Haishan Lin

Discovery of a cytokine and its receptor by functional screening of the extracellular proteome.

To understand the system of secreted proteins and receptors involved in cell-cell signaling, we produced a comprehensive set of recombinant secreted proteins and the extracellular domains of transmembrane proteins, which constitute most of the protein components of the extracellular space. Each protein was tested in a suite of assays that measured metabolic, growth, or transcriptional responses in diverse cell types. The pattern of responses across assays was analyzed for the degree of functional selectivity of each protein. One of the highly selective proteins was a previously undescribed ligand, designated interleukin-34 (IL-34), which stimulates monocyte viability but does not affect responses in a wide spectrum of other assays. In a separate functional screen, we used a collection of extracellular domains of transmembrane proteins to discover the receptor for IL-34, which was a known cytokine receptor, colony-stimulating factor 1 (also called macrophage colony-stimulating factor) receptor. This systematic approach is thus useful for discovering new ligands and receptors and assessing the functional selectivity of extracellular regulatory proteins.

Functional overlap but differential expression of CSF‐1 and IL‐34 in their CSF‐1 receptor‐mediated regulation of myeloid cells

CSF‐1 is broadly expressed and regulates macrophage and osteoclast development. The action and expression of IL‐34, a novel CSF‐1R ligand, were investigated in the mouse. As expected, huIL‐34 stimulated macrophage proliferation via the huCSF‐1R, equivalently to huCSF‐1, but was much less active at stimulating mouse macrophage proliferation than huCSF‐1. Like muCSF‐1, muIL‐34 and a muIL‐34 isoform lacking Q81 stimulated mouse macrophage proliferation, CSF‐1R tyrosine phosphorylation, and signaling and synergized with other cytokines to generate macrophages and osteoclasts from cultured progenitors. However, they respectively possessed twofold and fivefold lower affinities for the CSF‐1R and correspondingly, lower activities than muCSF‐1. Furthermore, muIL‐34, when transgenically expressed in a CSF‐1‐dependent manner in vivo, rescued the bone, osteoclast, tissue macrophage, and fertility defects of Csf1op/op mice, suggesting similar regulation of CSF‐1R‐expressing cells by IL‐34 and CSF‐1. Whole‐mount IL34 in situ hybridization and CSF‐1 reporter expression revealed that IL34 mRNA was strongly expressed in the embryonic brain at E11.5, prior to the expression of Csf1 mRNA. QRT‐PCR revealed that compared with Csf1 mRNA, IL34 mRNA levels were lower in pregnant uterus and in cultured osteoblasts, higher in most regions of the brain and heart, and not compensatorily increased in Csf1op/op mouse tissues. Thus, the different spatiotemporal expression of IL‐34 and CSF‐1 allows for complementary activation of the CSF‐1R in developing and adult tissues.

The CSF-1 receptor ligands IL-34 and CSF-1 exhibit distinct developmental brain expression patterns and regulate neural progenitor cell maintenance and maturation

The CSF-1 receptor (CSF-1R) regulates CNS microglial development. However, the localization and developmental roles of this receptor and its ligands, IL-34 and CSF-1, in the brain are poorly understood. Here we show that compared to wild type mice, CSF-1R-deficient (Csf1r-/-) mice have smaller brains of greater mass. They further exhibit an expansion of lateral ventricle size, an atrophy of the olfactory bulb and a failure of midline crossing of callosal axons. In brain, IL-34 exhibited a broader regional expression than CSF-1, mostly without overlap. Expression of IL-34, CSF-1 and the CSF-1R were maximal during early postnatal development. However, in contrast to the expression of its ligands, CSF-1R expression was very low in adult brain. Postnatal neocortical expression showed that CSF-1 was expressed in layer VI, whereas IL-34 was expressed in the meninges and layers II-V. The broader expression of IL-34 is consistent with its previously implicated role in microglial development. The differential expression of CSF-1R ligands, with respect to CSF-1R expression, could reflect their CSF-1R-independent signaling. Csf1r-/- mice displayed increased proliferation and apoptosis of neocortical progenitors and reduced differentiation of specific excitatory neuronal subtypes. Indeed, addition of CSF-1 or IL-34 to microglia-free, CSF-1R-expressing dorsal forebrain clonal cultures, suppressed progenitor self-renewal and enhanced neuronal differentiation. Consistent with a neural developmental role for the CSF-1R, ablation of the Csf1r gene in Nestin-positive neural progenitors led to a smaller brain size, an expanded neural progenitor pool and elevated cellular apoptosis in cortical forebrain. Thus our results also indicate novel roles for the CSF-1R in the regulation of corticogenesis.

Receptor-type Protein-tyrosine Phosphatase ζ Is a Functional Receptor for Interleukin-34

Background: IL-34 and the known IL-34 receptor, CSF-1R, are differentially expressed in mouse brain; thus, IL-34 may signal via an additional receptor(s). Results: IL-34 binds to PTP-ζ on U251 human glioblastoma cells to stimulate intracellular signaling and responses. Conclusion: PTP-ζ is an IL-34 receptor. Significance: CSF-1R-independent actions of IL-34 via PTP-ζ should be considered in evaluating IL-34 roles in development and disease. Interleukin-34 (IL-34) is highly expressed in brain. IL-34 signaling via its cognate receptor, colony-stimulating factor-1 receptor (CSF-1R), is required for the development of microglia. However, the differential expression of IL-34 and the CSF-1R in brain suggests that IL-34 may signal via an alternate receptor. By IL-34 affinity chromatography of solubilized mouse brain membrane followed by mass spectrometric analysis, we identified receptor-type protein-tyrosine phosphatase ζ (PTP-ζ), a cell surface chondroitin sulfate (CS) proteoglycan, as a novel IL-34 receptor. PTP-ζ is primarily expressed on neural progenitors and glial cells and is highly expressed in human glioblastomas. IL-34 selectively bound PTP-ζ in CSF-1R-deficient U251 human glioblastoma cell lysates and inhibited the proliferation, clonogenicity, and motility of U251 cells in a PTP-ζ-dependent manner. These effects were correlated with an increase in tyrosine phosphorylation of the previously identified PTP-ζ downstream effectors focal adhesion kinase and paxillin. IL-34 binding to U251 cells was abrogated by chondroitinase ABC treatment, and CS competed with IL-34 for binding to the extracellular domain of PTP-ζ and to the cells, indicating a dependence of binding on PTP-ζ CS moieties. This study identifies an alternate receptor for IL-34 that may mediate its action on novel cellular targets.

The mechanism of shared but distinct CSF-1R signaling by the non-homologous cytokines IL-34 and CSF-1.

Interleukin-34 (IL-34) and colony stimulating factor-1 (CSF-1) both signal through the CSF-1R receptor tyrosine kinase, but they have no sequence homology, and their functions and signaling activities are not identical. We report the crystal structures of mouse IL-34 alone and in complex with the N-terminal three immunoglobulin-like domains (D1-D3) of mouse CSF-1R. IL-34 is structurally related to other helical hematopoietic cytokines, but contains two additional helices integrally associated with the four shared helices. The non-covalently linked IL-34 homodimer recruits two copies of CSF-1R on the sides of the helical bundles, with an overall shape similar to the CSF-1:CSF-1R complex, but the flexible linker between CSF-1R D2 and D3 allows these domains to clamp IL-34 and CSF-1 at different angles. Functional dissection of the IL-34:CSF-1R interface indicates that the hydrophobic interactions, rather than the salt bridge network, dominate the biological activity of IL-34. To degenerately recognize two ligands with completely different surfaces, CSF-1R apparently takes advantage of different subsets of a chemically inert surface that can be tuned to fit different ligand shapes. Differentiated signaling between IL-34 and CSF-1 is likely achieved by the relative thermodynamic independence of IL-34 vs. negative cooperativity of CSF-1 at the receptor-recognition sites, in combination with the difference in hydrophobicity which dictates a more stable IL-34:CSF-1R complex compared to the CSF-1:CSF-1R complex.

Colony-stimulating factor (CSF) 1 receptor blockade reduces inflammation in human and murine models of rheumatoid arthritis

BackgroundCSF-1 or IL-34 stimulation of CSF1R promotes macrophage differentiation, activation and osteoclastogenesis, and pharmacological inhibition of CSF1R is beneficial in animal models of arthritis. The objective of this study was to determine the relative contributions of CSF-1 and IL-34 signaling to CSF1R in RA.MethodsCSF-1 and IL-34 were detected by immunohistochemical and digital image analysis in synovial tissue from 15 biological-naïve rheumatoid arthritis (RA) , 15 psoriatic arthritis (PsA) and 7 osteoarthritis (OA) patients . Gene expression in CSF-1- and IL-34-differentiated human macrophages was assessed by FACS analysis and quantitative PCR. RA synovial explants were incubated with CSF-1, IL-34, control antibody (Ab), or neutralizing/blocking Abs targeting CSF-1, IL-34, or CSF1R. The effect of a CSF1R-blocking Ab was examined in murine collagen-induced arthritis (CIA).ResultsCSF-1 (also known as M-CSF) and IL-34 expression was similar in RA and PsA synovial tissue, but lower in controls (P < 0.05). CSF-1 expression was observed in the synovial sublining, and IL-34 in the sublining and the intimal lining layer. CSF-1 and IL-34 differentially regulated the expression of 17 of 336 inflammation-associated genes in macrophages, including chemokines, extra-cellular matrix components, and matrix metalloproteinases. Exogenous CSF-1 or IL-34, or their independent neutralization, had no effect on RA synovial explant IL-6 production. Anti-CSF1R Ab significantly reduced IL-6 and other inflammatory mediator production in RA synovial explants, and paw swelling and joint destruction in CIA.ConclusionsSimultaneous inhibition of CSF1R interactions with both CSF-1 and IL-34 suppresses inflammatory activation of RA synovial tissue and pathology in CIA, suggesting a novel therapeutic strategy for RA.

THU0042 Colony-Stimulating Factor (CSF) Receptor 1 Blockade Overcomes Overlapping Effects of M-CSF and Interleukin-34 on Myeloid Differentiation and Gene Expression to Reduce Inflammation in Human and Murine Models of Rheumatoid Arthritis

Background Disease activity and response to therapy in RA correlates with changes in synovial macrophage numbers and their products. M-CSF or interleukin (IL)-34 stimulation of their common receptor CSFR1 promotes macrophage differentiation, activation and osteoclastogenesis, but M-CSF and IL-34 play distinct roles in murine development. Pharmacological inhibition of CSFR1 is beneficial in animal models of arthritis but the relative contributions of M-CSF, IL-34 and their receptor to inflammation in rheumatoid arthritis (RA) are unknown. Objectives To determine the roles of M-CSF and IL-34 in huma macrophage activation, and human and murine models of RA. Methods M-CSF, IL-34, and cellular markers were detected by immunohistochemistry and digital image analysis in synovial tissue from 18 biological-naïve RA patients, 14 PsA patients, and 4 controls without inflammatory disease (2 healthy donors and 2 OA patients). Gene expression in M-CSF and IL-34 –differentiated human macrophages was assessed by FACS analysis and q-PCR arrays. RA synovial explants were incubated with M-CSF, IL-34, control antibody (Ab), or a humanized CSFR1-blocking Ab. Prophylactic effects of control and CSFR1-blocking Ab were examined in murine collagen-induced arthritis (CIA). Results Expression of M-CSF and IL-34 was similar in RA and PsA synovial tissue, but lower in controls (p< 0.05). M-CSF expression was restricted to ECs and IL-34 was observed in sublining mononuclear cells and intimal lining layer cells. CXCL5, CXCL6, FN1, COL1A1, COL6A1-2, COL14A1, COL15A1, MMP7, MMP8 and MMP9 mRNA expression was significantly upregulated in macrophages differentiated in M-CSF compared to IL-34, while CXCL7, CXCL9, CXCL10, CXCL11, CXCL12, LAMA1, and MMP2 were downregulated. M-CSF or IL-34 had no effect on RA synovial explant IL-6 production, but anti-CSFR1 Ab dose-dependently reduced IL-6 production. Treatment with anti-CSFR1 Ab in CIA significantly reduced paw swelling and joint destruction. Conclusions M-CSF and IL-34 are expressed in topographically distinct regions of inflamed synovial tissue and differentially effect macrophage capacity to attract inflammatory cells and remodel tissue. Simultaneous inhibition of CSFR1 interactions with both M-CSF and IL-34 suppresses inflammatory activation of RA synovial tissue and pathology in CIA, suggesting a novel therapeutic strategy for RA. Disclosure of Interest S. Garcia: None Declared, L. Hartkamp: None Declared, M. W. Tang: None Declared, I. van Es: None Declared, H. Lin Shareholder of: stock options in Five Prime Therapeutics, Inc., Employee of: Five Prime Therapeutics, Inc., L. Long Shareholder of: stock options in Five Prime Therapeutics, Inc., Employee of: Five Prime Therapeutics, Inc., E. Masteller Shareholder of: stock options in Five Prime Therapeutics, Inc., Employee of: Five Prime Therapeutics, Inc., B. Wong Shareholder of: stock options in Five Prime Therapeutics, Inc., Employee of: Five Prime Therapeutics, Inc., P. Tak Employee of: GlaxoSmithKline, K. Reedquist Grant/research support from: Five Prime Therapeutics, Inc.