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. 2008 Dec 1;181(11):8018-26.
doi: 10.4049/jimmunol.181.11.8018.

SOCS1 regulates the IFN but not NFkappaB pathway in TLR-stimulated human monocytes and macrophages

Cecilia M Prêle  1 , Eleanor A Woodward, Jacqueline Bisley, April Keith-Magee, Sandra E Nicholson, Prue H Hart
Affiliations

SOCS1 regulates the IFN but not NFkappaB pathway in TLR-stimulated human monocytes and macrophages

Cecilia M Prêle et al. J Immunol. .

Abstract

SOCS1 can regulate TLR-mediated signal transduction, yet mechanistic studies in murine macrophages have been confusing and contradictory. This study has used an adenoviral transfection system to determine the role of SOCS1 in the regulation of TNF-alpha production by activated human monocytes. Monocytes were infected with AdV-SOCS1 or with an empty vector control, AdV-GFP, for 24 h before activation with the TLR4 ligand, LPS. SOCS1 did not regulate TNF-alpha mRNA or protein production within the first two hours of TLR4 activation. However, SOCS1 suppressed the sustained production of TNF-alpha by primary human monocytes and synovial fluid macrophages ex vivo. In addition, SOCS1 regulated the production of IL-6, but not IL-10, by monocytes. Analysis of the early signaling pathway downstream of TLR4 demonstrated that SOCS1 had no regulatory effect on the activation or on the DNA binding capacity of NFkappaB. The late effects of LPS are mediated in part through the MyD88-independent pathway activating IRF3 and initiating the production of IFN-beta. In response to adenoviral infection and before LPS exposure, monocytes expressed enhanced levels of IFN-beta and Myxovirus A mRNA, an anti-viral molecule characterizing IFN-beta activity. These two genes were reduced in AdV-SOCS1-infected cells. Further, SOCS1 regulated IFN-dependent pathways in LPS-activated cells as evidenced by reduced IFN-beta production and STAT1 phosphorylation. Using AdV-infection to dissect SOCS1 control of IFN-dependent pathways, this study suggests that SOCS1-regulation of the IFN-dependent component of the LPS-induced TLR4 signaling pathway may contribute to the down-regulation of inflammatory cytokine production by AdV-SOCS1-infected human monocytes.

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Figures

FIGURE 1
FIGURE 1. SOCS1 expression by AdV-SOCS1-infected human monocytes
A. LPS induced SOCS1 mRNA levels over 2 h in monocytes from each of 4 individual donors (mean ± SEM, n=3 replicates/donor). *p<0.05 compared to untreated (time 0), 0.5 and 1 h following LPS exposure. SOCS1 mRNA was normalized to the housekeeping gene UBE2D2. B. Infection efficiency (% GFP positive cells, determined using flow cytometry) of human monocytes infected with AdV-SOCS1 for 24 h. Infection with three batches of virus used in this study are shown. Batch A (open circles), batch B (closed circles), batch C (open squares). C. SOCS1 protein expression in cell lysates from human monocytes infected with AdV-SOCS1 and AdV-GFP for 24 h.
FIGURE 2
FIGURE 2. SOCS1 regulation of TNFα levels 24 h, but not 2 h following LPS-activation of human monocytes
A. TNFα levels in culture supernatants 2 h (n=9 donors for all groups except AdV-SOCS3, n=5 donors) and 24 h (n=18 donors for all groups except AdV-SOCS3, n=12 donors) after LPS (500 ng/ml) exposure. B. IL-6 and IL-10 levels assayed 24 h following LPS exposure. Each point represents the mean cytokine level calculated from triplicate cultures of cells from individual donors. * p<0.05, ns = not significant.
FIGURE 3
FIGURE 3. Dose-dependent control of TLR4-induced TNFα production by SOCS1
A, B. Increasing the MOI of AdV-SOCS1 significantly increased both the percentage of GFP+ cells and the MFI for GFP+ cells. Monocytes from a representative donor were infected with No virus, AdV-GFP (MOI 50), AdV-SOCS3 (MOI 50) or AdV-SOCS1 at MOI 1 to 100 for 24 h prior to analysis of the number of GFP positive cells or MFI by flow cytometry. C. After 24 h, the AdV-infected cells were incubated with LPS (50 or 500 ng/ml) for 24 h and TNFα levels measured in the culture supernatants. Data are shown as mean ± SEM for triplicate cultures. Significant differences between AdV-GFP- and AdV-SOCS1-infected cells are shown by *p<0.05, ** p<0.01. # represents a significant difference between uninfected and AdV -infected cells, p<0.05.
FIGURE 4
FIGURE 4. SOCS1 does not modulate the TLR-induced NFκB pathway
A. Activation of IκBα by phosphorylation on Ser32 and its subsequent degradation for 2 h following LPS exposure in AdV-GFP- and AdV-SOCS1-infected monocytes. Phospho-IκBα and IκBα levels for cells from 5 donors were quantified and normalized to β-tubulin; mean ± SEM. The kinetics and extent of IκBα-phosphorylationSer32 and IκBα-degradation were not significantly different between AdV-GFP- and AdV-SOCS1-infected monocytes. B. NFκB EMSA. Monocytes were left uninfected or infected with AdV-GFP or AdV-SOCS1 for 24 h. Cells were stimulated with or without LPS (500 ng/ml) or Pam3CSK4 (300 ng/ml) for 30 min and nuclear extracts prepared. Specificity of binding was determined by incubating nuclear lysates in the presence of a 50 fold excess of a cold NFκB probe (NFκB CP). C. Cumulative results for binding of the NFκB probe by nuclear extracts from 3 donors. The fold change in NFκB DNA binding capacity was determined by densitometry (ns = not significant). D. Nuclear lysates were prepared from CD14+ monocytes from a representative donor incubated for 24 h with no virus, AdV-GFP or AdV-SOCS1 prior to incubation with LPS for 30 min. The complexes binding to the NFκB probe were identified by supershifting with an anti-p65 (+p65) or anti-p50 antibody (+p50). The effect of addition of 50 fold excess of unlabelled NFκB (NFκB CP) or mutant NFκB (mNFκB CP) probe was determined.
FIGURE 5
FIGURE 5. Regulation by SOCS1 of AdV-induced IFN-dependent pathways
A. Relative levels of IFNβ, IRF-1 and IRF3 mRNA in AdV-GFP- and AdV-SOCS1-infected cells were determined for 3 individual donors using RT2 Profiler PCR Arrays (SABiosciences). Data from the 3 individual experiments are shown as mean fold change in mRNA expression relative to uninfected or AdV-SOCS1-infected monocytes. The dotted line indicates no change. B. MxA mRNA expression levels were measured by real time PCR and normalized to the expression of the housekeeping gene UBE2D2. C. Inflammatory cytokine mRNA levels, excluding IL-10 and IL-12, were significantly reduced in AdV-SOCS1-infected monocytes. D. Monocytes from a representative donor were left uninfected or infected with AdV-GFP, AdV-SOCS1 or AdV-SOCS3 for 24 h before exposure to LPS for up to 6 h. TNFα mRNA levels were measured by real time PCR and normalized to UBE2D2. E. IFNβ levels were measured in the culture supernatants of triplicate cultures after 6 h exposure to LPS. F. Activation of STAT1. STAT1 phosphorylationTyr701, in response to LPS exposure for 2 h, was determined for cells from a representative donor using Western blotting. Densitometric measures are shown upon normalization to the amount of total STAT1. At time 0, STAT1 activation was elevated in AdV-GFP-infected cells compared to cells incubated with no virus or AdV-SOCS1. After 2 h, STAT1-normalized levels of phosphorylated STAT1 were reduced in cells infected with AdV-SOCS1. All data are represented as mean ± SEM, significant differences are shown as *p<0.05 or ** p <0.01.
FIGURE 6
FIGURE 6. SOCS1-regulation of pro-inflammatory cytokine production by TLR-activated synovial fluid macrophages ex vivo
Mononuclear cells were isolated from synovial fluid of patients with inflammatory arthritis, incubated overnight in M-CSF, infected for 24 h with no virus, AdV-GFP and AdV-SOCS1 and then exposed to LPS (500 ng/ml) for 24 h. TNFα levels were determined in the culture supernatants, mean ± SEM for triplicate cultures.
FIGURE 7
FIGURE 7. Proposed regulation of LPS-induced TNFα production by SOCS1
A. In AdV-infected macrophages STAT1 activation, IFNβ production and MxA mRNA levels were enhanced. In AdV-SOCS1-infected cells relative to AdV-GFP-infected cells there was reduced IRF1 mRNA, IFNβ mRNA, STAT1 phosphorylation and reduced MxA mRNA levels, highlighting the capacity of SOCS1 to control AdV-mediated signaling. B. TLR4 signals via an early MyD88-dependent and a late MyD88-independent, TRIF-dependent pathway. The rapid production of TNFα following receptor activation (+ LPS early) can be attributed to the amplification of signals through the NFκB pathway with NFκB transcriptional activity driving the expression of proinflammatory genes. The sustained production of TNFα, in response to TLR4 activation (+LPS late), reflects the co-operative actions of the MyD88-dependent and MyD88-independent pathways. Recruitment of the adaptors TRIF and TRAM initiate the dimerization and translocation of IRF3 which regulates the production of type I IFN. IFNβ binds to and activates its receptor, IFNAR leading to the activation and nuclear translocation of STAT1 which may drive the expression of both IFN-inducible and pro-inflammatory genes. In addition, TRAF6 recruitment contributes to NFκB activation. Further, cross-talk between the IFN and NFκB pathways is proposed with both known and unknown targets of SOCS1 indicated.
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