Granulocyte-macrophage colony-stimulating factor negatively regulates early IL-10-mediated responses

Aim: Treatment of inflammatory disorders relies on the intervention in immune responses thereby restoring homeostasis. IL-10 is a cytokine with therapeutic potential, but until now has not been as successful as previously anticipated. A reason for this may be that IL-10 responsiveness depends on the environment of the inflamed tissue. In this study we investigated whether GM-CSF is able to influence IL-10-mediated responses. Methodology: Dendritic cells and macrophages were differentiated from mouse bone marrow and treated or depleted from GM-CSF prior to analyze their response to IL-10. Activity was assessed by measuring cytokine expression upon lipopolysaccharide stimulation, IL-10-induced signaling and down-stream gene expression. Conclusion: This study describes that GM-CSF negatively regulates IL-10-mediated responses.


Flow cytometry
BMDCs were stained in FACS buffer (phosphate buffered saline (PBS) containing 0.1% bovine serum albumin (BSA) and 5 mM EDTA) using the following monoclonal antibodies for cell surface markers (all obtained from eBioscience, Vienna, Austria): phycoerythrin (PE-conjugated anti-CD11b, allophycocyanin (APC)-conjugated anti-F4/80, PE-conjugated anti-CD11c, APC-conjugated MHC-II. Cells were first incubated with Fc receptor block (eBioscience) for 10 min to block any nonspecific binding and subsequent staining steps were performed for 20 min at 4 • C, followed by washing with FACS buffer. Stained cells were acquired using a Cyan-ADP Analyzer (Beckman Coulter, Woerden, The Netherlands) and analyzed with FlowJo software (Tree Star, Inc.).
LPS stimulation assays BMM s were differentiated in 96-well plates and BMDCs were seeded in 96-well plates at a density of 5 × 10 4 /well. Cells were pretreated for 15 min with IL-10 and subsequently stimulated with 100 ng/ml LPS by adding LPS-containing medium. After 2 h or overnight stimulation, supernatants were analyzed for TNF-α using the Ready-Set-Go! R ELISA kit (eBioscience, Vienna, Austria) according to the supplier's protocol.

IL-10-induced signaling
BMDCs were treated for 20 min with IL-10 (0, 1, 10 or 100 ng/ml). Cells were lyzed using 1× Cell Lysis Buffer (Cell Signaling Technology, Leiden, The Netherlands) and total soluble protein content in the lysates was determined by the BCA method (Pierce). Proteins were separated on 12% Bis-Tris gels (Life Technologies) followed by transfer to a PVDF membrane (Life Technologies) by a wet-blotting procedure. Thereafter the membrane was blocked in PBS (containing 0.1% v/v Tween-20 (Sigma-Aldrich, Zwijndrecht, The Netherlands) and 5% w/v non-fat dry milk powder) for 1 h at room temperature, followed by overnight incubation at 4 • C with monoclonal antibodies specific for STAT3, phospho-STAT3 Y705 or phospho-STAT3 S727 in PBS (containing 0.1% v/v Tween-20 (Sigma-Aldrich) and 1% w/v BSA). All STAT3 antibodies were obtained from Cell Signaling Technology. An HRP-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch, Suffolk, UK) was used as a secondary antibody. Band intensities were analyzed with Image J Software.
Cell lysates were also used to investigate the activation of 18 well-characterized signaling molecules using the PathScan Intracellular Signaling Array kit (Cell Signaling Technology) according to the manufacturer's protocol. Blots and arrays were visualized in the G:BOX Chemi System (VWR International, Amsterdam, The Netherlands).
Quantitative PCR BMM s were differentiated in six well plates and 3 × 10 6 BMDCs were seeded in six well plates and were treated for 2 h with IL-10 (0, 1, 10 or 100 ng/ml). Cells were then washed with PBS, and mRNA was isolated with the Maxwell R 16 Tissue LEV Total RNA Purification Kit and the Maxwell R 16 instrument (both from Promega, Leiden, The Netherlands). Then cDNA was synthesized using the GoScript™ Reverse Transcription System (Promega) according to the supplier's protocol. Samples were analyzed in triplicate for socs3 and hprt (reference gene) expression by quantitative PCR using ABsolute SYBR Green Fluorescein mix (Thermo Fisher Scientific, Etten-leur, The Netherlands). Fold induction of socs3 expression was determined by the Pfaffl method [16].

Data analysis
All data shown in the figures indicate the average of at least three biological replicates (n) that were determined by three technical replicates. In the figure legends n is indicated and error bars indicate the standard error. Significant differences between samples were calculated using the Student's t-test and regarded as significant when p < 0.05. Significant differences are indicated in the figures by asterisks (p < 0.05 [*] or p < 0.01 [**]).

IL-10-mediated signaling in dendritic cells & macrophages
Previously we observed cell-specific differences in the response to IL-10 in macrophages and dendritic cells [13]. Dendritic cells are strongly impaired in their ability to suppress LPS-induced TNF-α after 2 h of stimulation, whereas macrophages already inhibit TNF-α expression by approxiamtely 75% ( Figure 1A). We therefore investigated whether IL-10 signaling differs in these two cell types. First we focused on the activation of the transcription factor STAT3. As shown in Figure 1B, IL-10 induces strong tyrosine phosphorylation of STAT3 (Y705) in a dose-dependent manner in both cell types. Upon quantification of relative STAT3 activation, we observed a higher degree of STAT3 Y705 phosphorylation in macrophages than in dendritic cells ( Figure 1C). However, this was only significantly higher at a dose of 1 ng/ml IL-10. On the other hand, the degree of serine phosphorylation (S727) in macrophages was only significantly higher at a dose of 10 ng/ml ( Figure 1D). Yet, when comparing untreated cells with IL-10-treated cells we only observed dose-dependent STAT3 S727 phosphorylation in macrophages (p < 0.05, for all concentrations). Untreated dendritic cells already have a higher degree of STAT3 S727 phosphorylation, which is not further enhanced by IL-10 treatment. Serine phosphorylation of STAT3 could therefore be cell-type specific. Next, we assessed the activation of 18 well-characterized signaling molecules using the PathScan Intracellular Signaling Array kit. In Figure 1E representative arrays are given for macrophages and dendritic cells treated with 100 ng/ml IL-10 and their respective medium controls. The legend indicates which signaling molecules are differentially affected. Strikingly, we only detected phosphorylation of STAT3Y705 upon treatment with IL-10. Interestingly, we did observe differences in the activation of other signaling molecules between untreated macrophages and dendritic cells. Untreated dendritic cells seem to have a higher degree of phosphorylated 5' adenosine monophosphate-activated protein kinase α (AMPKα) and the ribosomal protein S6, which are indicators for cell-cycle progression and cellular growth. Furthermore, several signaling molecules downstream of the PI3K/Akt pathway (PRAS40, Bad and GSK-3β) are activated in untreated macrophages and dendritic cells, but the degree of GSK-3β phosphorylation (Ser9) is much stronger in dendritic cells. Phosphorylation of GSK-3β inhibits its activity and thereby promotes cell survival. Altogether we conclude that STAT3 is the major downstream signaling molecule for IL-10, but that there are no striking differences in the activation of this transcription factor between macrophages and dendritic cells. However, macrophages and dendritic are differentially affected in signaling molecules that regulate cellular growth and survival.
GM-CSF negatively regulates IL-10 activity Dendritic cells are differentiated from bone marrow cells by culturing them in the presence of the cytokine GM-CSF. In order to find out if GM-CSF could be responsible for the altered responses of dendritic cells to IL-10, we investigated whether GM-CSF could replicate this phenotype in macrophages. Macrophages and dendritic cells were differentiated from bone marrow, but now GM-CSF was added to macrophages or depleted from dendritic cells for the last 24 h of culture. Cells were then pretreated with IL-10 and subsequently challenged with LPS. Figure 2A-C reveals that GM-CSF is indeed able to alter the response of macrophages toward IL-10. IL-10 inhibits TNF-α expression in a dose-dependent matter in macrophages independent of GM-CSF treatment ( Figure 2A), but GM-CSF treatment lowers the maximum percentage of TNF-α inhibition at a lower dose of IL-10. Furthermore, GM-CSF treatment of macrophages significantly reduced early IL-10-mediated suppression of TNF-α by approximately 25% ( Figure 2B). We also observed that macrophages treated with GM-CSF produced significantly more TNF-α than untreated macrophages ( Figure 2C). These results indicate that GM-CSF is able to alter IL-10-mediated responses in macrophages as was observed for GM-CSF-differentiated dendritic cells.
Similarly, depletion of dendritic cells from GM-CSF enhanced their response toward IL-10. No differences were observed in the suppression of TNF-α expression after 24 h stimulation when dendritic cells were depleted from GM-CSF ( Figure 2D). However, early IL-10-mediated suppression of TNF-α expression increased significantly with almost 25% when dendritic cells were depleted from GM-CSF ( Figure 2E). Also, production of TNF-α was significantly reduced when dendritic cells were cultured in the absence of GM-CSF ( Figure 2F). We, therefore, conclude that GM-CSF is a key factor that modulates early IL-10-mediated responses in both macrophages and dendritic cells.
GM-CSF regulates IL-10 activity without affecting STAT3 activation Early IL-10-mediated responses seem to be controlled by the major anti-inflammatory pathway Jak1-STAT3-SOCS3. To investigate how GM-CSF influences early IL-10-mediated responses we first focused on the activation of STAT3. As shown in Figure 3A, IL-10 induces strong STAT3 Y705 phosphorylation regardless of GM-CSF treatment. Phosphorylation of STAT3 S727 seemed also not to be affected by GM-CSF treatment. GM-CSF seems to regulate early IL-10-mediated responses without affecting STAT3 activation.
As STAT3 phosphorylation was not affected by GM-CSF we continued to investigate the effect of GM-CSF on IL-10-induced socs3 expression. Relative socs3 transcript levels were determined by quantitative PCR in both macrophages and dendritic cells. As shown in Figure 3B, IL-10 induces socs3 expression by approximately 100fold in macrophages, whereas socs3 upregulation is significantly lower in dendritic cells (12-fold, p = 0.008). Unexpectedly, no significant differences were found for relative socs3 expression levels upon IL-10 treatment of normal and GM-CSF-treated macrophages. However, the IL-10-specific induction of socs3 expression was reduced approximately tenfold. This is because GM-CSF itself already enhances socs3 expression in macrophages prior to IL-10 treatment. Depletion of GM-CSF from dendritic cells only resulted in a twofold increase in socs3 expression. Therefore, GM-CSF seems to regulate early IL-10-mediated responses not only in a STAT3-independent manner, but also without affecting socs3 expression levels.
Next, we assessed whether GM-CSF treatment of macrophages alters IL-10-mediated signaling or other signaling pathways by using the PathScan Intracellular Signaling Array kit ( Figure 3C). As we observed previously, IL-10 only induces STAT3 Y705 phosphorylation in macrophages and dendritic cells, which is independent of GM-CSF pretreatment. GM-CSF itself induces an activation state in macrophages that is indistinguishable from dendritic cells. GM-CSF treatment of macrophages enhances the phosphorylation of AMPKα and the ribosomal protein S6, but most strongly induces the phosphorylation of GSK-3β. Phosphorylation of other signaling molecules downstream of PI3K/Akt (PRAS40 and Bad) is also enhanced. On the other hand, depletion of dendritic cells from GM-CSF relieves PRAS40 from phosphorylation, whereas GSK-3β remains strongly phosphorylated. We therefore conclude that GM-CSF alters the activation status of signaling molecules that regulate cellular growth and survival in macrophages and dendritic cells.

Discussion
IL-10 is an anti-inflammatory cytokine with promising therapeutic potential, but to date IL-10 therapy has not been as successful in the clinic as previously anticipated. A likely explanation for this phenomenon is that IL-10 activity is influenced by the cytokine milieu present in inflamed tissues [10][11][12]. Previously we have described the differential response of macrophages and dendritic cells toward IL-10 [13]. Dendritic cells are unable to respond rapidly to IL-10 as they are unable to suppress LPS-induced TNF-α at early stages. Furthermore, IL-10-induced socs3 expression was strongly reduced compared with macrophages. In this study we further investigated the mechanism that controls this differential response between macrophages and dendritic cells. Our study demonstrates that the cytokine GM-CSF, the cytokine used to differentiate dendritic cells, is a key factor that negatively regulates IL-10 activity. GM-CSF pretreatment of bone marrow-derived macrophages also reduced their ability to suppress LPS-induced TNF-α at 2 h after stimulation. Vice versa, depletion of dendritic cells from GM-CSF partially restored their early response to IL-10. Furthermore, both macrophages and dendritic cells cultured in the presence of GM-CSF produced significantly higher levels of TNF-α, which coincided with previous reports from Fleetwood et al. [17,18]. Increased TNF-α expression levels upon LPS stimulation are caused by increased basal TNF-α mRNA transcript levels upon GM-CSF treatment [18]. In our study GM-CSF-treated macrophages and dendritic cells do not produce significantly different levels of TNF-α, whereas both cell types respond significantly different toward IL-10. Differences in basal TNF-α expression levels cannot explain the observed differential response toward IL-10. Therefore, we continued to investigate IL-10-mediated signaling in macrophages and dendritic cells. IL-10 receptor engagement results in the activation of downstream Janus kinases Jak1 and Tyk2, which in turn phosphorylate the transcription factors STAT1, STAT3 and in some cell types STAT5 [19][20][21]. IL-10 is well known for its ability to activate the Jak-STAT pathway [22], but alternative signaling pathways have been described as well. Especially IL-10-mediated activation of the PI3K signaling pathway has been implicated in IL-10's survival promoting properties [23,24]. Zhou et al. also revealed that IL-10 induced the activation of extracellular signal-related kinase [25] one out of two in a PI3K-dependent manner in promyeloid cells [24]. Furthermore, IL-10 has been shown to activate p38 MAPK signaling to induce the expression of heme oxygenase-1 [26]. In our study we could only detect IL-10-mediated activation of the transcription factor STAT3 in bone marrow-derived macrophages and dendritic cells. This indicates that STAT3 is the major transcription factor required for IL-10-mediated responses in bone marrow-derived cells. However, we did observe subtle differences in the phosphorylation of STAT3 Y705 between regular macrophages and dendritic cells. More striking was the observation that IL-10 was not able to induce STAT3 S727 phosphorylation in a dose-dependent manner in dendritic cells. Serine phosphorylation of STAT3 has been reported previously for IL-6, IL-22, IFN-y and EGF [27][28][29][30] and enhances transcriptional activity of STAT3. The lack of STAT3 S727 phosphorylation might explain why IL-10-induced socs3 expression and early inhibition of TNF-α expression are impaired in dendritic cells.
Qasimi et al. previously described that SOCS3 is required by IL-10 to suppress TNF-α expression at early time points of stimulation [31]. We therefore investigated the role of SOCS3 in more detail. We observed a reduction in the ability of macrophages to respond rapidly to IL-10 upon GM-CSF treatment, but this seemed to be independent from upregulation of socs3 expression. Relative induction of socs3 expression by IL-10 was hardly affected by overnight treatment of macrophages with GM-CSF or depletion of dendritic cells from GM-CSF. This is in contrast to the difference in IL-10-induced socs3 expression between regular macrophages and dendritic cells. Furthermore, we also did not observe a difference in the phosphorylation of STAT3 Y705 and STAT3 S727 between regular macrophages or GM-CSF-treated macrophages. Therefore, GM-CSF seems to negatively regulate early IL-10-mediated responses in macrophages independent from STAT3-induced socs3 expression.
Interestingly, we did find major differences in the activation status of signaling molecules downstream of PI3K/Akt signaling in macrophages and dendritic cells. Furthermore, GM-CSF induced an activation state in macrophages that was indistinguishable from dendritic cells. Particularly the strong serine phosphorylation of GSK-3β S9 was a striking observation in GM-CSF-cultured dendritic cells or GM-CSF-treated macrophages. The degree of GSK-3β S9 phosphorylation also seems inversely correlated with early IL-10-mediated responses. A higher degree of GSK-3β S9 phosphorylation results in reduced suppression of LPS-induced TNF-α expression by IL-10. As IL-10 is also able to trigger the PI3K signaling pathway it is of great interest to investigate how these signaling pathways of IL-10 and GM-CSF are integrated and result in impaired IL-10-mediated responses.
Activation of STAT3 is regulated by a diverse set of post-translational modifications, including phosphorylation, acetylation and methylation [29,30,[32][33][34][35][36][37]. Every modification plays its own role in regulating optimal STAT3 dimerization, DNA binding activity and transcriptional activity [38]. Involvement of the PI3K signaling pathway in the activation of STAT3 has also been reported previously. Spencer et al. revealed that the cytomegalovirus homolog of IL-10 was capable of inducing serine phosphorylation of STAT3 S727 in a PI3K-dependent manner [39]. Furthermore, acetylation of STAT3 L685 by IL-6 was also shown to depend on PI3K/Akt activation [40]. However, whether IL-10-induced STAT3 transcriptional activation requires acetylation and/or methylation and whether the PI3K pathway is involved, need further investigation. Interestingly, Waitkus et al. recently reported on a novel mechanism of activation of STAT3, which was mediated by GSK-3α/β [38]. GSK-3α/β was shown to directly phosphorylate STAT3 at the residues S727 and T714. The requirement of GSK-3α/β in IL-10-induced STAT3 activation is therefore of great interest as this study shows that GM-CSF strongly inhibits GSK-3β by phosphorylation of serine residue-9. GSK-3α/β might therefore be a key signaling node that is able to control IL-10-mediated responses.
Previously, Hart et al. already reported that IL-10 was unable to suppress MHC-II expression in GM-CSFcultured monocytes [41]. Our study now also demonstrates that GM-CSF is able to alter IL-10-mediated suppression of TNF-α expression in both macrophages and dendritic cells. Furthermore, we and others show that GM-CSF-cultured cells produce significantly higher levels of the proinflammatory cytokine TNF-α [18], but also the secretion of IL-12p70 and IL-23 is significantly enhanced by GM-CSF-treated macrophages [18]. IL-23 has recently been identified as an important proinflammatory cytokine driving both innate and T-cell-induced intestinal inflammation [42]. Therefore, GM-CSF seems to induce a cytokine environment favorable for chronic inflammation. Over the last couple of years GM-CSF has been identified as a key contributor to the development of chronic inflammation in animal models of intestinal inflammation, multiple sclerosis and rheumatoid arthritis [43][44][45].

Conclusion
Taking together the key role of GM-CSF in the development of chronic inflammation with the results obtained in this study might explain why IL-10 therapy has not been as effective as previously anticipated. Our study demonstrates that the proinflammatory cytokine GM-CSF is able to negatively regulate IL-10-mediated responses in macrophages and dendritic cells.

Future perspective
Future research has to elucidate the exact mechanisms by which GM-CSF alters IL-10 signaling pathways and result in impaired cellular responses toward IL-10. It would be interesting to investigate whether GM-CSF alters the phenotype of dendritic cells and macrophages or how GM-CSF signaling interacts with the IL-10 signaling pathway. Ultimately this knowledge could provide us with new therapeutic strategies to treat inflammatory disorders.

Executive summary
• IL-10 is a promising immunoregulatory cytokine to be used to treat inflammatory disorders, but its application in the clinic is not as successful as previously anticipated.
• The local inflammatory cytokine environment might affect the response of immune cells toward IL-10.
• Previously we observed that GM-CSF-differentiated dendritic cells from mouse bone marrow respond differently toward IL-10 than bone marrow-derived macrophages.
• Treatment of macrophages with GM-CSF negatively affects their responses toward IL-10.
• Depletion of dendritic cells from GM-CSF restores their response toward IL-10.
• GM-CSF affects early IL-10-mediated responses in particular and does so independent from STAT3 activation and socs3 expression.

Financial & competing interests disclosure
This work was partly funded by Synthon BV, Nijmegen, The Netherlands. There was no additional external funding received for this study. Synthon BV supported this project financially but never interfered in the research approach. Furthermore, no patents or commercial products are under development based on this manuscript. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research
The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

Open access
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