterized for peripheral B cell developmental stages in spleens, including AA4.1+ (I and J), AA4.1− (I and K), transitional type 1 (T1) (B220+AA4.1+CD23−IgM+) (L and M), transitional type 2 (B220+AA4.1+CD23+IgM+) (L and N), and transitional type 3 (B220+AA4.1+CD23+IgM−) (L and O), marginal zone (MZ) B cells (B220+CD93−CD23−IgM+) (P and Q), and mature/follicular B cells (FoB) (B220+CD93−CD23+IgM+) (P and R) of total B220+ B cells. These data are representative of two independent experiments (four to five mice in each experiment). Each symbol in each panel represents a mouse. Statistical analysis was performed by unpaired, nonparametric Mann–Whitney Student t test. ns, nonsignificant.

STAT1-pS727 is dispensable for foreign Ag, gut microbiota, or dietary Ag–driven GC, Tfh, and Ab responses

Given the impact of STAT-pS727 on the regulation of autoimmune responses, we investigated the requirement of STAT-pS727 in immune responses to foreign Ags, including viral infection. We first immunized mice with the T cell–dependent Ag NP-KLH. Surprisingly, GC and Tfh responses were similar between Sle1b.STAT1-SA and B6.Sle1b control mice 14 d postimmunization (Fig. 3A). The GC size, GC number, and 4-hydroxy-3-nitrophenol (NP)-specific GC B cell frequency were also comparable between the strains (Fig. 3B–E). Likewise, we observed comparable high (NP4) and low (NP29) affinity NP-specific Ab responses between Sle1b.STAT1-SA and control mice (Fig. 3F). B cells have recently been shown to be the dominant APCs involved in the induction of Tfh differentiation in virus-derived nanoparticle immunization (23). Thus, next we analyzed the B cell responses to VLP. Upon VLP immunization, Sle1b.STAT1-SA mice showed identical GC and Tfh frequencies compared with control mice (Fig. 3G–L). The VLP-specific (QB+) total and GC B cells were also similar between the groups (Fig. 3J). To further explore B cell responses to a pathogen, we used an infection model, in which we infected mice intracerebrally with muPyV. muPyV also induced comparable GC and Tfh responses in both the groups (Fig. 3K, 3L). Virus specific Ab titers (Fig. 3M), cytokine-producing and virus specific effector CD8+ T cells, and viral titers in spleen and brain were similar between the groups (data not shown). Additionally, gut microbiota and dietary Ag–driven GC and Tfh responses in Peyer’s patches and mesenteric lymph nodes in Sle1b.STAT1-SA mice were comparable to B6.Sle1b control groups (Fig. 3N). Together, these data using multiple models of foreign antigenic challenge or gut microbiota highlight no significant role for STAT-pS727 in foreign Ag–, gut microbiota–, or dietary Ag–driven GC B cell, Tfh, and Ab responses.

FIGURE 3.
FIGURE 3.

STAT1-pS727 deficiency does not alter immune response to T cell–dependent Ags, VLPs, and viral infection. (A–F) These data were generated from spleens of NP-KLH immunized B6.Sle1b and Sle1b.STAT1-SA female mice. (A) Quantification of B220+GL7hiCD95hi GC B cells and CD4+CD44hiPD-1hiCXCR5hi Tfh cells that were gated on total B220+ B and CD4+ T cells, respectively. Representative histological images show GCs (B), GC area (C), and GC frequency (D) in spleen sections. (E) Percentage of NP18-specific B220+GL7hiCD95hi GC B cells that were gated on total B220+ B cells. (F) NP4- and NP29-specific IgG titers were measured by ELISA. (G–J) These data were derived from VLP-immunized B6.Sle1b and Sle1b.STAT1-SA female mice. (G) Flow cytometry data depict the frequency of splenic B220+GL7hiCD95hi GC B cells and CD4+CD44hiPD-1hiCXCR5hi Tfh cells that were gated on total B220+ B and CD4+ T cells, respectively. Quantification of GC area (H) and GC frequency (I) in spleen sections. (J) Percentage of splenic VLP-specific Qβ+ B and GC B cells. (K–M) These data were derived from mPyV-infected B6.Sle1b and Sle1b.STAT1-SA female mice. (K) Frequency of splenic B220+GL7hiCD95hi GC B cells and CD4+CD44hiPD-1hiCXCR5hi Tfh cells. (L) Quantification of GC area and GC frequency in spleen sections. (M) VP1-specific IgG titers measured by ELISA. (N) Quantitation of GC B cells and Tfh cells from total B220+ B and CD4+ T cells, respectively, in Peyer’s patches (PPs) and mesenteric lymph nodes (MLNs) in unimmunized mice. These data represent two to three experiments, and each symbol indicates an individual mouse or GC. Three to five mice were analyzed in each experiment. Statistical analysis was performed by two-way ANOVA, with a follow-up Sidak multiple comparison test (F and M) or unpaired, nonparametric Mann–Whitney Student t test. *p < 0.05. ns, nonsignificant.

B cell–intrinsic STAT1-pS727 regulates autoimmune AFC, GC, and Tfh responses

Next, to define the B cell–intrinsic role of STAT1-pS727 in regulating autoimmune AFC, GC, and Tfh responses, we generated mixed BM chimeras by reconstituting lethally irradiated B6.μMT mice, which lack mature B cells, with a mixture of BM cells from B6.μMT mice and B6.Sle1b or Sle1b.STAT1-SA mice as described (4, 5). Analysis of chimeras post–BM transfer revealed a significantly lower percentage of GC B cells and Tfh in Sle1b.STAT1-SA +μMT > μMT mice than B6.Sle1b +μMT > μMT control mice (Fig. 4A, 4B). Sle1b.STAT1-SA +μMT > μMT mice had a lower frequency of and smaller GCs than those in B6.Sle1b +μMT > μMT control mice (Fig. 4C–E). Sle1b.STAT1-SA +μMT > μMT mice had significantly decreased autoantibody-producing AFCs (Fig. 4F), serum autoantibody titers (Fig. 4G), and ANA seropositivity (Fig. 4H) than B6.Sle1b +μMT > μMT control chimeras. These data demonstrate an important B cell–intrinsic role for STAT1-pS727 in the regulation of autoimmune AFC, GC, and Tfh responses.

FIGURE 4.
FIGURE 4.

B cell–intrinsic role of STAT1-pS727 in autoimmune AFC, GC, and Tfh responses. Quantitation of B220+GL7hiCD95hi GC B cells gated on total B220+ B cells (A) and CD4+CD44hiPD-1hiCXCR5hi Tfh cells gated on total CD4+ T cells (B) in splenocytes isolated from BM chimeric B6.Sle1b + μMT > μMT and Sle1b.STAT1-SA + μMT > μMT female mice 10 wk post–BM cell transfer. Representative histological images of splenic GCs (C), and GC area (D) and frequency (E) were quantified. dsDNA- and nucleosome-specific splenic AFCs (F), serum dsDNA- and nucleosome-specific Abs (G), and serum ANA reactivity (H) are shown. Each symbol represents a mouse (A, B, and F) or a GC (D and E). These data represent one experiment with six recipient mice per group. Statistical analysis was performed by two-way ANOVA, with a follow-up Sidak multiple comparison test (G) or unpaired, nonparametric Mann–Whitney Student t test. *p < 0.05, **p < 0.01, ****p < 0.0001.

STAT1-pS727 regulates TLR7-accelerated autoimmune AFC, GC, and autoantibody responses

Having demonstrated the requirement of STAT1-pS727 in regulating moderate levels of autoimmune responses in B6.Sle1b mice, which do not develop SLE disease, we investigated the role of STAT1-pS727 in TLR7-accelerated autoimmune AFC and GC responses in the Sle1bYaa SLE disease model. We crossed Sle1b.STAT1-SA mice to the Sle1bYaa SLE disease model in which male mice overexpress TLR7 because of a translocation of a section of the X chromosome containing the Tlr7 gene (Sle1bYaa.STAT1-SA) (24–26). Male Sle1bYaa.STAT1-SA mice showed reduced splenomegaly compared with Sle1bYaa control mice (Fig. 5A, 5B). The reduction in splenomegaly was accompanied by a lower frequency of and reduced size of GCs (Fig. 5C, 5D) in Sle1bYaa.STAT1-SA mice compared with Sle1bYaa control mice. The number of autoantibody-producing splenic (Fig. 5E) and BM (Fig. 5F) AFCs, serum ANA reactivity (Fig. 5G), and autoantibody titers (Fig. 5H) in Sle1bYaa.STAT1-SA mice were also much lower than Sle1bYaa control mice.

FIGURE 5.
FIGURE 5.

STAT1-pS727 promotes TLR7-accelerated systemic autoimmune responses. Representative spleen size (A) and weight of spleens (B) from 6-mo-old Sle1bYaa and Sle1bYaa.STAT1-SA male mice. Representative histological images of splenic GCs (C), and quantified GC area and their frequency (D) are shown. Numbers of dsDNA-, nucleosome-, and SmRNP-specific splenic (E) and BM (F) AFCs; ANA reactivity (G); serum Abs against dsDNA, nucleosome, and SmRNP (H) were measured by ELISA. The percentages of splenic B220+CD11b+CD11c+ ABCs (I), GC B cells in Peyer’s patches (J), and mesenteric lymph nodes (K) that were pregated on total B220+ B cells from 6-mo-old mice were measured by flow cytometry analysis. These data represent two to four experiments, and each symbol indicates an individual mouse (B and E–K) or a GC (D). Four to five mice were analyzed in each experiment. Statistical analysis was performed by two-way ANOVA, with a follow-up Sidak multiple comparison test (H) or unpaired, nonparametric Mann–Whitney Student t test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Ns, nonsignificant.

TLR7-driven accumulation of age-associated B cells (ABCs) has recently been shown to be important for the development of SLE-like autoimmunity (27). Interestingly, we observed a reduced percentage of ABCs in Sle1bYaa.STAT1-SA mice compared with control mice (Fig. 5I). However, the gut microbiota– and dietary Ag–driven GC (Fig. 5J, 5K) and Tfh (data not shown) responses in Peyer’s patches and mesenteric lymph nodes of Sle1bYaa.STAT1-SA mice were similar to Sle1bYaa control mice. To further validate our findings, we used the TLR7 ligand IMQ treatment model, in which female mice were treated epicutaneously to accelerate autoimmune responses in the B6.Sle1b model following a protocol previously described (17–19). We found reduced splenomegaly (Supplemental Fig. 1A, 1B) and a significantly lower number of dsDNA- and nucleosome-specific splenic (Supplemental Fig. 1C) and BM (Supplemental Fig. 1D) AFCs in IMQ-treated Sle1b.STAT1-SA mice than IMQ-treated B6.Sle1b control mice. IMQ-treated Sle1b.STAT1-SA mice also had more reduced serum autoantibody titers (Supplemental Fig. 1E) than treated B6.Sle1b control mice. These findings together demonstrate that STAT1-pS727 regulates TLR7-accelerated autoimmune AFC and GC responses and autoantibody production.

STAT1-pS727 promotes TLR7-accelerated SLE pathogenesis

Having demonstrated the role of STAT1-pS727 in autoimmune GC and AFC responses and autoantibody production, we next investigated the importance of STAT1-pS727 in SLE pathogenesis, focusing on kidney pathology using TLR7-accelerated Sle1bYaa SLE disease model. We found that reduced autoimmune GC, AFC, ABC, and autoantibody responses in Sle1bYaa.STAT1-SA mice strongly correlated with reduced immune complex deposition in the kidney glomeruli of Sle1bYaa.STAT1-SA mice compared with Sle1bYaa control mice as evaluated by immunofluorescent staining of kidney sections for anti-C3 and anti-IgG (Fig. 6A). Consistent with reduced immune complex deposition, we observed significantly reduced glomerulonephritis in Sle1bYaa.STAT1-SA mice compared with Sle1bYaa control mice (Fig. 6B, 6C). These findings demonstrate that STAT1-pS727 regulates the TLR7-accelerated development of SLE.

FIGURE 6.
FIGURE 6.

STAT1-pS727 promotes TLR7-accelerated lupus nephritis. (A) Kidney sections from 6- to 8-mo-old Sle1bYaa and Sle1bYaa.STAT1-SA male mice were stained with anti-C3 (green) and anti-IgG (red). Representative images (B) of periodic acid–Schiff–stained kidney sections and the glomerulonephritis (GN) score (C) are shown from these mice. These data represent seven to eight mice from each group that was collected from two independent experiments. Statistical analysis was performed by two-way ANOVA, with a follow-up Sidak multiple comparison test or unpaired, nonparametric Mann–Whitney Student t test. **p < 0.01. Ns, nonsignificant.

Discussion

Our findings in this study provide novel insights into the role of STAT1-pS727 in promoting systemic autoimmunity and SLE disease development by regulating AFC, GC, Tfh, and autoantibody responses. Through the generation of B cell–specific BM chimeras, we demonstrated the B cell–intrinsic role of STAT1-pS727 in autoimmune AFC and GC responses, promoting autoantibody production. In a marked contrast, we found no contribution of STAT1-pS727 to foreign Ag– or pathogen-driven GC, Tfh, and Ab responses. STAT1-pS727 also did not play a significant role in GC and Tfh responses in GALT in B6.Sle1b mice, which were previously shown to be mediated by gut microbiota and dietary Ags (28–30). Together, these data collectively highlight the importance of STAT1-pS727 in promoting SLE-associated AFC, GC, and SLE development, and the dispensability of this mechanism for GC B cell, Tfh, and Ab responses to foreign Ags, including pathogenic infection or gut microbiota and dietary Ags. Importantly, our findings indicate a differential requirement for STAT1-pS727 between autoimmune and pathogen-driven responses, which may be the ideal scenario for the implementation of targeted SLE therapeutics that preserve antimicrobial immunity.

Although STAT1 downstream of type I and II IFN signaling promotes systemic autoimmunity and SLE disease, it also plays an important role in antipathogen responses (31). A number of patients with STAT1 deficiencies are highly susceptible to viral infections (8). Mice deficient in STAT1 are also highly sensitive to microbial and viral infections (32). STAT1 is expressed as α and β isoforms. Unlike STAT1α, STAT1β lacks the C-terminal TAD including serine-727 and shows attenuated function compared with STAT1α. Attenuated function of the β isoform is likely due to the ability of the TAD in the α isoform to interact with other transcriptional coregulators and promote maximal transcriptional activity of STAT1 (10, 11, 33). STAT1 deficiency in the TAD showed a significant attenuation of its interactions with other proteins (34, 35). Therefore, targeting entire STAT1 or the STAT1 TAD for treating SLE may predispose patients to lethal infections and death. We found significantly reduced autoimmune responses and alleviated SLE pathogenesis but intact immune responses to foreign Ags including muPyV infection in S727A mutant mice. These data suggest that the suboptimal activity of STAT1 in the absence of STAT1-pS727 is sufficient for mounting antipathogen but not autoimmune responses. Although inhibition of STAT1-pS727 by flavopiridol or by S727A mutation in a previous in vitro study did not change the amount of promoter-bound STAT1, it affected the expression of 40–50% of IFN-γ–induced genes (14). Previously published data and our current data together suggest that STAT1-pS727 is required to regulate the expression of genes that are important for autoimmune responses but dispensable for foreign Ag–driven responses.

In addition to IFN signaling, other factors were also previously shown to induce and regulate STAT1-pS727 function. One of the critical regulatory mechanisms that inhibits B cell responses is the inhibitory Fc receptor, FcγRIIb (36). Concurrent engagement of FcγRIIb and BCR with Ag and Ab complexes recruits the FcγRIIb into the BCR signaling complex to negatively regulate BCR signaling (37). Polymorphisms in the Fcgr2b gene or the absence of FcγRIIb signaling contributes to SLE development (38). Interestingly, BCR stimulation induces sustained STAT1-pS727 in B cells, whereas FcγRIIb inhibits BCR-induced STAT1-pS727 (39). Previous studies also highlighted the role of TLRs in inducing STAT1-pS727 in myeloid cells independent of IFN signaling (40), although TLR-mediated induction of STAT1-pS727 in B cells was previously not well explored. Using an in vitro stimulation system, a previous study demonstrated the role of TLR7 in the induction of phosphorylation of STAT1 at both Y701 and S727 (41). It was suggested that STAT1-pY701 but not STAT1-pS727, downstream of TLR7 and Bank1 in type I IFN response in B cells, contributed to SLE development in the TLR7-accelerated Sle1bYaa model (41). Consistent with this previous report, we have identified a role for TLR7 stimulation in the induction of STAT1-pS727 in B cells independent of IFN (both type I and II IFN) and IL-21R signaling (S.B. Chodisetti and Z.S.M. Rahman, unpublished observations). However, as opposed to the previous report (41), our findings demonstrate a role for STAT1-pS727 in TLR7-promoted SLE autoimmunity and disease development. These data indicate that STAT1-pS727 is at the intersection of several critical signaling pathways involved in immune cell function and signaling, and BCR, TLR, and IFN signaling in part promote autoimmunity and SLE disease development through regulation of STAT1-pS727. Furthermore, the kinase(s) responsible for STAT1-pS727 following activation of these pathways and the subcellular location of this phosphorylation event are unclear at this time (12, 14). Our future studies will be focused on pursuing a deeper mechanistic understanding of differential regulation of antipathogen and autoimmune responses by STAT1 serine 727 phosphorylation.

In conclusion, our data highlight the importance of STAT1-pS727 downstream of several signaling pathways, such as BCR, TLR, and IFN signaling in autoimmune AFC, GC, and Tfh responses, leading to autoantibody production and development of SLE pathogenesis. Our data further indicate that STAT1-pS727 is not required for foreign Ag–driven responses, including pathogens. Future efforts should focus on the identification of kinase(s) involved in STAT1 serine phosphorylation and the development of therapeutics to block STAT1-pS727 as a treatment for SLE that can maintain protective immunity to pathogens in SLE patients.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank the Penn State University Hershey Medical Center flow cytometry core facility for their assistance. We thank the Penn State University Hershey Medical Center Department of Comparative Medicine for animal housing and care.

Footnotes

  • This work was supported by National Institutes of Health National Institutes of Allergy and Infectious Diseases RO1AI091670 (to Z.S.M.R.), Lupus Research Alliance Grant 548931 (to Z.S.M.R.), and the Finkelstein Memorial award (to S.B.C.).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    ABC
    age-associated B cell
    AFC
    Ab-forming cell
    ANA
    antinuclear Ab
    BM
    bone marrow
    GC
    germinal center
    IMQ
    imiquimod
    muPyV
    mouse polyomavirus
    NP
    4-hydroxy-3-nitrophenol, NP-KLH, 4-hydroxy-3-nitrophenol-keyhole-limpet-hemocyanin
    SA
    streptavidin
    Sle1b.STAT1-SA
    B6.Sle1b.STAT1-S727A
    Sle1bYaa
    B6.Sle1b.yaa
    SLE
    systemic lupus erythematosus
    smRNP
    Smith/ribonucleoprotein
    STAT1-pS727
    STAT1 serine-727 phosphorylation
    STAT1-pY701
    STAT1 tyrosine-701 phosphorylation
    TAD
    transactivation domain
    Tfh
    T follicular helper cell
    VLP
    virus-like particle.

  • Received February 13, 2020.
  • Accepted March 18, 2020.

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