Suppressor of cytokine signaling-1 mimetic peptides attenuate lymphocyte activation in the MRL/lpr mouse autoimmune model

Autoimmune diseases are driven largely by a pathogenic cytokine milieu produced by aberrantly activated lymphocytes. Many cytokines, including interferon gamma (IFN-γ), utilize the JAK/STAT pathway for signal propagation. Suppressor of Cytokine Signaling-1 (SOCS1) is an inducible, intracellular protein that regulates IFN-γ signaling by dampening JAK/STAT signaling. Using Fas deficient, MRL/MpJ-Faslpr/J (MRL/lpr) mice, which develop lupus-like disease spontaneously, we tested the hypothesis that a peptide mimic of the SOCS1 kinase inhibitory region (SOCS1-KIR) would inhibit lymphocyte activation and modulate lupus-associated pathologies. Consistent with in vitro studies, SOCS1-KIR intraperitoneal administration reduced the frequency, activation, and cytokine production of memory CD8+ and CD4+ T lymphocytes within the peripheral blood, spleen, and lymph nodes. In addition, SOCS1-KIR administration reduced lymphadenopathy, severity of skin lesions, autoantibody production, and modestly reduced kidney pathology. On a cellular level, peritoneal SOCS1-KIR administration enhanced Foxp3 expression in total splenic and follicular regulatory T cells, reduced the effector memory/naïve T lymphocyte ratio for both CD4+ and CD8+ cells, and reduced the frequency of GL7+ germinal center enriched B cells. Together, these data show that SOCS1-KIR treatment reduced auto-reactive lymphocyte effector functions and suggest that therapeutic targeting of the SOCS1 pathway through peptide administration may have efficacy in mitigating autoimmune pathologies.


Materials and methods
Mice. Female MRL/MpJ-Fas lpr /J (MRL/lpr) mice were acquired from the Jackson Laboratory (Bar Harbor, ME) and housed in specific pathogen free conditions at the University of Florida Cancer and Genetics Animal Care Facility, in strict accordance of approved protocols by the Institutional Animal Care and Use Committee-accredited Association of Assessment and Accreditation for Laboratory Animal Care, and in accordance to ARRIVE guidelines.
Peptide synthesis. The SOCS1-KIR mimetic peptide ( 53 DTHFRTFRSHSDYRRI), SOCS1-KIR dimeric variant (DTHFRTFRSHSDYRRIGGGGGDTHFRTFRSHSDYRRI), or pJAK2 ( 1001 LPQDKEYYKVKEP) was generated in-house using Applied Biosystems 431a automated peptide synthesizer (Applied Biosystems, Carlsbad, CA) by conventional fluorenylmethylcarbonyl chemical methods as described 37,38 , or purchased from Gen-Script (Piscataway, NJ) at 95% purity. A palmitoyl-lysine (a lipophilic group) was added to the N-terminus of the peptides during the final step to assist in cell penetration. Peptides were characterized by high-performance liquid chromatography (HPLC) and mass spectrometry. Peptides were dissolved dropwise in DMSO (Thermo Scientific, Rockford, IL), then suspended to final administration volume in sterile PBS (Sigma Aldrich St. Louis, MO), or dissolved in Barnstead Nanopure water prior to use.
Animal treatments. MRL/lpr mice, aged 8 (cohort 1) or 12 (cohort 2) weeks, were randomized through computer algorithm before receiving intraperitoneal injections of SOCS1-KIR peptide (10 μg/g animal weight) or PBS carrier 3 times per week. Treatments began 1 week after animal facility acclimation and extended through the end of experiments at 26 weeks, or animal sacrifice due to morbidity. For cohort 3, 8-week-old MRL/lpr mice received intraperitoneal injections of SOCS1-KIR peptide (60 μg/animal), SOCS1-KIR dimer (60 μg/animal), or carrier once daily. Treatments began 1 week after animal facility acclimation and extended through the end of experiments at 15 weeks. Mice were weighed weekly and general health evaluated using the standard body score index. Onset of lymphadenopathy was assessed based on a caliper scoring system from 1 to 4 where the following values were assigned: (1) No lymphadenopathy, (2) lymphadenopathy at multiple sites (< 2 cm diameter),  , and anti-CD138-APC (281-2). All antibodies were purchased from BDBiosciences or eBioscienes, unless otherwise indicated. Intracellular expression of IFN-γ was measured after cell activation as previously described 40 . Briefly, cells were incubated in a cocktail of PMA, ionomycin, and brefeldin A for 4 h, then fixed in a 2% paraformaldehyde solution. A total of 50,000 live events were collected by LSRII (BD Bioscience). All flow cytometry analysis was performed using FlowJo v10 (Tree Star, San Carlos, CA).

RNA isolation and RT-qPCR.
Total RNA was extracted as previously described 41 from both blood derived cells and lymph nodes of age-matched MRL/lpr mice. First-strand cDNA synthesis was completed using iScript Kit (Bio-Rad). iQ SYBR Green Supermix (Bio-Rad) and gene-specific primers (Table 1) were utilized to amplify relative amounts of cDNA on a CFX Connect Real Time System (Bio-Rad). The fold change expression was calculated using the value 2 −∆∆CT method, with Bio-Rad software and Gapdh as the reference gene.
Renal pathology. Kidneys were embedded in paraffin in week 15. Paraffin-embedded kidney samples were sectioned and stained with hematoxylin and eosin (H&E). Digitized H&E images were analyzed using Aperio ImageScope as previously described 42 . The area was measured in at least 40 glomeruli per sample by blinded reviewers. Kidney pathology was scored and measured on a semi-quantitative scale of 0-3+ as previously described 43 . Statistical analysis. GraphPad Prism v8 was used to calculate statistical significance between various groups using Student's t-test and ANOVA coupled with Dunnett's or Sidak's multiple comparison tests. p values ≤ 0.05 was considered significant as indicated within each figure.

SOCS1-KIR mimetic peptides mitigate MRL/lpr T lymphocyte activation and cytokine production in vitro.
To assess the ability of SOCS1 mimetic peptides (SOCS1-KIR) to modulate T lymphocyte activation, we cultured a single cell suspension from total axillary, brachial, cervical, and inguinal LN isolated from 8-week old MRL/lpr mice with anti-CD3 or anti-CD3/anti-CD28 antibodies, in the presence of SOCS1- www.nature.com/scientificreports/ KIR monomeric or a dimeric peptide variants (SOCS1-KIR dimer) for 5 days. As a control, activated cultures were also incubated with a peptide corresponding to the region of JAK2 previously shown to interact with endogenous SOCS1 thereby acting as a SOCS1 antagonist (pJAK2 1001-1013) 44 . anti-CD3 stimulation yielded a modest upregulation of CD25, CD69, and CD44 in CD4 + ( Fig. 1A-C) and CD8 + (Fig. 1D-F) T lymphocytes. As expected, anti-CD3/anti-CD28 co-stimulation yielded twofold to fivefold increases in the frequency of the highly activated CD4 + and CD8 + lymphocytes bearing increased size (based on FSC) and elevated surface levels of CD25, CD69, and CD44 when compared to unstimulated cells. The co-incubation with the SOCS1-KIR dimer reduced all three of these activation markers, while SOCS1-KIR reduced the frequency of CD4 + and CD8 + T lymphocytes bearing high surface expression of CD44 and the frequency of CD25 + CD8 + T lymphocytes. Conversely, co-incubation with pJAK2 (1001-1013) failed to reduce activation. Since IFN-γ production is associated with lupus progression [45][46][47][48] and it is modulated by endogenous SOCS1 12,49,50 , we next assessed IFN-γ production following incubation with SOCS1 mimetic or antagonist peptides. Remarkably, co-incubation with SOCS1 mimetic, but not pJAK2 (1001-1013), significantly reduced the frequency of CD4 + and CD8 + CD25 + IFN-γ + T lymphocytes ( Fig. 2A,B). The SOCS1-KIR mimetic also reduced IFN-γ mRNA expression and protein secretion in LN cells cultured in these conditions (Fig. 2C,D). Consistent with MHC class II upregulation by IFN-γ, H2-Aa expression levels were significantly reduced in stimulatory conditions co-cultured with SOCS1-KIR, compared to stimulatory conditions alone (Fig. 2E). In summary, in vitro administration of SOCS1-KIR to MRL/lpr leukocytes inhibited T lymphocyte activation and production of IFN-γ.
Peptide administration decreases the frequency of memory T lymphocytes within peripheral blood and secondary lymphoid organs, while decreasing splenomegaly and lymphadenopathy. MRL/lpr mice experience pronounced lympho-accumulation that often requires euthanasia. Using a cohort of four SOCS1-KIR treated and four PBS control 8-week-old MRL/lpr mice, we initially assessed the effect of the SOCS1 mimetic peptide on lymphadenopathy. 75% of the PBS treated animals in experimental cohort 1 required premature euthanasia after 20 weeks compared to only 25% in the SOCS1-KIR treated group ( Figure S1). This result provided insight into the possible efficacy of SOCS1-KIR in the MRL/lpr mouse model. We next assessed SOCS1-KIR mitigation of lymphadenopathy using a larger sample size. Although SOCS1-KIR administration did not affect the overall weight of the mice, the lymph nodes and spleens from SOCS1-KIR treated animals were significantly smaller (Fig. 3A). Together, these results suggested that SOCS1-KIR treatment may reduce lymphadenopathy and splenomegaly in the MRL/lpr mouse.
Given that dysregulated IFN-γ production by memory T cells facilitates disease pathogenesis in MRL/lpr mice 51 , and that endogenous SOCS1 inhibits IFN-γ production ( 12,49,50 and our in vitro results), we hypothesized that SOCS1-KIR peptide administration could inhibit the presence of IFN-γ-producing memory T lymphocytes (CD44 + ) within the peripheral blood of MRL/lpr mice. At 9 weeks of age, there was a negligible amount of CD44 + IFN-γ + T lymphocytes within the peripheral blood of PBS treated (control) MRL/lpr mice that markedly increased over the next 7 weeks (Fig. 3B). In contrast, the frequency of CD44 + IFN-γ + CD4 + and CD8 + T lymphocytes within the peripheral blood of SOCS1-KIR treated mice were significantly reduced in comparison to control mice over the same time period (Fig. 3B and Figure S2). At sacrifice, the frequency of CD44 + cells was reduced in both CD4 + and CD8 + T lymphocyte populations in the spleen and LN of mice treated with SOCS1-KIR, while the frequency of CD44 + cells in CD4 − CD8 − double negative T cells (often associated with SLE and ALPS progression) was reduced only in lymph nodes (Fig. 3C). Together, these data show that SOCS1-KIR treatment reduced cellular accumulation within the secondary lymphoid organs of MRL/lpr mice, possibly through the reduction of memory T cells.
SOCS-1 mimetic peptides reduce spontaneous skin lesions, anti-dsDNA IgG, and glomerular enlargement in MRL/lpr mice. We next examined the ability of the peptide to regulate spontaneous lesions and antibody production in MRL/lpr mice. Monomeric SOCS1-KIR, the dimeric variant SOCS1-KIR dimer (previously shown to be superior in vitro in Fig. 1), or PBS-carrier were administered at 8-weeks of age until sacrifice at week 15. Although there was no statistical difference in renal pathology scores (Fig. 4B), there was a significant reduction in glomerulus size, which is another indication of kidney damage 42 (Fig. 4A). There was also a reduction in the level of serum anti dsDNA IgG (Fig. 4C) that was present in SOCS1-KIR dimer treated mice. Finally, as can be seen in Fig. 4D, SOCS1-KIR mimetic peptide treated mice had reduced severity in spontaneous skin lesions (Fig. 4D). Together, these results suggest that animals receiving SOCS1-KIR treatments had reduced immune-mediated pathology.

SOCS1 KIR and KIR dimer treatment increases Foxp3 expression in Tregs and follicular
Tregs. Treg suppressive abilities directly correlate with Foxp3 expression 52 . While high Foxp3 expression is generally an indicator of a stable suppressive phenotype, reduced Foxp3 expression is associated with decreased suppressive function and autoimmunity 52 . As we have previously shown that partial rescue of SOCS1 −/− mouse perinatal lethality by SOCS1-KIR mimetic peptide administration was correlated to enhanced Treg function and an overall increase in Foxp3 expression in CD4 + T cells 12 , we next evaluated both total and follicular Tregs in the spleen of mice treated with SOCS1-KIR monomer, dimer, or control by flow cytometry. Using the gating scheme present in Figure S3, we found that while there was no difference in total numbers of conventional Tregs (CD4 + Foxp3 + ) or follicular Tregs (CD4 + PD1 + CXCR5 + Bcl6 + Foxp3 + ) (Fig. S4A), there was a greater than 50% and 30% increase in Foxp3 mean fluorescent intensity (MFI) in the total and follicular Treg populations, respectively (Fig. 5A,B) www.nature.com/scientificreports/ www.nature.com/scientificreports/ function 12,30,31 , together these results suggest that the administration of SOCS-KIR peptide may serve to stabilize the phenotype of peripheral and follicular Tregs.

SOCS1 KIR mimetic peptide treatment attenuates T cell activation and reduces CD19 + GL7 + B cell populations enriched for germinal center B cells in vivo.
In therapeutic cohort 1 we observed a reduction in CD44 + IFN-γ + CD4 + and CD44 + IFN-γ + CD8 + memory T lymphocytes circulating in the blood of mice treated with the SOCS1-KIR monomer at 15 weeks of age (Fig. 3A), which corresponded to reduced memory CD4 + and CD8 + T cells within the spleen and lymph nodes (Fig. 3B). To better understand the immunomodulatory effects of SOCS1-KIR on T lymphocyte activation, we next evaluated changes within specific T lymphocyte populations present in the spleens of treated and control mice of cohort 3 at 15 weeks of age. Although total CD4 + and CD4 + effector memory cell populations were unaffected by mimetic treatments, the percentage of naïve CD4 T cells were consistently higher within SOCS1 mimetic peptide treated mice, reaching statistical significance in monomeric SOCS1-KIR treated mice ( Figure S4B). In addition, monomeric SOCS1-KIR significantly reduced the CD4 + effector memory/naïve T lymphocyte ratio (Fig. 5C). The percentage and total number of naive splenic CD8 + T lymphocytes were increased by mimetic administration, reaching significance with the SOCS1-KIR dimeric peptide ( Figure S4B). Indeed, in similar fashion to the CD4 + T lymphocytes, the CD8 + T effector memory/naïve cell ratio was significantly reduced by the mimetic peptide treatments (Fig. 5C). As we saw SOCS1-KIR mimetic peptide mediated reductions in antibody production, we next evaluated peptide mediated changes in splenic B cells. While many B cell subsets appeared unaffected ( Figure S5), there was significant reduction (~ 40%) in the frequency of GL7 + B cells, which are enriched in germinal center B cells, within the SOCS1-KIR monomer treated group (Fig. 5D). This result aligns with two previous studies where tofacitinib, which also targets JAK/STAT signaling, was shown to reduce germinal center B cells post immunization in BALB/c mice and anti-dsDNA concentration in MRL/lpr mice respectively 53,54 . As the germinal center (GC) is the major source of high affinity class switched IgGs 36 , these data suggest that SOCS1-KIR regulation of GC B cell populations can in part explain lower anti-nucleic acid autoantibodies and reduced kidney-associated pathology in treated mice. www.nature.com/scientificreports/ SOCS1-KIR mediated lymphocyte changes correlated to reduced anti-DNA IgG production. Since anti-DNA IgG production is a common clinical immunopathology associated with lupus progression, we next analyzed the correlation between anti-DNA IgG production and SOCS1-KIR mediated leukocyte changes using the 1:1000, 1:3000, and 1:9000 serum dilutions. Foxp3 MFI in both total splenic and follicular Tregs trended towards a negative correlation with anti-dsDNA IgG production, reaching statistical significance at 1:3000 and 1:9000 serum dilutions, respectively (Supplemental Figure 6A,B). Notably, the frequency of naïve CD8 + , but not CD4 + T lymphocytes-shown to be enhanced by SOCS1 peptide treatment (Fig. 6, supplemental Figure S6C), was also negatively correlated to anti-dsDNA IgG production at all serum dilutions. In addition, glomerular area trended towards positive correlation with anti-dsDNA IgG production, achieving statistical www.nature.com/scientificreports/ significance at the 1:9000 serum dilution (Supplemental Figure S6D), Together, these results suggest a clinical benefit was mediated by the SOCS1-KIR driven changes in T lymphocytes in vivo.

Discussion
Although medical advances have significantly improved the quality of life in patients with autoimmune diseases, there remains a critical need for novel therapeutic options as many patients remain refractory or intolerant to www.nature.com/scientificreports/ existing strategies. The clinical manifestations observed in autoimmune diseases (such as SLE, ALPS, or Sjogrens syndrome) arise from defective tolerance mechanisms. In addition to regulatory T cells and tight regulation of pro-inflammatory cytokine/cytokine receptor levels, the suppressor of cytokine signaling family of proteins (SOCS) is also critically involved in the maintenance of self-tolerance 9,19 . While complete knockout of SOCS1 results in lethal auto-inflammatory disease, inadequate SOCS1 signaling is associated with lupus-like disease in several rodent models 50,55,56 . Recent studies have also associated variants in SOCS1 related genes, or defects in expression, with lupus and other auto-inflammatory diseases in humans 13,17,[57][58][59] . Notably, the KIR of SOCS1 has a potent immuno-modulatory effect, made evident by the increased survival of SOCS1 −/− mice made transgenic to express KIR devoid of the SOCS box 27 . In this study, we show that administration of peptide variants of SOCS1-KIR to MRL/lpr mice mitigated skin lesions, lymphadenopathy, anti-dsDNA IgG levels, and reduced lupus associated kidney pathology. In addition, SOCS1-KIR administration enhanced Foxp3 expression in both total splenic Tregs and follicular Tregs, while reducing GL7 + B cells which are enriched for germinal center B cells.
Although clinical manifestations of SLE are largely driven by auto-antibody production by B lymphocytes, activated T lymphocytes are critical in SLE pathogenesis and a strong indicator of disease activity in patients [60][61][62][63] . CD4 + , CD8 + , and CD4 − CD8 − T lymphocytes promote a pro-inflammatory environment conducive to lupus pathogenesis and generation of pathogenic anti-DNA Ig secreting B cells. Notably, interferon gamma, produced by CD4 + and CD8 effector T lymphocytes, plays an indispensable role in promoting the availability of autoantigens and the development of TLR7 dependent autoreactive B cells that drive systemic autoimmunity [64][65][66] . In this study, we showed that SOCS1-KIR reduced the frequency anti-CD3/anti-CD28 activated, IFN-γ producing, CD4 + and CD8 + T lymphocytes from MRL/lpr mice in vitro. Moreover, in vivo administration of SOCS1-KIR reduced the frequencies of circulating and splenic T lymphocytes, and prevented the acquisition of the IFN-γ effector function. Notably, the peripheral CD4 + and CD8 + T lymphocytes in SOCS1-KIR treated mice were more biased towards a naïve phenotype compared to control MRL/lpr mice as indicated by the memory/naïve T cell ratio. These results could bear importance to human autoimmune diseases, such as SLE and ALPS, as the JAK/ STAT pathway has been strongly implicated in driving T cell mediated immunopathogenesis [67][68][69] . Therefore, it is likely that one mechanism of action by SOCS1-KIR is the regulation of autoimmune-promoting effector functions by T lymphocytes. Future studies will further elucidate the mechanistic contribution of SOCS1-KIR to direct T cell activation, or indirect antigen presenting cell regulation.
The ability of follicular helper T cells to drive antibody production by B lymphocytes is tightly regulated by T follicular regulatory cells [3][4][5] . Accumulating evidence has implicated dysregulated follicular helper and regulatory T cell interactions in the generation of auto antibodies by B lymphocytes. We and others have shown that SOCS1 is critical for the stability of Foxp3 + Tregs under inflammatory conditions 12,30 , but a specific role in follicular regulatory T cells has not been explored to date. In this manuscript we shown that SOCS1 KIR treatment increased Foxp3 expression in both splenic and follicular Tregs, which is consistent with our previous studies showing that peritoneal administration of SOCS1-KIR can stabilize the Foxp3 + Treg population and facilitate the production of immunomodulatory cytokines 12,14,28 . We also observed decreased germinal center B cells and anti-dsDNA IgG production in mice treated with SOCS1-KIR mimetic peptides. Future work will assess the ability of SOCS1-KIR mimetic peptides to modulate marginal cell B cells, as they are also a relevant B cell population in MRL/lpr mice 70,71 . Notably, our renal results are supported by a previous paper which showed that peritoneal injection of SOCS1-KIR peptide abated renal pathology in a rodent model of type 2 diabetes 72 . The difference in effects between the monomer and dimeric variants could be due to increased valency of dimer while monomer being smaller and having better flexibility. However, further studies would have to be done to elaborate exact mechanistic differences between the peptide variants.
There has been reluctance to pursue therapeutic peptide use for the treatment of auto-inflammatory diseases by the pharmaceutical industry because of high degradation rates, poor target site delivery, and low stability 73 . However, therapeutic peptide use has been shown advantageous as they can be highly specific, safe, and well tolerated by humans. Peritoneal injected SOCS1-KIR has previously shown peak levels within the serum and plasma of injected animals around 6 h with effective clearance at 18 h 74 . The SOCS1-KIR mimetic peptide has a palmitoyl group facilitating intracellular delivery into target cells, thereby possessing a mechanism of action that is distinct to that of decoy receptors or antibodies that act extracellularly. It is therefore tempting to speculate that intracellular-targeting SOCS1-KIR peptides could be used in combination with other biologics or steroids, potentially lowering the effective dose and/or toxicity. This study extends our understanding of the use of SOCS www.nature.com/scientificreports/ mimetic peptides in the treatment of inflammatory disease by showing the regulation of lymphocytes and amelioration of lupus-associated pathologies. Our current study, combined with previous work, suggests that the use of peptide mimetics of SOCS proteins should be evaluated as a therapeutic strategy for the treatment of human autoimmune diseases.