In this study TNFα production by human monocytes was controlled by several interacting pathways. In the absence of adenoviral infection, two mechanisms for LPS-induction of TNFα synthesis exist; an early MyD88-dependent and a later MyD88-independent, TRIF-dependent, pathway (25
). These processes interact to enhance levels of TNFα produced after several hours of LPS exposure. In cells infected with the adenoviral vectors for 24 h prior to TLR activation, a significant increase in IFNβ and MxA mRNA was detected in the absence of LPS. IFNβ protein expression was detected 6 h after LPS exposure. As previously reported (26
), IRF3 mRNA levels were not induced in human monocytes in response to adenoviral infection, presumably due to constitutive expression. The contribution of IFN to TLR-induced pro-inflammatory cytokine production is unclear. It is notable that minimal TNFα was produced by AdV-infected cells in the absence of TLR signaling. The AdV-induced pathway, like the MyD88-independent pathway, was dependent on the activity of IFNβ. It has been recently suggested that IRF3, like STAT1, can also bind to, and transactivate the human TNFα promoter (27
). In one study the MyD88-independent pathway required IRF3-dependent expression of TNFα to activate NFκB (28
Enhanced SOCS1 expression following exposure of murine macrophages to LPS had suggested SOCS1 may control LPS-mediated signaling in a negative feedback loop (13
). Up-regulated IκB-, p38- and STAT1-phosphorylation in SOCS1-deficient macrophages, together with reduced nitric oxide synthesis and elevated TNFα production had implicated SOCS1 in direct regulation of TLR4 signaling (14
). There were reports that SOCS1 could block MyD88-mediated signaling by binding to the adaptor protein Mal following its tyrosine phosphorylation, thus targeting it for proteosomal degradation (15
). An additional study reported that SOCS1 interacted directly with the p65 subunit of the NFκB complex (17
Using an adenoviral vector for SOCS1 overexpression in human monocytes, SOCS1 suppressed LPS-induced TNFα production at 24 h without affecting TNFα levels after 2 h. These data suggest that SOCS1 targets in human monocytes the later MyD88-independent (TRIF-dependent) pathway (summarized in ), although some later regulation of an NFκB pathway cannot be ruled out. The SOCS1-mediated reduction in AdV-induced IFNβ, IRF1, MxA mRNA and IFNβ production suggested that the SOCS1-control of TNFα production by primary human monocytes occurred, at least in part by an indirect mechanism notably, via regulation of type I IFN production. Furthermore, in the PCR arrays, there was no change in the mRNA expression of components of the NFκB pathway (p50, p65) in AdV-infected cells before LPS exposure and suggested that the NFκB pathway was not a target for AdV-actions (data not shown). No supportive data of SOCS1 control of NFκB signaling were found in a study in human keratinocytes (31
). Similar to our investigation, reduced inflammatory cytokine production by SOCS1-infected cells was observed and SOCS1 did not alter the NFκB pathway over the 24 h time course studied (31
). These observations support a series of studies performed by Dalpke and colleagues (reviewed in (32
)) which propose both direct and indirect mechanisms of TLR-associated IFNβ-dependent signaling, each displaying different susceptibility to SOCS1 regulation. Effects of IFNβ on the duration, and not the magnitude of the NFκB activation were also questioned (32
). In another study in CpG DNA-stimulated macrophages that failed to produce IFNβ, STAT1 did not promote the production of inflammatory cytokines but instead limited their production (33
). This mechanism of altered transcription factor activity may help to explain the dramatic decrease in TNFα production by IFNβ-compromised, AdV-SOCS1-infected monocytes. However, we cannot rule out the possibility that regulation of IFN-signaling alone is not sufficient for this effect. Perhaps, the combined actions of SOCS1 on IFN-mediated pathways and on unknown targets may be required for the potent regulatory effect on TNFα production observed ().
Proposed regulation of LPS-induced TNFα production by SOCS1
Our data using primary human monocytes suggest that SOCS1 expression has no direct effect on the NFκB pathway early after LPS stimulation. There was no effect on IκBα-phosphorylation and in keeping with a recent study of GM-CSF and M-CSF treated murine bone marrow-derived macrophages (34
), both p50 homodimer and p65-p50 heterodimer complexes were identified in nuclear extracts from M-CSF cultured human monocytes. Furthermore, the relative amounts of these complexes did not alter between uninfected, AdV-GFP- or AdV-SOCS1-infected cells before or after LPS exposure. It had been reported that the nuclear translocation of the p65-p50 heterodimer and p50 homodimer complexes were important for LPS-induced TNFα and IL-10 production, respectively (35
). However, with regulation of TNFα but not IL-10 in this study, and the regulation of TNFα occurring independently of the NFκB pathway, this conclusion could not be confirmed in human monocytes.
These data suggest that control of TLR signaling differs between primary human and murine macrophages. This is not the first study with a similar conclusion; the adaptor protein Mal has been reported to be involved in p65-NFκB activation in manipulated cell lines and mouse but not in human macrophages (36
). Different cell types may also have different adaptor protein use by TLR receptors (36
The regulatory effects of SOCS1 on TNFα and IL-6 production were specific and were not observed in AdV-GFP- or AdV-SOCS3-infected cells. Furthermore AdV-SOCS1-mediated effects on TNFα production could be titrated. The regulation of IL-6 production in AdV-SOCS1-infected cells may be due to impaired TNFα signaling as a consequence of reduced TNFα production.
IL-10 production in response to TLR-stimulation was not modulated in monocytes by overexpression of SOCS1. Furthermore, in the PCR arrays, the IFN-dependent pathway operating in response to AdV-infection did not affect IL-10 levels. This result suggests that IRF3 and type I IFNs do not regulate IL-10 expression and would explain outcomes of acute inflammatory arthritis in Socs1−/−Ifnγ−/−
). The severity of synovial inflammation and joint destruction at the peak of disease was greater in the absence of SOCS1, although disease resolution, a process dependent on IL-10 activity, occurred normally.
In this study, we propose that the effects of SOCS1 on pro-inflammatory cytokine production by human monocytes reflect the control by SOCS1 of type I IFN production. A role for SOCS1 in regulating the production and actions of type I IFN, is not new (38
). In murine macrophages SOCS1 can control the production and actions of IFNβ by interacting with the IFN (alpha, beta and omega) receptor-1 (IFNAR1) chain blocking downstream signaling (13
). SOCS1 control of the MyD88-independent pathway, rather than the NFκB-dependent pathway is further supported by the observation that SOCS proteins target tyrosine phosphorylated proteins. The TLR signaling cascades are predominately regulated by serine/threonine phosphorylation (4
) which potentially limits possible SOCS1-interacting partners within the TLR pathway signaling intermediates (19
). Furthermore, TLR4 signaling was not modulated in Socs1−/−
Recently 4-1BBL/TNFα superfamily member-9 was implicated in the regulation of LPS-induced TNFα production, by interacting with the TLR complex responsible for sustained TNFα production, i.e. via regulation of type I IFN (40
). SOCS1 may interact with 4-1BBL and target 4-1BBL for degradation. Alternatively, SOCS1 may be associated with the receptor itself and prevent association of the 4-1BBL-TLR complex required for the late production of TNFα.
It has been suggested that SOCS1-regulation of IFNβ is important for keeping the innate immune response in check and provides a mechanism for preventing an exaggerated response to activation (32
). In a study comparing the effects of an empty vector control and an adenoviral vector encoding SOCS1, Sakurai and collegues demonstrated the capacity of SOCS1 to limit adenoviral-mediated activation of the innate immune response (41
). These effects of SOCS1 are not limited to immune cells. SOCS1 regulates the type I IFN-mediated anti-viral effects triggered by influenza A virus exposures in human respiratory epithelial cells (42
Low levels of expression combined with rapid turnover have made studies investigating the role of SOCS1 in human monocytes and macrophages difficult. Further human monocytes as non-proliferative, phagocytic cells are difficult to transfect to high efficiency. In this study primary human monocytes were infected to greater than 50% efficiency with an AdV encoding human SOCS1, introduced into the cells by spinofection (21
). The adenoviral infection approach enhanced the type I IFN levels but fortuitously provided a mechanism to examine the IFN-dependent pathway of TLR. Use of adenoviral vectors to express a siRNA to SOCS1 would have had the same vector-induced, IFN-dependent effects. Further, while successful silencing of the Socs1
gene has been previously demonstrated in murine dendritic cells (43
), siRNA are now known to non-specifically activate TLR7 and TLR8, initiating an immune response which makes analysis of TLR-ligand-specific responses impossible (45
This study demonstrated that SOCS1 regulates the MyD88-independent, TRIF-dependent pathway in human monocytes and macrophages. The capacity of SOCS1 to regulate sustained TNFα production is of potential therapeutic benefit for the treatment of chronic inflammatory conditions, such as rheumatoid arthritis and Crohn’s disease. This study makes an important contribution to our understanding of the regulatory capacity of SOCS1 in human cells at a time when SOCS1 mimetics are being considered for therapeutic use (46