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A CSF-1 receptor kinase inhibitor targets effector functions and inhibits pro-inflammatory cytokine production from murine macrophage populations

Katharine M. Irvine^*, Christopher J. Burns, Andrew F. Wilks, Stephen Su, David A. Hume^* and Matthew J. Sweet^*,,1

^* Cooperative Research Centre for Chronic Inflammatory Diseases and Special Research Centre for Functional and Applied Genomics, Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland, Australia;

Cytopia Research Pty Ltd., Baker Heart Research Institute Building, Victoria, Australia; and

School of Molecular and Microbial Sciences, The University of Queensland, Brisbane, Queensland, Australia

¹Correspondence: Institute for Molecular Bioscience, The University of Qld, St. Lucia, Qld, 4072, Australia. E-mail: m.sweet{at}imb.uq.edu.au

SPECIFIC AIMS

Macrophage colony-stimulating factor (M-CSF/CSF-1) regulates macrophage survival, proliferation, and function through the receptor tyrosine kinase CSF-1R. The importance of the CSF-1/CSF-1R system has been clearly demonstrated in experimental animal models of inflammatory and metastatic diseases, thus implicating the CSF-1R as a potential therapeutic target. We therefore set out to identify selective and potent kinase inhibitors of the CSF-1R that could potentially be used in therapeutic applications. In addition, we aimed to use such inhibitors as a means of dissecting the biology of the CSF-1/CSF-1R signaling system in macrophages.

PRINCIPAL FINDINGS

1. CYC10268 is a novel tyrosine kinase inhibitor that shows specificity for the CSF-1R
CYC10268 blocked the kinase activity of the CSF-1R in in vitro kinase assays (Table 1 ) in cell-based assays in which the CSF-1R was ectopically expressed and in primary macrophage cultures. CYC10268 did not inhibit the activity of a range of other tyrosine kinases, and was 5- to 15-fold more potent on CSF-1R than on closely related type III receptor tyrosine kinases (Table 1) . CYC10268 had greater potency and selectivity on the CSF-1R than imatinib mesylate, which also targets the receptor. Notably, whereas imatinib targets the Abl kinase, CYC10268 did not. In addition, CYC10268 was less potent than imatinib on Kit and the platelet-derived growth factor (PDGF) receptor. CYC10268 was equally effective at inhibiting human and mouse CSF-1R, and, in cell-based assays, was effective in cells that were already proliferating in CSF-1. In mouse bone marrow-derived macrophages (BMM), CYC10268 ablated CSF-1-mediated cell survival but did not affect survival in response to bacterial LPS.

2. CYC10268 regulates effector functions and inhibits production of proinflammatory cytokines from macrophages
The CSF-1/CSF-1R system regulates a number of key macrophage effector functions including migration, tissue remodeling, and cytokine production. It does so via its actions on multiple signaling pathways [e.g., MAPK ERK-1/2, c-Jun NH2-terminal kinase (JNK), and Akt], which in turn regulate the expression of genes such as urokinase plasminogen activator (uPA) and Toll-like Receptor (TLR) 9, which influence macrophage function. CYC10268 blocked CSF-1-induced receptor autophosphorylation and downstream signaling cascades in BMM. Consequently, CYC10268 prevented CSF-1 from inducing expression of uPA mRNA and from repressing expression of apolipoprotein E and TLR9 mRNAs (Fig. 1 ). We also used CYC10268 to show that CSF-1 greatly amplified production of the proinflammatory mediators IL-6, IL-12, and TNF- from macrophages in response to LPS (Fig. 1) . Although CYC10268 blocked signaling downstream of CSF-1, it did not inhibit CSF-1-mediated internalization of the CSF-1R. Thus, CSF-1R kinase activity is not required for this response.

View larger version (31K): [in this window] [in a new window]   Figure 1. CYC10268 inhibits gene expression downstream of CSF-1R and CSF-1 immunomodulation in BMM. A) CSF-1-starved BMM were treated with CSF-1 and/or 5 µM CYC10268 over 24 h. Total RNA was collected at the indicated time points and reverse transcribed to cDNA. uPA, tlr9, and apolipoprotein E mRNA levels were estimated by real-time polymerase chain reaction (PCR) using SYBR green amplification and an ABI7000. Gene expression levels were normalized to hprt expression. B) BMM, pretreated with vehicle or 5 µM CYC10268, were primed with CSF-1 or IFN for 20 h before treatment with 10 ng/ml LPS for 24 h. ELISAs were performed on supernatants for TNF, IL-6, and IL-12. Data points represent the mean of triplicates, and SD are displayed. These data are representative of at least 3 independent experiments.

3. The inflammatory phenotype of thioglycollate-elicited peritoneal macrophages (TEPM) was inhibited by CYC10268
Unlike BMM, TEPM are not dependent on exogenous CSF-1 for survival. This may be because TEPM produce low levels of endogenous CSF-1 or receive an alternative survival signal. We used CYC10268 to assess the dependence of TEPM on autocrine CSF-1. CYC10268, at doses that were not toxic to CSF-1-starved BMM, reduced TEPM viability 2-fold in the absence of CSF-1. We also examined the effect of CYC10268 on the expression of known CSF-1-regulated genes in TEPM. In the absence of CSF-1, TEPM expressed high basal levels of uPA mRNA, and CYC10268 down-regulated uPA expression, consistent with the theory that basal uPA expression in TEPM is maintained by autocrine CSF-1. CSF-1 primes murine macrophage populations for production of proinflammatory cytokines in response to LPS (Fig. 1) . Consistent with our hypothesis that TEPM behave as a CSF-1 "primed " macrophage population, CYC10268 dramatically impaired LPS-induced inflammatory cytokine production in TEPM in the absence of CSF-1. Finally, we showed that, in TEPM, csf-1 mRNA was expressed at levels comparable to those in osteoblasts that produce biologically active CSF-1. The levels of csf-1 mRNA in BMM, which require exogenous CSF-1 for survival, were 20-fold lower.

CONCLUSIONS AND SIGNIFICANCE

CSF-1 has been implicated as a key mediator of chronic inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, kidney disease, and cardiovascular disease, as well as cancers of the breast, uterus, and colon. Its involvement in such a diverse array of diseases highlights the pleiotropic effect of CSF-1 on macrophage function. As well as regulating macrophage survival, differentiation, and proliferation; CSF-1 also primes responses to LPS, is a potent chemoattractant, triggers the production of extracellular matrix (ECM) degrading enzymes, and suppresses antigen-specific immune responses. In this study, we have identified a novel and potent inhibitor of CSF-1R kinase activity, CYC10268. CSF-1R inhibitors such as CYC10268 provide insight into the role of the CSF-1/CSF-1R signaling system in mature macrophage effector function in health and disease. CYC10268 was equally potent on mouse and human CSF-1R, so this compound has potential in experimental animal disease models as well as in clinical applications. The effect of CYC10268 on CSF-1R activity was demonstrated in a range of biological assays, and it was more potent on the CSF-1R than on other closely related kinases [PDGF-R, Kit, Flt3, vascular endothelial growth factor (VEGF)-R; Table 1 ].

CYC10268 is one of only a handful of small molecule inhibitors demonstrated to target the CSF-1R, the others being imatinib mesylate (the clinically successful Abl/Kit/PDGF-R inhibitor), GW2580 (a relatively selective CSF-1R inhibitor), and the broad-spectrum inhibitor SU11248. These inhibitors are structurally diverse and have different specificity profiles. At this stage, it is not possible to predict which of these inhibitors will be most efficacious and safe in therapeutic applications. While imatinib is already used in the clinic, it is important to recognize that it has been used clinically for only a relatively short time (5 years), and its long-term safety and efficacy profile is not yet clear. Indeed, cases of imatinib resistance have already arisen. Alternative, more selective inhibitors such as CYC10268 may have enhanced therapeutic benefit in conditions in which CSF-1 is central to pathology. Moreover, it is possible that some of the therapeutic benefits associated with imatinib are attributable to its action on the CSF-1R rather than Abl. Compounds like CYC10268 represent useful tools to analyze the extent of such a contribution.

We show in this report that CYC10268 inhibited several outcomes of CSF-1 signaling that are central to the pathologies described above. Our compound blocked macrophage proliferation and CSF-1-dependent priming for production of proinflammatory cytokines that are associated with chronic inflammation. In addition, the clear link between CSF-1/CSF-1R and metastatic potential is thought to primarily involve expression of enzymes such as uPA, which degrade the ECM. Since CYC10268 blocked CSF-1-induced expression of uPA, it could also have potential in malignant disease. CYC10268 might also be utilized for enhancing vaccine efficacy. CSF-1 is immunosuppressive for T cell responses in vitro and in vivo, and a recent report showed that imatinib enhanced antigen-presenting cell function. In addition, CSF-1 repressed expression of TLR9, which is required for responses to CpG DNA. CpG DNA has been used widely as a vaccine adjuvant. Hence, targeting CSF-1 during vaccine delivery may enhance efficacy by promoting antigen presentation as well as increasing the adjuvant activity of CpG DNA.

CYC10268 also provides a powerful tool to dissect the role of CSF-1/CSF-1R signaling in mature macrophage effector functions. In this study, we have used CYC10268 to assess several aspects of CSF-1 signaling. First, we show that CSF-1-dependent CSF-1R internalization is not dependent on CSF-1R kinase activity. We also show that LPS-mediated survival of BMM does not occur via a CSF-1-dependent pathway. Hence, although LPS can induce CSF-1 expression in certain cell populations and in vivo, it acts independently of CSF-1 to trigger survival in BMM. We also show that TEPM, which do not require exogenous CSF-1 for survival, behave as CSF-1 primed cells. TEPM are often used as a model for inflammatory macrophages but are, in fact, a relatively ill-defined population. TEPM proliferation and colony-forming capacity in the presence of TEPM-conditioned medium, prior to the identification of CSF-1, led to suggestions that TEPM cultures were autocrine for an essential growth factor. We show that TEPM constitutively express both c-fms and csf-1 mRNAs, and treatment of TEPM with CYC10268 regulated the basal expression of the CSF-1 target gene, uPA. Furthermore, TEPM produced high levels of proinflammatory cytokines in response to LPS. CSF-1 did not enhance this response, but CYC10268 potently inhibited LPS-induced cytokine production. This supports the notion that constitutive CSF-1 "priming " is part of the TEPM inflammatory phenotype and that blockade of CSF-1 signaling is likely to be an effective way to suppress macrophage effector functions.

Our report clearly demonstrates the potency and selectivity of a novel inhibitory compound that targets the CSF-1R. This compound and its derivatives will be useful in understanding the biology of CSF-1 action and will have a broad range of potential therapeutic applications. A comparison of the efficacies and tolerance of the different CSF-1R kinase inhibitors in different models of inflammatory and metastatic disease should be a focus of future research.

View larger version (33K): [in this window] [in a new window]   Figure 2. Schematic diagram summarizing CSF-1-dependent control points in macrophage function that could be targeted therapeutically. CSF-1 controls macrophage production and survival, but also regulates many functions of mature macrophages. CYC10268, via its actions on the CSF-1R, may have efficacy in inflammatory disease, cancer, and vaccine design.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-5848fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-5848fjev1 20/11/1921    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Irvine, K. M. Articles by Sweet, M. J. Search for Related Content PubMed PubMed Citation Articles by Irvine, K. M. Articles by Sweet, M. J.