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Drug Discovery Today Volume 00, Number 00 June 2014
Targeting IL-34 in chronic inflammation Emma L. Masteller and Brian R. Wong Q1 Five Prime Therapeutics, 2 Corporate Drive, South San Francisco, CA 94080, USA
A second ligand for colony-stimulating factor-1 receptor (CSF-1R) with distinct biologic activities had long been implicated but not appreciated until the recent discovery of interleukin (IL)-34. IL-34 and CSF1 signal through this common receptor to mediate the biology of mononuclear phagocytic cells. Aberrant macrophage activation by CSF-1 and/or IL-34 is associated with numerous diseases, and Q2 clinical therapies targeting this pathway are being tested. Although IL-34 and CSF1 have distinct activities under physiologic conditions, they appear functionally redundant in various disease states. Thus, blocking the activity of both might be necessary for maximal efficacy.
Introduction
Discovery of IL-34
Cells of the mononuclear phagocytic lineage including monocytes, macrophages, Langerhans cells, osteoclasts and microglia are involved in tissue development, homeostasis and repair and serve as key regulators of immune function. However, dysregulation of these cells contributes to a variety of diseases including inflammation, cancer, fibrosis and bone disease. Colony-stimulating factor-1 (CSF-1) signaling through the CSF1 receptor (CSF-1R), a type III receptor tyrosine kinase, regulates mononuclear phagocytic cell biology and has been well studied since its discovery over three decades ago. It had long been postulated that there was a second ligand for CSF-1R, because the phenotypes of the CSF-1 and CSF-1R knockout mice were not entirely complementary. Recently, interleukin (IL)-34 was identified as a potent activator of monocytes and macrophages, signaling through CSF-1R with a distinct tissue distribution from CSF-1 [1]. IL-34 and CSF-1 share many functional properties; however, notable differences suggest these two cytokines serve complementary rather than redundant roles in supporting macrophage homeostasis. In this review we discuss the most recent advances in understanding the role of IL-34 in macrophage biology and the potential for targeting IL-34 and the CSF-1R pathway in autoimmune and inflammatory diseases.
The possibility of a second ligand for CSF-1R had been predicted by the more severe phenotype seen in CSF-1R-deficient (CSF-1R / ) mice compared with CSF-1-deficient (CSF-1op/op) mice, which harbor an inactivating mutation in the csf-1 gene [2]. CFS-1op/op mice had an osteopetrotic phenotype at birth characterized by a profound loss of bone osteoclasts and reduction of macrophages in some tissues that were partially restored with age. Microglia numbers were only modestly reduced and Langerhans cell development in the skin appeared relatively normal. By contrast, CSF1R / mice were severely depleted of macrophages and lacked Langerhans cells, microglia and osteoclasts throughout their lifespan. Therefore, it appeared that some activities of CSF-1R were mediated independently of CSF-1, possibly through a second ligand. IL-34 was identified by screening a comprehensive human protein library containing 3400 secreted and extracellular domain proteins in a human monocyte viability assay [1]. C16orf77, a hypothetical protein in the public database, supported the Q3 survival of human peripheral blood monocytes and was designated as IL-34. The activity of IL-34 appeared specific to the monocyte and macrophage lineage because it did not affect 28 other cell types tested in a variety of assays. IL-34 lacked appreciable sequence similarity to every other cytokine or protein. The receptor for IL-34 was identified by screening a library of extracellular domains of transmembrane proteins for the ability to block IL-34 activity in the monocyte viability screen. The identified
Corresponding author: Masteller, E.L. ([email protected])
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receptor was CSF-1R. Further functional studies confirmed the specific activity of IL-34 through CSF-1R on monocyte-lineage cells.
IL-34 biology
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In many aspects IL-34 functions similarly to CSF-1, however some differences between the two cytokines have been identified. IL-34 supports the growth and survival of primary human monocytes and promotes the formation of macrophage colonies from human bone marrow equivalently to CSF-1 and was found to be able to substitute for CSF-1 in receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis [3,4]. In vivo, the mouse IL-34 gene expressed transgenically under the control of the mouse CSF-1 promoter was able to rescue the defects of the CSF-1op/op mice [5]. Similar to CSF-1, IL-34 stimulation of CSF-1R Q4 leads to phosphorylation of ERK1/2 in human monocytes [1]. However, IL-34 binds to CSF-1R with higher affinity than CSF-1 and was found to produce a stronger but more transient tyrosine phosphorylation of CSF-1R and downstream mediators, suggesting the two cytokines differ somewhat in signaling activity [6]. Studies on human monocytes determined that 68% of the IL-34- or CSF-1-induced gene expression changes were similar for the two stimuli, whereas only 32% differed [7]. In general, IL-34 induced transcriptional changes of a lesser magnitude than CSF-1. The most notable difference between IL-34 and CSF-1 is their expression patterns in embryonic and adult tissues. CSF-1 has relatively broad expression whereas IL-34 expression is more restricted. Strikingly, IL-34 mRNA was strongly expressed in the embryonic brain before the appearance of CSF-1 mRNA expression, and IL-34 was expressed more strongly in most areas of the postnatal and adult brain in largely nonoverlapping regions to CSF-1 [5,8]. The expression data suggest that IL-34 and CSF-1 serve complimentary nonredundant roles in the developing central nervous system. To identify IL34-specific effects two groups produced IL-34deficient reporter mice. Mice deficient in IL-34 displayed a marked reduction of Langerhans cells and a decrease in microglia, whereas monocytes, tissue macrophages and dendritic cells were largely unaffected [9,10]. IL-34 was expressed primarily by keratinocytes in the epidermis and neurons in the brain. IL-34 was essential for embryonic development of Langerhans cells and for their homeostasis in the adult in the steady state. Under conditions of inflammation in the skin, IL-34 was not required for the recruitment of blood-borne precursors and their differentiation into Langerhans cells but was crucial for their maintenance after inflammation was resolved. Reductions of Langerhans cells in the IL34-deficient mice resulted in reduced sensitivity to chemical hapten- and Candidaalbicans-mediated contact hypersensitivity [10]. IL-34 was found to be essential for microglia homeostasis in specific areas of the adult brain but was not required for embryonic development of microglia [9]. The reduction of microglia in the IL-34-deficient mice did not cause a substantially altered phenotype in the central nervous system under steady-state conditions but did negatively affect the survival of mice infected intracranially with an attenuated strain of West Nile virus [10]. In addition to expression in skin and brain, IL-34 transcripts are also detected in various tissues including the spleen, kidney and testes. IL-34 is abundantly expressed in human spleen and studies 2
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in CSF-1-deficient mice suggest that IL-34 plays a pivotal part in maintaining the splenic reservoir of osteoclast precursor cells [11]. A gene expression study of lymph node stromal cells found IL-34 expressed by fibroblast reticular cells, suggesting IL-34 could regulate myeloid cell function in lymph node immune responses [12]. A group studying the role of follicular dendritic cells (FDCs) in controlling B cell fate in germinal centers found that a mouse FDC line produced IL-34 that promoted the differentiation of a particular type of mononuclear phagocytic cell (designated as FDMC) Q5 that was capable of stimulating activated B cell proliferation [13]. The FDC line produced CSF-1 in addition to IL-34; however, IL-34 but not CSF-1 was reported to be responsible for FDC-mediated generation of the FDMCs. Additional studies in IL-34-deficient mice are needed to confirm IL-34 specificity in promoting B cell follicle-associated macrophage differentiation and to determine the role of FDMCs in germinal center reactions. It was observed that IL-34 and CSF-1R showed differential expression in the brain, suggesting that IL-34 could signal via an alternative receptor. This led to the discovery that IL-34 also interacts with receptor-type protein-tyrosine phosphatase z (PTPz), a cell surface chondroitin sulfate (CS) proteoglycan [14]. PTP-z is primarily expressed on neural progenitors and glial cells and possesses multiple ligands. IL-34 binding inhibits the constitutive phosphatase activity of PTP-z leading to increased tyrosine phosphorylation of downstream targets. To date, no CSF-1R-independent functions of IL-34 have been identified. It is possible that PTPz and CSF-1R cooperate to mediate IL-34 activity, although further studies are needed to identify CSF-1R-dependent or -independent IL-34 signaling through PTP-z. CSF-1R expression is restricted primarily to cells of the mononuclear phagocytic lineage. This includes macrophage precursors in the bone marrow, monocytes, osteoclasts and tissue macrophages including Kupffer cells in the liver and microglia in the brain. In non-hematopoietic cells CSF-1R is expressed on trophoblasts. Different groups have also reported CSF-1R expression on epithelial cells of colonic crypts, on subsets of neurons and on renal proximal tubule cells. However, not all studies support these observations and the expression of CSF-1R on some non-monocyte lineage cells remains controversial: discussed in [15].
Structural basis of CSF-1R activation by IL-34 The discovery that IL-34 and CSF-1 could activate CSF-1R raised the question of how these two disparate cytokines structurally engaged CSF-1R. IL-34 and CSF-1 were shown to compete for binding to CSF-1R, and anti-CSF-1R monoclonal antibodies (mAbs) were identified that could block CSF-1 but not IL-34 suggesting the two cytokines share overlapping but distinct binding sites [5,6]. Although IL-34 shares no appreciable sequence similarity with any other protein it was proposed by fold recognition to be a four-helix bundle cytokine belonging to the same family as CSF-1 [16]. Recently, the crystallographic structures of truncated versions of human and mouse IL-34 were solved and IL34 was confirmed to be a four-helix bundle cytokine, albeit with some unique features [17,18]. IL-34 forms a noncovalently linked dimer whereas CSF-1 contains an intersubunit disulfide bond. The structure of the IL-34–CSF-1R complex shows a similar ligand– receptor assembly to that of CSF-1–CSF-1R. Dimeric IL-34 and
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CSF-1 bind the same general region of CSF-1R by interacting with overlapping but distinct epitopes on CSF-1R [17–19].
Preclinical studies Benefits of blocking the CSF-1R pathway have been shown in numerous animal models of chronic disease. CSF-1-deficient mice are protected from arthritis, bone loss, nephritis, cutaneous lupus and lung fibrosis, and these disorders are also reduced in the setting of CSF-1 or CSF-1R blockade, reviewed in [20,21]. Results in inflammatory bowel disease models were mixed depending on the model used. Although numerous animal studies demonstrate the benefit of blocking the CSF-1R pathway in inflammation not all studies support this. A study using a particular anti-CSF-1R antibody found no inhibitory effect in acute inflammation models, discussed in [20,21]. This is in contrast to results reported in a second study using similar models but a different anti-CSF-1R antibody. This discrepancy could be explained by different modes of action of these two antibodies caused by either differences in epitope binding, and thus differential CSF-1 versus IL-34 blockade, or differences in Fc effector function. Additionally, the models studied were acute inflammatory models, and the relevance of the CSF1R pathway might differ between acute and chronic disease models. Targeting the CSF-1R pathway in chronic inflammatory disease models has been shown to be efficacious [20]. More-recent studies demonstrated efficacy of CSF-1R inhibition in mouse models of cancer. A blocking anti-CSF-1R mAb reduced anti-inflammatory tumor-associated macrophages resulting in reduced tumor growth and prolonged survival [22]. In a glioblastoma model, small molecule inhibition of CSF-1R resulted in tumor growth inhibition and polarizing tumor-associated macrophage to a proinflammatory phenotype [23]. Thus, depending on the disease context, and probably the subsets of macrophage involved, CSF-1R pathway inhibition could have beneficial anti-inflammatory or proinflammatory effects.
IL-34 pathology in rheumatoid arthritis and other inflammatory diseases Rheumatoid arthritis (RA) is a chronic autoimmune disorder of the joints characterized by synovial inflammation and hyperplasia leading to progressive cartilage and bone destruction. Activated macrophages are the predominant infiltrating cell type in the inflamed synovium and promote inflammation through production of inflammatory cytokines such as tumor necrosis factor (TNF)a and IL-6 [24]. Factors from the inflamed synovium drive the differentiation of monocytic lineage cells to osteoclasts, which mediate bone destruction. The CSF-1R pathway is thought to play a predominant part by promoting the differentiation and survival of monocytes and macrophages and osteoclasts. In support of this, activation of CSF-1R has been detected in RA synovium and CSF-1 expression is upregulated in RA [25]. To understand the role of IL-34 in RA several groups have looked at the expression of IL-34 in tissue and blood from RA patients. Immunohistochemistry studies demonstrated that IL-34 is increased in RA synovium compared with osteoarthritis and healthy controls [26,27]. Chemel et al. reported a positive association between synovial IL-34 expression levels and synovitis severity [26]. Several groups also reported elevated levels of IL-34 in RA
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blood and synovial fluid [26–29]. Some caution is needed in interpreting the IL-34 ELISA results because it is not clear if all groups used methods to eliminate the interference of rheumatoid factor (RF). Moon et al. did use methods to block RF interference and detected elevated levels of IL-34 in RA serum and synovial fluid and found an association between IL-34 levels and RF and anticyclic citrullinated peptide (CCP) antibody titers [28]. Functionally, isolated RA-derived fibroblast-like synovial cells and osteoblasts were found to produce IL-34 in response to TNFa [27,30]. As discussed above, IL-34 can promote osteoclastogenesis in combination with RANKL and induce bone destruction [3,4]. IL34 was also shown capable of inducing proinflammatory cytokines and chemokines such as IL-6, IL-8 and CCL2 in human whole Q6 blood [31]. Together the data suggest that IL-34 is involved in RA pathogenesis. Given that IL-34 and CSF-1 are simultaneously expressed in RA synovium it will probably be necessary to block the activity of both to produce a therapeutic benefit. To test the effect of targeting the CSF-1R pathway in RA, we determined the effect of blocking CSF-1R in RA synovial explants (Garcia, S. et al. (2012) American College of Rheumatology Annual Q7 Meeting, Washington DC, Abstr. 901). Synovial biopsy samples were obtained by arthroscopy from sites with active inflammation and cultured without dissociation ex vivo. This system maintains the synovial architecture and cell–cell contacts and therefore reflects more closely the in vivo joint environment. Intact synovial biopsy samples continue to produce cytokines and chemokines without the addition of exogenous stimulus. Treatment with an anti-CSF-1R antibody that blocks IL-34 and CSF-1 signaling significantly reduced IL-6 levels compared with samples treated with an isotype control antibody. These results suggest that targeting the CSF-1R pathway will have clinical benefit in RA. In addition to RA, targeting the CSF-1R pathway could have clinical benefit in other inflammatory diseases. IL-34 was found to be overexpressed in inflamed salivary glands of patients with Sjogren’s syndrome (SS), a chronic immune disorder typically affecting exocrine glands [32]. Macrophages are recognized as an important component of SS lesions with increased levels of macrophages appearing in severe lesions [33]. IL-34 expression was associated with increased expression of TNFa, IL-1b and IL-17, suggesting that IL-34 could regulate monocytes and/or macrophages involved in the pathogenesis of salivary gland inflammation [32]. Intriguingly, a study aimed at identifying shared inflammatory mechanisms of lupus identified IL-34 among a limited number of cytokines and chemokines that were upregulated in kidneys of three different mouse models of lupus nephritis (LN) [34]. LN is associated with inflammation caused by deposition of immune complexes in the kidney. Macrophages are activated by these immune complexes through Fc receptors and contribute to the chronic renal inflammation and tissue damage associated with nephritis. CSF-1 is also upregulated in blood and urine of patients with LN [35]. The presence of IL-34, CSF-1 and activated macrophage suggest that targeting the CSF-1R pathway in LN might provide clinical benefit.
Strategies for blocking CSF-1R pathway activity There are several strategies for inhibiting CSF-1R pathway activity (Fig. 1). CSF-1R is part of a family of receptor tyrosine kinases with
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Drug Discovery Today Volume 00, Number 00 June 2014
IL-34
CSF-1
Antibodies neutralizing CSF-1 or IL-34 FPA008
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Antibodies blocking CSF-1R
CSF-1R (M-CSF receptor)
Small molecule kinase inhibitors
Monocytes/macrophages
Inflammation bone destruction Drug Discovery Today
FIGURE 1
Q13 Strategies to block colony-stimulating factor-1 receptor (CSF-1R) activation. CSF-1R is a receptor tyrosine kinase that is activated by interleukin (IL)-34 or CSF-1. CSF-1R signaling can be blocked by small molecule inhibitors targeting the kinase domain, by neutralizing antibodies targeting either or both IL-34 and CSF-1 or by blocking antibodies targeting CSF-1R. FPA008 is a humanized antibody that blocks CSF-1 and IL-34 with high potency.
intracellular domains that are very closely related. Small molecules inhibiting the kinase activity of CSF-1R have been developed and are being tested in the clinic. These inhibitors range from being highly selective for CSF-1R to possessing dual activity to related Q8 kinases such as KIT or FLT3 [36]. Antibodies against the receptor that block binding of IL-34 and CSF-1 or block the dimerization of the receptor have also been developed and are in early clinical testing. Dual blockade of both cytokines could also be achieved through bi-specific antibodies directed against the ligands or a CSF-1 receptor-trap, although neither of these is reported to be in the clinic. Blockade of either cytokine alone could be achieved through anticytokine antibodies or, because the two ligands interact with unique epitopes on the CSF-1R, antibodies directed at
the CSF-1R with selective blocking activity could be developed. Pfizer has tested an anti-CSF-1 mAb in early clinical trials and in non-human primates. In non-human primates the antibody resulted in selective reduction of CD16+ peripheral monocytes and a loss of Kupffer cells in the liver [37]. The reduction of Kupffer cells was associated with in an increase in serum enzymes with no signs of hepatic or skeletal muscle injury. The rise in serum enzymes was attributed to reduced clearance caused by decreased Kupffer cell numbers and not as a result of hepatic or skeletal injury. This conclusion was supported by additional studies in rats where treatment with clodronate liposomes to deplete Kupffer cells led to increased serum enzyme levels, again without evidence of tissue injury. In addition, CSF-1-deficient mice, which have
TABLE 1
Clinical trials targeting the colony-stimulating factor-1 receptor (CSF-1R) pathway Target
Molecule
Drug type
Company
Indication
Phase
CSF-1
MCS110
Antibody
Novartis
Cancer Pigmented villonodular synovitis
I/II II
CSF-1
PD0360324
Antibody
Pfizer
Rheumatoid arthritis (RA) Cutaneous lupus Pulmonary sarcoidosis
I I I
CSF-1R
AMG820
Antibody
Amgen
Cancer
I
CSF-1R
ARRY382
Small molecule
Array
Cancer
I
CSF-1R
FPA008
Antibody
Five Prime
RA
I
CSF-1R
IMCCS4
Antibody
ImClone/Eli Lilly
Cancer
I
CSF-1R
JNJ40346527
Small molecule
Johnson & Johnson
RA Cancer
II I
CSF-1R
PLX5622
Small molecule
Plexxikon/Daiichi Sankyo
RA
I
4
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reduced levels of Kupffer cells, were found to have higher levels of serum enzymes compared with wild-type littermates. In humans the anti-CSF-1 mAb also specifically reduced CD16+ peripheral blood monocytes [38]. CD16+ monocytes are considered to be inflammatory and are specifically upregulated in many inflammatory conditions including in RA [39]. Pfizer has completed a Phase I clinical trial in RA but has not reported the data yet. Several other compounds targeting the CSF-1R pathway are in the clinic for autoimmune diseases or cancer (Table 1).
The discovery of IL-34 has added a new layer of complexity to this pathway but has also provided answers to outstanding questions. In disease settings where IL-34, CSF-1 and CSF-1R are all present, such as the inflamed synovium in RA, it will probably be necessary to inhibit the activity of both ligands to achieve efficacy. More studies are needed to understand the contribution of IL-34 to other diseases where macrophages are implicated in the pathology (such as fibrosis and inflammatory bowel disease) and to identify new opportunities for targeting this new cytokine for clinical benefit.
Concluding remarks
Conflicts of interest
CSF-1 and CSF-1R involvement in disease pathology and their potential as therapeutic targets have been well studied for years.
Emma Masteller and Brian Wong are employees of Five Prime Therapeutics, and possess stock options in the company.
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21 Hume, D.A. and MacDonald, K.P. (2012) Therapeutic applications of macrophage colony-stimulating factor-1 (CSF-1) and antagonists of CSF-1 receptor (CSF-1R) signaling. Blood 119, 1810–1820 22 Fend, L. et al. (2013) Therapeutic effects of anti-CD115 monoclonal antibody in mouse cancer models through dual inhibition of tumor-associated macrophages and osteoclasts. PLoS One 8, e73310 23 Pyonteck, S.M. et al. (2013) CSF-1R inhibition alters macrophage polarization and blocks glioma progression. Nat. Med. 19, 1264–1272 24 Hamilton, J.A. and Tak, P.P. (2009) The dynamics of macrophage lineage populations in inflammatory and autoimmune diseases. Arthritis Rheum. 60, 1210–1221 25 Paniagua, R.T. et al. (2010) c-Fms-mediated differentiation and priming of monocyte lineage cells play a central role in autoimmune arthritis. Arthritis Res. Ther. 12, R32 26 Chemel, M. et al. (2012) Interleukin 34 expression is associated with synovitis severity in rheumatoid arthritis patients. Ann. Rheum. Dis. 71, 150–154 Q12 27 Hwang, S.J. et al. (2012) Interleukin-34 produced by human fibroblast-like synovial cells in rheumatoid arthritis supports osteoclastogenesis. Arthritis Res. Ther. 14, R14 28 Moon, S.J. et al. (2013) Increased levels of interleukin 34 in serum and synovial fluid are associated with rheumatoid factor and anticyclic citrullinated peptide antibody titers in patients with rheumatoid arthritis. J. Rheumatol. 40, 1842–1849 29 Tian, Y. et al. (2013) Elevated serum and synovial fluid levels of interleukin-34 in rheumatoid arthritis: possible association with disease progression via interleukin17 production. J. Interferon Cytokine Res. 33, 398–401 30 Eda, H. et al. (2011) Proinflammatory cytokines, IL-1beta and TNF-alpha, induce expression of interleukin-34 mRNA via JNK- and p44/42 MAPK-NF-kappaB pathway but not p38 pathway in osteoblasts. Rheumatol. Int. 31, 1525–1530 31 Eda, H. et al. (2010) Macrophage-colony stimulating factor and interleukin-34 induce chemokines in human whole blood. Cytokine 52, 215–220 32 Ciccia, F. et al. (2013) IL-34 is overexpressed in the inflamed salivary glands of patients with Sjogren’s syndrome and is associated with the local expansion of proinflammatory CD14(bright)CD16+ monocytes. Rheumatology 52, 1009–1017 33 Mavragani, C.P. and Moutsopoulos, H.M. (2014) Sjogren’s syndrome. Annu. Rev. Pathol. 9, 273–285 34 Bethunaickan, R. et al. (2013) Comparative transcriptional profiling of 3 murine models of SLE nephritis reveals both unique and shared regulatory networks. PLoS One 8, e77489 35 Menke, J. et al. (2009) Circulating CSF-1 promotes monocyte and macrophage phenotypes that enhance lupus nephritis. J. Am. Soc. Nephrol. 20, 2581–2592 36 Zhang, C. et al. (2013) Design and pharmacology of a highly specific dual FMS and KIT kinase inhibitor. Proc. Natl. Acad. Sci. U. S. A. 110, 5689–5694 37 Radi, Z.A. et al. (2011) Increased serum enzyme levels associated with kupffer cell reduction with no signs of hepatic or skeletal muscle injury. Am. J. Pathol. 179, 240–247 38 Korkosz, M. et al. (2012) Monoclonal antibodies against macrophage colonystimulating factor diminish the number of circulating intermediate and nonclassical (CD14(++)CD16(+)/CD14(+)CD16(++)) monocytes in rheumatoid arthritis patient. Blood 119, 5329–5330 39 Wong, K.L. et al. (2012) The three human monocyte subsets: implications for health and disease. Immunol. Res. 53, 41–57
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Drug Discovery Today Volume 00, Number 00 June 2014
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Drug Discovery Today Volume 00, Number 00 June 2014
Targeting IL-34 in chronic inflammation Emma L. Masteller and Brian R. Wong Q1 Five Prime Therapeutics, 2 Corporate Drive, South San Francisco, CA 94080, USA
A second ligand for colony-stimulating factor-1 receptor (CSF-1R) with distinct biologic activities had long been implicated but not appreciated until the recent discovery of interleukin (IL)-34. IL-34 and CSF1 signal through this common receptor to mediate the biology of mononuclear phagocytic cells. Aberrant macrophage activation by CSF-1 and/or IL-34 is associated with numerous diseases, and Q2 clinical therapies targeting this pathway are being tested. Although IL-34 and CSF1 have distinct activities under physiologic conditions, they appear functionally redundant in various disease states. Thus, blocking the activity of both might be necessary for maximal efficacy.
Introduction
Discovery of IL-34
Cells of the mononuclear phagocytic lineage including monocytes, macrophages, Langerhans cells, osteoclasts and microglia are involved in tissue development, homeostasis and repair and serve as key regulators of immune function. However, dysregulation of these cells contributes to a variety of diseases including inflammation, cancer, fibrosis and bone disease. Colony-stimulating factor-1 (CSF-1) signaling through the CSF1 receptor (CSF-1R), a type III receptor tyrosine kinase, regulates mononuclear phagocytic cell biology and has been well studied since its discovery over three decades ago. It had long been postulated that there was a second ligand for CSF-1R, because the phenotypes of the CSF-1 and CSF-1R knockout mice were not entirely complementary. Recently, interleukin (IL)-34 was identified as a potent activator of monocytes and macrophages, signaling through CSF-1R with a distinct tissue distribution from CSF-1 [1]. IL-34 and CSF-1 share many functional properties; however, notable differences suggest these two cytokines serve complementary rather than redundant roles in supporting macrophage homeostasis. In this review we discuss the most recent advances in understanding the role of IL-34 in macrophage biology and the potential for targeting IL-34 and the CSF-1R pathway in autoimmune and inflammatory diseases.
The possibility of a second ligand for CSF-1R had been predicted by the more severe phenotype seen in CSF-1R-deficient (CSF-1R / ) mice compared with CSF-1-deficient (CSF-1op/op) mice, which harbor an inactivating mutation in the csf-1 gene [2]. CFS-1op/op mice had an osteopetrotic phenotype at birth characterized by a profound loss of bone osteoclasts and reduction of macrophages in some tissues that were partially restored with age. Microglia numbers were only modestly reduced and Langerhans cell development in the skin appeared relatively normal. By contrast, CSF1R / mice were severely depleted of macrophages and lacked Langerhans cells, microglia and osteoclasts throughout their lifespan. Therefore, it appeared that some activities of CSF-1R were mediated independently of CSF-1, possibly through a second ligand. IL-34 was identified by screening a comprehensive human protein library containing 3400 secreted and extracellular domain proteins in a human monocyte viability assay [1]. C16orf77, a hypothetical protein in the public database, supported the Q3 survival of human peripheral blood monocytes and was designated as IL-34. The activity of IL-34 appeared specific to the monocyte and macrophage lineage because it did not affect 28 other cell types tested in a variety of assays. IL-34 lacked appreciable sequence similarity to every other cytokine or protein. The receptor for IL-34 was identified by screening a library of extracellular domains of transmembrane proteins for the ability to block IL-34 activity in the monocyte viability screen. The identified
Corresponding author: Masteller, E.L. ([email protected])
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receptor was CSF-1R. Further functional studies confirmed the specific activity of IL-34 through CSF-1R on monocyte-lineage cells.
IL-34 biology
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In many aspects IL-34 functions similarly to CSF-1, however some differences between the two cytokines have been identified. IL-34 supports the growth and survival of primary human monocytes and promotes the formation of macrophage colonies from human bone marrow equivalently to CSF-1 and was found to be able to substitute for CSF-1 in receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis [3,4]. In vivo, the mouse IL-34 gene expressed transgenically under the control of the mouse CSF-1 promoter was able to rescue the defects of the CSF-1op/op mice [5]. Similar to CSF-1, IL-34 stimulation of CSF-1R Q4 leads to phosphorylation of ERK1/2 in human monocytes [1]. However, IL-34 binds to CSF-1R with higher affinity than CSF-1 and was found to produce a stronger but more transient tyrosine phosphorylation of CSF-1R and downstream mediators, suggesting the two cytokines differ somewhat in signaling activity [6]. Studies on human monocytes determined that 68% of the IL-34- or CSF-1-induced gene expression changes were similar for the two stimuli, whereas only 32% differed [7]. In general, IL-34 induced transcriptional changes of a lesser magnitude than CSF-1. The most notable difference between IL-34 and CSF-1 is their expression patterns in embryonic and adult tissues. CSF-1 has relatively broad expression whereas IL-34 expression is more restricted. Strikingly, IL-34 mRNA was strongly expressed in the embryonic brain before the appearance of CSF-1 mRNA expression, and IL-34 was expressed more strongly in most areas of the postnatal and adult brain in largely nonoverlapping regions to CSF-1 [5,8]. The expression data suggest that IL-34 and CSF-1 serve complimentary nonredundant roles in the developing central nervous system. To identify IL34-specific effects two groups produced IL-34deficient reporter mice. Mice deficient in IL-34 displayed a marked reduction of Langerhans cells and a decrease in microglia, whereas monocytes, tissue macrophages and dendritic cells were largely unaffected [9,10]. IL-34 was expressed primarily by keratinocytes in the epidermis and neurons in the brain. IL-34 was essential for embryonic development of Langerhans cells and for their homeostasis in the adult in the steady state. Under conditions of inflammation in the skin, IL-34 was not required for the recruitment of blood-borne precursors and their differentiation into Langerhans cells but was crucial for their maintenance after inflammation was resolved. Reductions of Langerhans cells in the IL34-deficient mice resulted in reduced sensitivity to chemical hapten- and Candidaalbicans-mediated contact hypersensitivity [10]. IL-34 was found to be essential for microglia homeostasis in specific areas of the adult brain but was not required for embryonic development of microglia [9]. The reduction of microglia in the IL-34-deficient mice did not cause a substantially altered phenotype in the central nervous system under steady-state conditions but did negatively affect the survival of mice infected intracranially with an attenuated strain of West Nile virus [10]. In addition to expression in skin and brain, IL-34 transcripts are also detected in various tissues including the spleen, kidney and testes. IL-34 is abundantly expressed in human spleen and studies 2
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in CSF-1-deficient mice suggest that IL-34 plays a pivotal part in maintaining the splenic reservoir of osteoclast precursor cells [11]. A gene expression study of lymph node stromal cells found IL-34 expressed by fibroblast reticular cells, suggesting IL-34 could regulate myeloid cell function in lymph node immune responses [12]. A group studying the role of follicular dendritic cells (FDCs) in controlling B cell fate in germinal centers found that a mouse FDC line produced IL-34 that promoted the differentiation of a particular type of mononuclear phagocytic cell (designated as FDMC) Q5 that was capable of stimulating activated B cell proliferation [13]. The FDC line produced CSF-1 in addition to IL-34; however, IL-34 but not CSF-1 was reported to be responsible for FDC-mediated generation of the FDMCs. Additional studies in IL-34-deficient mice are needed to confirm IL-34 specificity in promoting B cell follicle-associated macrophage differentiation and to determine the role of FDMCs in germinal center reactions. It was observed that IL-34 and CSF-1R showed differential expression in the brain, suggesting that IL-34 could signal via an alternative receptor. This led to the discovery that IL-34 also interacts with receptor-type protein-tyrosine phosphatase z (PTPz), a cell surface chondroitin sulfate (CS) proteoglycan [14]. PTP-z is primarily expressed on neural progenitors and glial cells and possesses multiple ligands. IL-34 binding inhibits the constitutive phosphatase activity of PTP-z leading to increased tyrosine phosphorylation of downstream targets. To date, no CSF-1R-independent functions of IL-34 have been identified. It is possible that PTPz and CSF-1R cooperate to mediate IL-34 activity, although further studies are needed to identify CSF-1R-dependent or -independent IL-34 signaling through PTP-z. CSF-1R expression is restricted primarily to cells of the mononuclear phagocytic lineage. This includes macrophage precursors in the bone marrow, monocytes, osteoclasts and tissue macrophages including Kupffer cells in the liver and microglia in the brain. In non-hematopoietic cells CSF-1R is expressed on trophoblasts. Different groups have also reported CSF-1R expression on epithelial cells of colonic crypts, on subsets of neurons and on renal proximal tubule cells. However, not all studies support these observations and the expression of CSF-1R on some non-monocyte lineage cells remains controversial: discussed in [15].
Structural basis of CSF-1R activation by IL-34 The discovery that IL-34 and CSF-1 could activate CSF-1R raised the question of how these two disparate cytokines structurally engaged CSF-1R. IL-34 and CSF-1 were shown to compete for binding to CSF-1R, and anti-CSF-1R monoclonal antibodies (mAbs) were identified that could block CSF-1 but not IL-34 suggesting the two cytokines share overlapping but distinct binding sites [5,6]. Although IL-34 shares no appreciable sequence similarity with any other protein it was proposed by fold recognition to be a four-helix bundle cytokine belonging to the same family as CSF-1 [16]. Recently, the crystallographic structures of truncated versions of human and mouse IL-34 were solved and IL34 was confirmed to be a four-helix bundle cytokine, albeit with some unique features [17,18]. IL-34 forms a noncovalently linked dimer whereas CSF-1 contains an intersubunit disulfide bond. The structure of the IL-34–CSF-1R complex shows a similar ligand– receptor assembly to that of CSF-1–CSF-1R. Dimeric IL-34 and
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CSF-1 bind the same general region of CSF-1R by interacting with overlapping but distinct epitopes on CSF-1R [17–19].
Preclinical studies Benefits of blocking the CSF-1R pathway have been shown in numerous animal models of chronic disease. CSF-1-deficient mice are protected from arthritis, bone loss, nephritis, cutaneous lupus and lung fibrosis, and these disorders are also reduced in the setting of CSF-1 or CSF-1R blockade, reviewed in [20,21]. Results in inflammatory bowel disease models were mixed depending on the model used. Although numerous animal studies demonstrate the benefit of blocking the CSF-1R pathway in inflammation not all studies support this. A study using a particular anti-CSF-1R antibody found no inhibitory effect in acute inflammation models, discussed in [20,21]. This is in contrast to results reported in a second study using similar models but a different anti-CSF-1R antibody. This discrepancy could be explained by different modes of action of these two antibodies caused by either differences in epitope binding, and thus differential CSF-1 versus IL-34 blockade, or differences in Fc effector function. Additionally, the models studied were acute inflammatory models, and the relevance of the CSF1R pathway might differ between acute and chronic disease models. Targeting the CSF-1R pathway in chronic inflammatory disease models has been shown to be efficacious [20]. More-recent studies demonstrated efficacy of CSF-1R inhibition in mouse models of cancer. A blocking anti-CSF-1R mAb reduced anti-inflammatory tumor-associated macrophages resulting in reduced tumor growth and prolonged survival [22]. In a glioblastoma model, small molecule inhibition of CSF-1R resulted in tumor growth inhibition and polarizing tumor-associated macrophage to a proinflammatory phenotype [23]. Thus, depending on the disease context, and probably the subsets of macrophage involved, CSF-1R pathway inhibition could have beneficial anti-inflammatory or proinflammatory effects.
IL-34 pathology in rheumatoid arthritis and other inflammatory diseases Rheumatoid arthritis (RA) is a chronic autoimmune disorder of the joints characterized by synovial inflammation and hyperplasia leading to progressive cartilage and bone destruction. Activated macrophages are the predominant infiltrating cell type in the inflamed synovium and promote inflammation through production of inflammatory cytokines such as tumor necrosis factor (TNF)a and IL-6 [24]. Factors from the inflamed synovium drive the differentiation of monocytic lineage cells to osteoclasts, which mediate bone destruction. The CSF-1R pathway is thought to play a predominant part by promoting the differentiation and survival of monocytes and macrophages and osteoclasts. In support of this, activation of CSF-1R has been detected in RA synovium and CSF-1 expression is upregulated in RA [25]. To understand the role of IL-34 in RA several groups have looked at the expression of IL-34 in tissue and blood from RA patients. Immunohistochemistry studies demonstrated that IL-34 is increased in RA synovium compared with osteoarthritis and healthy controls [26,27]. Chemel et al. reported a positive association between synovial IL-34 expression levels and synovitis severity [26]. Several groups also reported elevated levels of IL-34 in RA
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blood and synovial fluid [26–29]. Some caution is needed in interpreting the IL-34 ELISA results because it is not clear if all groups used methods to eliminate the interference of rheumatoid factor (RF). Moon et al. did use methods to block RF interference and detected elevated levels of IL-34 in RA serum and synovial fluid and found an association between IL-34 levels and RF and anticyclic citrullinated peptide (CCP) antibody titers [28]. Functionally, isolated RA-derived fibroblast-like synovial cells and osteoblasts were found to produce IL-34 in response to TNFa [27,30]. As discussed above, IL-34 can promote osteoclastogenesis in combination with RANKL and induce bone destruction [3,4]. IL34 was also shown capable of inducing proinflammatory cytokines and chemokines such as IL-6, IL-8 and CCL2 in human whole Q6 blood [31]. Together the data suggest that IL-34 is involved in RA pathogenesis. Given that IL-34 and CSF-1 are simultaneously expressed in RA synovium it will probably be necessary to block the activity of both to produce a therapeutic benefit. To test the effect of targeting the CSF-1R pathway in RA, we determined the effect of blocking CSF-1R in RA synovial explants (Garcia, S. et al. (2012) American College of Rheumatology Annual Q7 Meeting, Washington DC, Abstr. 901). Synovial biopsy samples were obtained by arthroscopy from sites with active inflammation and cultured without dissociation ex vivo. This system maintains the synovial architecture and cell–cell contacts and therefore reflects more closely the in vivo joint environment. Intact synovial biopsy samples continue to produce cytokines and chemokines without the addition of exogenous stimulus. Treatment with an anti-CSF-1R antibody that blocks IL-34 and CSF-1 signaling significantly reduced IL-6 levels compared with samples treated with an isotype control antibody. These results suggest that targeting the CSF-1R pathway will have clinical benefit in RA. In addition to RA, targeting the CSF-1R pathway could have clinical benefit in other inflammatory diseases. IL-34 was found to be overexpressed in inflamed salivary glands of patients with Sjogren’s syndrome (SS), a chronic immune disorder typically affecting exocrine glands [32]. Macrophages are recognized as an important component of SS lesions with increased levels of macrophages appearing in severe lesions [33]. IL-34 expression was associated with increased expression of TNFa, IL-1b and IL-17, suggesting that IL-34 could regulate monocytes and/or macrophages involved in the pathogenesis of salivary gland inflammation [32]. Intriguingly, a study aimed at identifying shared inflammatory mechanisms of lupus identified IL-34 among a limited number of cytokines and chemokines that were upregulated in kidneys of three different mouse models of lupus nephritis (LN) [34]. LN is associated with inflammation caused by deposition of immune complexes in the kidney. Macrophages are activated by these immune complexes through Fc receptors and contribute to the chronic renal inflammation and tissue damage associated with nephritis. CSF-1 is also upregulated in blood and urine of patients with LN [35]. The presence of IL-34, CSF-1 and activated macrophage suggest that targeting the CSF-1R pathway in LN might provide clinical benefit.
Strategies for blocking CSF-1R pathway activity There are several strategies for inhibiting CSF-1R pathway activity (Fig. 1). CSF-1R is part of a family of receptor tyrosine kinases with
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Drug Discovery Today Volume 00, Number 00 June 2014
IL-34
CSF-1
Antibodies neutralizing CSF-1 or IL-34 FPA008
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Antibodies blocking CSF-1R
CSF-1R (M-CSF receptor)
Small molecule kinase inhibitors
Monocytes/macrophages
Inflammation bone destruction Drug Discovery Today
FIGURE 1
Q13 Strategies to block colony-stimulating factor-1 receptor (CSF-1R) activation. CSF-1R is a receptor tyrosine kinase that is activated by interleukin (IL)-34 or CSF-1. CSF-1R signaling can be blocked by small molecule inhibitors targeting the kinase domain, by neutralizing antibodies targeting either or both IL-34 and CSF-1 or by blocking antibodies targeting CSF-1R. FPA008 is a humanized antibody that blocks CSF-1 and IL-34 with high potency.
intracellular domains that are very closely related. Small molecules inhibiting the kinase activity of CSF-1R have been developed and are being tested in the clinic. These inhibitors range from being highly selective for CSF-1R to possessing dual activity to related Q8 kinases such as KIT or FLT3 [36]. Antibodies against the receptor that block binding of IL-34 and CSF-1 or block the dimerization of the receptor have also been developed and are in early clinical testing. Dual blockade of both cytokines could also be achieved through bi-specific antibodies directed against the ligands or a CSF-1 receptor-trap, although neither of these is reported to be in the clinic. Blockade of either cytokine alone could be achieved through anticytokine antibodies or, because the two ligands interact with unique epitopes on the CSF-1R, antibodies directed at
the CSF-1R with selective blocking activity could be developed. Pfizer has tested an anti-CSF-1 mAb in early clinical trials and in non-human primates. In non-human primates the antibody resulted in selective reduction of CD16+ peripheral monocytes and a loss of Kupffer cells in the liver [37]. The reduction of Kupffer cells was associated with in an increase in serum enzymes with no signs of hepatic or skeletal muscle injury. The rise in serum enzymes was attributed to reduced clearance caused by decreased Kupffer cell numbers and not as a result of hepatic or skeletal injury. This conclusion was supported by additional studies in rats where treatment with clodronate liposomes to deplete Kupffer cells led to increased serum enzyme levels, again without evidence of tissue injury. In addition, CSF-1-deficient mice, which have
TABLE 1
Clinical trials targeting the colony-stimulating factor-1 receptor (CSF-1R) pathway Target
Molecule
Drug type
Company
Indication
Phase
CSF-1
MCS110
Antibody
Novartis
Cancer Pigmented villonodular synovitis
I/II II
CSF-1
PD0360324
Antibody
Pfizer
Rheumatoid arthritis (RA) Cutaneous lupus Pulmonary sarcoidosis
I I I
CSF-1R
AMG820
Antibody
Amgen
Cancer
I
CSF-1R
ARRY382
Small molecule
Array
Cancer
I
CSF-1R
FPA008
Antibody
Five Prime
RA
I
CSF-1R
IMCCS4
Antibody
ImClone/Eli Lilly
Cancer
I
CSF-1R
JNJ40346527
Small molecule
Johnson & Johnson
RA Cancer
II I
CSF-1R
PLX5622
Small molecule
Plexxikon/Daiichi Sankyo
RA
I
4
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reduced levels of Kupffer cells, were found to have higher levels of serum enzymes compared with wild-type littermates. In humans the anti-CSF-1 mAb also specifically reduced CD16+ peripheral blood monocytes [38]. CD16+ monocytes are considered to be inflammatory and are specifically upregulated in many inflammatory conditions including in RA [39]. Pfizer has completed a Phase I clinical trial in RA but has not reported the data yet. Several other compounds targeting the CSF-1R pathway are in the clinic for autoimmune diseases or cancer (Table 1).
The discovery of IL-34 has added a new layer of complexity to this pathway but has also provided answers to outstanding questions. In disease settings where IL-34, CSF-1 and CSF-1R are all present, such as the inflamed synovium in RA, it will probably be necessary to inhibit the activity of both ligands to achieve efficacy. More studies are needed to understand the contribution of IL-34 to other diseases where macrophages are implicated in the pathology (such as fibrosis and inflammatory bowel disease) and to identify new opportunities for targeting this new cytokine for clinical benefit.
Concluding remarks
Conflicts of interest
CSF-1 and CSF-1R involvement in disease pathology and their potential as therapeutic targets have been well studied for years.
Emma Masteller and Brian Wong are employees of Five Prime Therapeutics, and possess stock options in the company.
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