SOCS1 KIR mimetics reduce TLR7-induced inflammatory mediators
To gain greater insight on the regulation of TLR7 signaling by the kinase inhibitory region of SOCS1, the RAW264.7 murine macrophage cell line was stimulated with TLR7 agonist Resiquimod (R848) for 24 hours in the presence of SOCS1 KIR peptide mimetics (SOCS1 KIR and SOCS1 KIR dimer 10 µM), or AG490, a known JAK1/2 inhibitor. Additionally, a peptide corresponding to region 1001–1019 of murine JAK2 (pJAK2(1001–1019)) was used as a peptide control. Multiplex cytokine analysis showed that while both SOCS1 KIR (p = 0.0053) and AG490 (p = 0.0006) significantly reduced IL-6 secretion, SOCS1 KIR dimer failed to do so (Fig. 1A). The SOCS1 KIR dimer significantly reduced TNFa production (p = 0.0013), by 70% on average while TNFa secretion was not significantly impacted by either AG490 or SOCS1 KIR (Fig. 1B). Neither SOCS1 mimetic modulated IL-10 secretion, while AG490 reduced IL-10 to baseline levels (p = 0.0002) (Fig. 1C). pJAK2 (1001–1019), previously shown to antagonize endogenous SOCS1 function (50, 51), significantly enhanced IL-6 production almost two-fold beyond R848 stimulation but had no additional effect on TNFa or IL-10 production (Fig. 1A-C). Together, these results suggested regulation of TLR7-mediated effector functions in macrophages by SOCS1 KIR and SOCS1 KIR dimer, which prompted additional experiments.
Considering differences between the effects of the monomeric and dimeric variants of SOCS1 KIR mimetics on TNFα production, it was likely that dosing of these mimetics had not been optimized. As such, we next ran a dose response experiment with the mimetics, and measured TNFa secretion by ELISA (Fig. 1D). Although SOCS1 KIR failed to inhibit TNFa production initially (Fig. 1B), increasing concentrations of SOCS1 KIR were effective in reducing TNFa production. Consistent with the result in Fig. 1B, the SOCS1 KIR dimer had a lower effective half max compared to SOCS1 KIR; however, SOCS1 KIR, SOCS1 KIR dimer, and AG490 all inhibited TNFa in a dose dependent manner reaching maximum effective dose at 33µM (Fig. 1D). At the newly defined maximum dose (33µM), SOCS1 KIR [RAW264.7, p = 0.0029 and PECs, p = 0.0932)] and SOCS1 KIR dimer [RAW264.7, p < 0.0001 and PECs, p = 0.0518] reduced TNFα secretion induced by R848 in all macrophages to near unstimulated levels. (Fig. 1E, 1G). Considering that previous reports using SOCS1 deficient macrophages demonstrated the importance SOCS1 in the regulation of LPS-induced TNFα (52, 53), we assessed the ability of the SOCS1 KIR mimetics to modulate LPS stimulation. We observed that both SOCS1 KIR mimetics were effective in inhibiting LPS-induced TNFα (Fig. 1F), while the control peptide had no effect on R848 (TLR7) or LPS (TLR4) mediated activation of the RAW264.7 cell line or PECs (Fig. 1D-G). Significantly, minimal cellular toxicity was observed with the SOCS1 mimetic peptides, which contrasted with cellular cytotoxicity by AG490 and Stattic, measured by lactate dehydrogenase (Fig. S1). Together these results showed inhibition of TLR induced TNFa production in a dose-dependent manner by the SOCS1 KIR mimetic peptide, which was comparable to the effect of AG490, but without toxicity.
Although chemokine and nitric oxide synthase production upon TLR stimulation play a critical role in the trafficking of leucocytes to the site of infection and subsequent microorganism clearance, overproduction of these molecules is associated with autoimmunity and cancer(54). As such, we next examined the effect of SOCS1 KIR mimetics on TLR-7 mediated chemokine and Nos2 transcript profiles in RAW264.7 cells and PECs through qPCR [primers in Table 1]. As expected, chemokines ccl2, ccl3, ccl5, cxcl10, and nos2 transcripts were minimally expressed in either RAW264.7 or PECs in the absence of TLR-7 agonist R848 stimulation (as indicated by the white blocks), but their levels were markedly elevated upon R848 administration (as denoted by the conversion of transcriptomic levels to black) (Fig. 2A, B). Consistent with Fig. 1, R848 stimulation also enhanced tnfa and il6 transcript levels in RAW264.7 cells and murine macrophages (Fig. 2A, B). The administration of either SOCS1 KIR, or SOCS1 KIR dimer, attenuated the TLR-7 agonist mediated transcript levels of ccl2, ccl3, cxcl10, nos2, and tnfa in both macrophage populations. Also consistent with the results shown in Fig. 1, tnfa inhibition was more potent with the SOCS1 KIR dimer compared to SOCS1 KIR in both macrophage populations. The quantitative graphical representations show that while chemokine and nos2 down-modulation was consistently more pronounced by the SOCS1 KIR dimer within PECs, as compared to SOCS1 KIR, the results were more variable in the RAW264.7 cell line (Fig. 2A, B, and S2A). Indeed, SOCS1 KIR dimer administration reduced ccl2, cxcl10, and nos2 transcripts to near baseline in PECs (Fig. 2B). SOCS1 KIR dimer-mediated reduction of R848-induced ifnb and ccl5 was also more pronounced in the RAW264.7 cell line compared to SOCS1 KIR, while the level of ccl4 reductions were comparable (Fig. 2A, B, and S2B). It is also notable that SOCS1 KIR regulated R848-induced il6 transcript levels in RAW264.7 macrophages, but not in PECs. In contrast, SOCS1 KIR dimer effectively mitigated il6 transcript levels in both the cell line and primary macrophages. As expected, control peptide had no effect on R848-induced transcript up-regulation. Additionally, it should be noted that TLR7 stimulation with R848 had no effect on TATA binding protein (TBP), which served as a control non-TLR inducible gene. (Fig. S2C). Taken together, this data indicates that while SOCS1 KIR dimer and SOCS1 KIR both attenuated TLR7-induced inflammatory genes, the SOCS1 KIR dimer demonstrated a higher attenuation efficacy, particularly in primary macrophages.
SOCS1 KIR mimetics attenuate STAT1 transcriptional activity
IFNγ canonically signals through JAK2-mediated phosphorylation of STAT1 and is also a potent activator of macrophage effector functions such as phagocytosis, antigen presentation, and NO synthesis (55–57). SOCS1 KIR has been previously shown to bind JAK2 and attenuate STAT1 activation (46, 58) however, the regulation of downstream transcriptional activity by both the monomeric and dimeric variants remains poorly understood. To better understand the effects of the SOCS1 mimetics on STAT1 transcriptional activity, we utilized a HeLa-reporter cell line transfected with a STAT1 responsive luciferase construct. The reporter HeLa cells were treated with IFNγ for 30min and were intracellularly stained for Y701-STAT1 phosphorylation followed by flow cytometry (Fig. 3A). The effect of the SOCS1 mimetic peptides on the inhibition of Y701 phosphorylation was assessed by western blot analysis (Fig. 3B). Statistically significant inhibition was observed for SOCS1 KIR dimer (p = 0.0263) and AG490 (p = 0.0111) (Fig. 3B). To assess the effect of SOCS1 mimetics on STAT1 transcriptional activity, we treated the HeLa reporter cells with IFNγ for 24 hours and co-treated with inhibitors or control peptide. We observed a dose-dependent inhibitory effect in relative luminescence after treatment with the SOCS1 mimetics (Fig. 3C), with the highest concentration of SOCS1 KIR inhibiting 50% (p < 0.0001) luminescence compared to 70% with SOCS1 KIR dimer (Fig. 3D).
These results provided an impetus to more closely evaluate the effect of SOCS1 mimetics on the regulation of STAT1-induced genes mediated by IFNg stimulation. We treated RAW264.7 cells and PECs with IFNγ in the presence or absence of SOCS1 KIR dimer and SOCS1 KIR and analyzed selected STAT1 targeted genes. In the absence of IFNγ stimulation, our panel of STAT1 targeted transcripts were minimally activated in RAW264.7 and PECs as denoted by the lightly colored heat map panel which became near black in response to IFNγ signaling (Fig. 4A, B). Although some distinct transcripts were utilized between RAW264.7 macrophages and PECs, based on differential expression levels, the administration of the SOCS1 KIR mimetics reduced the color intensity of the heat map, indicative of reduced transcript levels for multiple targets in both RAW264.7 cells and PECs. The quantitative graphical representations revealed statistically significant reductions in Nos2 and CD274 (PDL1) by SOCS1 KIR peptides within both RAW264.7 and primary macrophages. In contrast to the overall results obtained from TLR stimulation, SOCS1 mimetic peptide efficacy varied between primary and RAW264.7 macrophages after IFNg stimulation. While Irf1 and Stat1 were consistently reduced upon administration of the SOCS1 KIR mimetics, statistical significance was only achieved by SOCS1 KIR dimer mediated reduction of stat1 transcripts in primary macrophages. Within PECs, the SOCS1 KIR dimer significantly attenuated the expression of Cxcl9, Nos2, and Stat1. Notably SOCS1 KIR effects were more modest within PECs compared to RAW264.7 cell line; however, a similar trend of inhibition could still be seen. Together, these results highlight the inhibitory effect of the SOCS1 KIR mimetics on STAT1-induced transcription in response to interferon gamma stimulation.
SOCS1 KIR dimer promotes enhanced inhibition of IFNγ-mediated STAT1 & STAT3 signaling
Given our previously published preclinical data showing enhanced reduction of SLE associated pathologies by the SOCS1 KIR dimer compared to SOCS1 KIR,(50) combined with our current data showing enhanced efficacy of the dimer in mitigating R848 and IFNg signaling, we next tested the hypothesis that the attenuation of R848- and IFNγ-induced inflammatory markers in macrophages would be associated with reduced STAT phosphorylation levels. We assessed Y701 and Y705 phosphorylation status on STAT1 and STAT3 respectively in RAW264.7 cells and peritoneal macrophages after co-incubation with IFNγ and the SOCS1 mimics. As can be seen, the SOCS1 KIR dimer, but not SOCS1 KIR, significantly reduced IFNg mediated STAT1 and STAT3 phosphorylation at Y701 and Y705 in the RAW264.7 cell line. (Fig. 5A, B). IFNg-mediated STAT1 and STAT3 phosphorylation at tyrosine Y701 and Y705 was not reduced through SOCS1 KIR administration, while SOCS1 KIR dimer had a trending, though not statistically significant, effect within PECs (Fig. 5C, D). The control, AG490, caused significant reductions in IFNg mediated STAT1 and STAT3 phosphorylation at Y701and Y705 in both the RAW264.7 cell line and in PECs (Fig. 5A-D). Together these results show that although the SOCS1 KIR and SOCS1 KIR dimer consistently decreased IFNγ-mediated STAT1 activation at Y701, and SOCS1 KIR dimer consistently decreased IFNγ-mediated STAT3 activation at Y705 in RAW264.7 macrophages, only SOCS1 KIR dimer inhibited IFNγ-mediated activation of STAT1 and STAT3 to levels comparable to AG490 in primary peritoneal macrophages.
SOCS1 KIR dimer regulates IL-6 mediated STAT1 and STAT3 phosphorylation in macrophages.
IL-6 activates Jak2, promoting the phosphorylation and activation of transcription factors STAT1 and STAT3 (59). Since SOCS1 was originally discovered as a regulator of IL-6 signaling (60–62), we next investigated whether the peptide mimics of the KIR region of SOCS1 could inhibit STAT3 activation with respect to IL-6 signaling. To test this, we first treated the U3A reporter cell line, transfected with a STAT3-responsive luciferase construct, with IL-6. SOCS1 KIR (p < 0.0027), SOCS1 KIR dimer (p < 0.0072), and AG490 comparably reduced IL-6 mediated STAT3 transcriptional activity and STAT3 phosphorylation (Fig. 6A, B). Stattic, a STAT3 irreversible competitive inhibitor used as a positive control, reduced IL-6 mediated transcriptional levels and STAT3 phosphorylation at Y705 to near background. We also tested the STAT1 transcriptional activity using the HeLa STAT1-luciferase reporter cells. While the luminescence increase was only two-fold with IL-6 (Fig. 6D), compared to more than 15-fold with IFNγ (Fig. 3D), we observed a dose dependent inhibitory effect with SOCS1 KIR dimer, but not with SOCS1 KIR (Fig. 6C). The SOCS1 KIR dimer had a strong and significant effect (p < 0.0001) on IL-6 induced STAT1 transcriptional activity at the highest concentration tested that was consistently more pronounced than AG490 (Fig. 6D). Next, we analyzed the inhibitory activity of the SOCS1 mimetics on macrophages treated with IL-6. While the reporter assays suggested regulation of IL-6 mediated STAT3 but not STAT1 activation by SOCS1 KIR, the SOCS1 mimetic monomer failed to inhibit either IL-6 mediated STAT1 or STAT3 phosphorylation in either RAW264.7 cells or PECs (Fig. 6E-H). AG490 treatment significantly reduced Y701-STAT1 phosphorylation in PECs but not RAW264.7, while inhibition of Y705-STAT3 failed to reach significance in either cell type. In contrast to SOCS1 KIR, SOCS1 KIR dimer significantly inhibited both IL-6 mediated Y701-pSTAT1 and Y705-pSTAT3 in RAW264.7 cells (Fig. 6E, F) and PECs (Fig. 6G, H). These data suggest that SOCS1 KIR dimer, and not SOCS1 KIR, effectively inhibited IL-6 mediated Y701-pSTAT1 and Y705-pSTAT3 phosphorylation. The inhibition by SOCS1 KIR dimer was also consistently more potent than Jak inhibitor AG490, while lacking any cytotoxicity observed with AG490 at equivalent concentration (Fig. S1).
SOCS1 KIR inhibits TLR7 and combined IFNγ-induced expression of IFNγ gene signature
While phosphorylation of Y701 drives STAT1 nuclear translocation (63), full transcriptional and biological activity requires phosphorylation of S727. Additionally, it has been shown that the induction of effector functions, such as TNFa secretion and Nos2 expression, in RAW264.7 by TLR signaling is dependent on S727-STAT1 phosphorylation (30, 64). We next assessed the ability of the SOCS1 mimetics to regulate this process. As such, we stimulated RAW264.7 and murine primary macrophages with either R848 or IFNγ in the presence or absence of SOCS1 KIR, SOCS1 KIR dimer, a control peptide, or AG490. As can be seen within the representative western blots, and graphically compiled from several experiments, treatment of RAW264.7 and PECs with either R848 or IFNγ significantly enhanced the phosphorylation of STAT1 at S727 (Fig. 7A-D). While SOCS1 KIR, SOCS1 KIR dimer, and AG490 significantly reduced R848 induced S727 phosphorylation in RAW264.7 cells, only SOCS1 KIR dimer and AG490 consistently reduced R848-induced S727 phosphorylation in PECS (Fig. 7A-D). Notably IFNγ induced phosphorylation of S727 was unaffected by either SOCS1 mimetic or AG490. As expected, the control peptide had no effect on IFNγ or R848 stimulation in either the RAW264.7 cells or the primary macrophages (Fig. 7A-D).
Given that TLR signaling can modulate cellular responsiveness to IFNg in a S727-STAT1 dependent manner (65), we next assessed the ability of the SOCS1 mimetic peptides to regulate the combinatorial effects of TLR7 and IFNγ signaling within primary murine macrophages. As expected, IFNγ, but not TLR7 stimulation, promoted the phosphorylation of Y701 on STAT1. Additionally, Y701 phosphorylation by the combination of R848 and IFNγ stimulation was statistically indistinct from IFNγ stimulation alone (Fig. 7E). In contrast, while both R848 and IFNγ enhanced S727-STAT1 phosphorylation individually, western blot data clearly show enhanced phosphorylation with the combination of R848 and IFNγ treatment (Fig. 7E). Notably, murine macrophages cultured in the presence of the combinatorial stimulation consistently had lower levels of Y701 and S727 stimulation in the presence of the SOCS1 KIR mimetic peptides, which reached statistical significance with tyrosine 701 phosphorylation when co-cultured with SOCS1 KIR dimer. While the control peptide had no effect on either Y701 or S727 phosphorylation in response to combinatorial stimulation, AG490 only significantly inhibited S727 phosphorylation. Together these data show that the SOCS1 KIR mimetics, in particular SOCS1 KIR dimer, mediated inhibitory effects on the phosphorylation of S727 within macrophages in response to R848, combined R848 and IFNγ stimulation, but not IFNγ alone.
We next assessed the ability of the SOCS1 mimetic peptides to modulate transcriptional activity induced by combined R848 and IFNγ stimulation by designing a panel of responsive transcripts (Fig. S3). Baseline transcript levels for Stat1, Nos2, Cd274 (PDL1), Socs1, and Socs3 were more elevated in primary macrophages than the RAW264.7 macrophage cell line, as can be seen with darker shading of grey within the PECs compared to the RAW264.7 cells. Except for Socs3 transcript levels within primary macrophages, the addition of 30 µM R848 minimally affected other transcripts. The addition of 1ng/ml of IFNg alone induced transcript changes within RAW264.7 cells after 24 hours of culture. However, the combined treatment of 1ng/ml IFNγ and 30 µM R848 mediated statistically significant up-regulation of Stat1, Nos2, Cd274, Socs1, and Socs3 in both primary macrophages and the RAW264.7 cell line (Fig. 8A, B). The enhanced transcriptional activity was consistent with the increased S727-STAT1 phosphorylation in response to the combined treatment (Fig. 7E), suggesting S727-STAT1 dependent enhancement of IFNg responsiveness by TLR signaling(30, 65). The administration of SOCS1 KIR dimer, but not the monomer consistently reduced Stat1 transcription in both the RAW264.7 cell line and primary macrophages. Nos2, Cd274, Socs1, and Socs3 gene transcription was significantly reduced in the macrophage cell line and consistently reduced in PECs (Fig. 8A, B). As expected, the control peptide had no effect on the transcription activity mediated by the combinatorial stimulation. Notably, the reduction in transcription by SOCS1 KIR dimer was comparable to the reduction mediated by AG490. However, unlike AG490, the transcriptional inhibition mediated by SOCS1 KIR dimer was at a concentration that did not promote cellular toxicity. To dissect the role of the S727 residue on STAT1 in this dual signal-enhanced transcription, we transfected RAW264.7 cells with a S727A-STAT1 plasmid and evaluated transcript levels in response to R848 and IFNγ co-treatment. An approximate 50% reduction in all transcript levels except PDL1 was noted, highlighting at least a partial role of the S727-STAT1 residue in mediating R848 and IFNγ-mediated transcription (Fig. S4). Together, these data show that SOCS1 KIR dimer administration ameliorated transcriptional activity promoted by TLR7/IFNg.