Cytokine producing B-cells and their capability to polarize macrophages in giant cell arteritis.

OBJECTIVE: The lack of disease-specific autoantibodies in giant cell arteritis (GCA) suggests an alternative role for B-cells readily detected in the inflamed arteries. Here we study the cytokine profile of tissue infiltrated and peripheral blood B-cells of patients with GCA. Moreover, we investigate the macrophage skewing capability of B-cell-derived cytokines. METHODS: The presence of various cytokines in B-cell areas in temporal artery (n = 11) and aorta (n = 10) was identified by immunohistochemistry. PBMCs of patients with GCA (n = 11) and polymyalgia rheumatica (n = 10), and 14 age- and sex-matched healthy controls (HC) were stimulated, followed by flow cytometry for cytokine expression in B-cells. The skewing potential of B-cell-derived cytokines (n = 6 for GCA and HC) on macrophages was studied in vitro. RESULTS: The presence of IL-6, GM-CSF, TNFα, IFNγ, LTß and IL-10 was documented in B-cells and B-cell rich areas of GCA arteries. In vitro, B-cell-derived cytokines (from both GCA and HC) skewed macrophages towards a pro-inflammatory phenotype with enhanced expression of IL-6, IL-1ß, TNFα, IL-23, YKL-40 and MMP-9. In vitro stimulated peripheral blood B-cells from treatment-naïve GCA patients showed an enhanced frequency of IL-6+ and TNFα+IL-6+ B-cells compared to HCs. This difference was no longer detected in treatment-induced remission. Erythrocyte sedimentation rate positively correlated with IL-6+TNFα+ B-cells. CONCLUSION: B-cells are capable of producing cytokines and steering macrophages towards a pro-inflammatory phenotype. Although the capacity of B-cells in skewing macrophages is not GCA specific, these data support a cytokine-mediated role for B-cells in GCA and provide grounds for B-cell targeted therapy in GCA.

CD163 and CD206 expression define distinct macrophage subsets involved in active ANCA-associated glomerulonephritis.

INTRODUCTION: Macrophages are key players in the immunopathology of anti-neutrophil cytoplasmic antibody (ANCA) mediated-vasculitis (AAV) with glomerulonephritis (ANCA GN). Different macrophage phenotypes are expected to play distinct roles in ANCA GN. Macrophages expressing CD163 and CD206 are found in lesions associated with ANCA GN. Hence, we aimed to investigate the clinicopathological significance of CD206 and CD163 in ANCA GN in a multicenter retrospective cohort study. MATERIAL AND METHODS: Patients with ANCA-associated vasculitis, with clinical data, serum and urine samples were included from three cohorts. Serum soluble CD206 (ssCD206) and urinary soluble CD163 (usCD163) levels were measured. Human kidney tissue samples (n = 53) were stained for CD206 and CD163 using immunohistochemistry and immunofluorescence, and findings were correlated with clinical and pathological data. RESULTS: In total, 210 patients were included (i.e., ANCA GN, n = 134; AAV without GN, n = 24; AAV in remission n = 52). Increased levels of both ssCD206 and usCD163 were seen in ANCA GN. High levels of ssCD206 declined after reaching remission, however, ssCD206 did not improve the accuracy of usCD163 to detect ANCA GN. Soluble markers correlated with histopathological findings. CD163+CD206- macrophages were found in the glomerulus and may play pivotal roles in glomerulonephritis, whereas CD206+CD163- and CD206+CD163+ macrophages were located tubulointerstitially and likely play a more prominent role in ANCA-associated tubulointerstitial inflammation. In ANCA GN patients increasing levels of ssCD206 increased the risk for end-stage renal disease and mortality. CONCLUSIONS: Our results confirm and extend the notion that CD206+ and CD163+ macrophages are prominent components of the cellular infiltrate in ANCA GN. We found distinct macrophage phenotypes that may play distinct roles in the immunopathology of ANCA GN and elaborate on a potential mechanism underlying the findings of this study. usCD163 remains an excellent marker to detect active ANCA GN, whereas ssCD206 seems a more prominent marker for risk prediction.

Phenotypic, transcriptomic and functional profiling reveal reduced activation thresholds of CD8+ T cells in giant cell arteritis.

OBJECTIVES: Evidence from temporal artery tissue and blood suggests involvement of CD8+ T cells in the pathogenesis of GCA, but their exact role is poorly understood. Therefore, we performed a comprehensive analysis of circulating and lesional CD8+ T cells in GCA patients. METHODS: Circulating CD8+ T cells were analysed for differentiation status (CD45RO, CCR7), markers of activation (CD69 and CD25) and proliferation (Ki-67) in 14 newly diagnosed GCA patients and 18 healthy controls by flow cytometry. Proliferative capacity of CD8+ T cells upon anti-CD3 and anti-CD3/28 in vitro stimulation was assessed. Single-cell RNA sequencing of peripheral blood mononuclear cells of patients and controls (n = 3 each) was performed for mechanistic insight. Immunohistochemistry was used to detect CD3, CD8, Ki-67, TNF-α and IFN-γ in GCA-affected tissues. RESULTS: GCA patients had decreased numbers of circulating effector memory CD8+ T cells but the percentage of Ki-67-expressing effector memory CD8+ T cells was increased. Circulating CD8+ T cells from GCA patients demonstrated reduced T cell receptor activation thresholds and displayed a gene expression profile that is concurrent with increased proliferation. CD8+ T cells were detected in GCA temporal arteries and aorta. These vascular CD8+ T cells expressed IFN-γ but not Ki-67. CONCLUSION: In GCA, circulating effector memory CD8+ T cells demonstrate a proliferation-prone phenotype. The presence of CD8+ T cells in inflamed arteries seems to reflect recruitment of circulating cells rather than local expansion. CD8+ T cells in inflamed tissues produce IFN-γ, which is an important mediator of local inflammatory responses in GCA.

Plasma Pyruvate Kinase M2 as a marker of vascular inflammation in giant cell arteritis.

OBJECTIVES: GCA is a large vessel vasculitis in which metabolically active immune cells play an important role. GCA diagnosis is based on CRP/ESR and temporal artery biopsies (TABs), in combination with 18F-fluorodeoxyglucose ([18F]FDG)-PET/CT relying on enhanced glucose uptake by glycolytic macrophages. Here, we studied circulating Pyruvate Kinase M2 (PKM2), a glycolytic enzyme, as a possible systemic marker of vessel wall inflammation in GCA. METHODS: Immunohistochemical detection of PKM2 was performed on inflamed (n = 12) and non-inflamed (n = 4) TABs from GCA patients and non-GCA (n = 9) patients. Dimeric PKM2 levels were assessed in plasma of GCA patients (n = 44), age-matched healthy controls (n = 41), metastatic melanoma patients (n = 7) and infection controls (n = 11). CRP, ESR and macrophage markers calprotectin and YKL-40 were correlated with plasma PKM2 levels. To detect the cellular source of plasma PKM2 in tissue, double IF staining was performed on inflamed GCA TABs. [18F]FDG-PET scans of 23 GCA patients were analysed and maximum standard uptake values and target to background ratios were calculated. RESULTS: PKM2 is abundantly expressed in TABs of GCA patients. Dimeric PKM2 plasma levels were elevated in GCA and correlated with CRP, ESR, calprotectin and YKL-40 levels. Elevated plasma PKM2 levels were downmodulated by glucocorticoid treatment. PKM2 was detected in both macrophages and T cells at the site of vascular inflammation. Circulating PKM2 levels correlated with average target to background ratios PET scores. CONCLUSION: Elevated plasma PKM2 levels reflect active vessel inflammation in GCA and may assist in disease diagnosis and in disease monitoring.

Evaluation of Hydra HALT-1 as a toxin moiety for recombinant immunotoxin.

BACKGROUND: Immunotoxin is a hybrid protein consisting of a toxin moiety that is linked to a targeting moiety for the purpose of specific elimination of target cells. Toxins used in traditional immunotoxins are practically difficult to be produced in large amount, have poor tissue penetration and a complex internalization process. We hypothesized that the smaller HALT-1, a cytolysin derived from Hydra magnipapillata, can be used as the toxin moiety in construction of a recombinant immunotoxin. RESULTS: In this study, pro-inflammatory macrophage was selected as the target cell due to its major roles in numerous inflammatory and autoimmune disorders. We aimed to construct macrophage-targeted recombinant immunotoxins by combining HALT-1 with anti-CD64-scFv in two orientations, and to assess whether their cytotoxic activity and binding capability could be preserved upon molecular fusion. The recombinant immunotoxins, HALT-1-scFv and scFv-HALT-1, were successfully constructed and expressed in Escherichia coli (E. coli). Our data showed that HALT-1 still exhibited significant cytotoxicity against CD64+ and CD64- cell lines upon fusion with anti-CD64 scFv, although it had half cytotoxic activity as compared to HALT-1 alone. As positioning HALT-1 at N- or C-terminus did not affect its potency, the two constructs demonstrated comparable cytotoxic activities with IC50 lower in CD64+ cell line than in CD64- cell line. In contrast, the location of targeting moieties anti-CD64 scFv at C-terminal end was crucial in maintaining the scFv binding capability. CONCLUSIONS: HALT-1 could be fused with anti-CD64-scFv via a fsexible polypeptide linker. Upon the successful production of this recombinant HALT-1 scFv fusion protein, HALT-1 was proven effective for killing two human cell lines. Hence, this preliminary study strongly suggested that HALT-1 holds potential as the toxin moiety in therapeutic cell targeting.

Altered Plasma Levels and Tissue Expression of Fibroblast Activation Protein Alpha in Giant Cell Arteritis.

OBJECTIVE: Giant cell arteritis (GCA) is characterized by granulomatous inflammation of the medium- and large-sized arteries accompanied by remodeling of the vessel wall. Fibroblast activation protein alpha (FAP) is a serine protease that promotes both inflammation and fibrosis. Here, we investigated the plasma levels and vascular expression of FAP in GCA. METHODS: Plasma FAP levels were measured with enzyme-linked immunosorbent assay in treatment-naive patients with GCA (n = 60) and polymyalgia rheumatica (PMR) (n = 63) compared with age- and sex-matched healthy controls (HCs) (n = 42) and during follow-up, including treatment-free remission (TFR). Inflamed temporal artery biopsies (TABs) of patients with GCA (n = 9), noninflamed TABs (n = 14), and aorta samples from GCA-related (n = 9) and atherosclerosis-related aneurysm (n = 11) were stained for FAP using immunohistochemistry. Immunofluorescence staining was performed for fibroblasts (CD90), macrophages (CD68/CD206/folate receptor beta), vascular smooth muscle cells (desmin), myofibroblasts (α-smooth muscle actin), interleukin-6 (IL-6), and matrix metalloproteinase-9 (MMP-9). RESULTS: Baseline plasma FAP levels were significantly lower in patients with GCA compared with patients with PMR and HCs and inversely correlated with systemic markers of inflammation and angiogenesis. FAP levels decreased even further at 3 months on remission in patients with GCA and gradually increased to the level of HCs in TFR. FAP expression was increased in inflamed TABs and aorta of patients with GCA compared with control tissues. FAP was abundantly expressed in fibroblasts and macrophages. Some of the FAP+ fibroblasts expressed IL-6 and MMP-9. CONCLUSION: FAP expression in GCA is clearly modulated both in plasma and in vessels. FAP may be involved in the inflammatory and remodeling processes in GCA and have utility as a target for imaging and therapeutic intervention.

Current evidence on the role of fibroblasts in large-vessel vasculitides: From pathogenesis to therapeutics.

Large-vessel vasculitides (LVV) comprise a group of chronic inflammatory diseases of the aorta and its major branches. The most common forms of LVV are giant cell arteritis (GCA) and Takayasu arteritis (TAK). Both GCA and TAK are characterized by granulomatous inflammation of the vessel wall accompanied by a maladaptive immune and vascular response that promotes vascular damage and remodeling. The inflammatory process in LVV starts in the adventitia where fibroblasts constitute the dominant cell population. Fibroblasts are traditionally recognized for synthesizing and renewing the extracellular matrix thereby being major players in maintenance of normal tissue architecture and in tissue repair. More recently, fibroblasts have emerged as a highly plastic cell population exerting various functions, including the regulation of local immune processes and organization of immune cells at the site of inflammation through production of cytokines, chemokines and growth factors as well as cell-cell interaction. In this review, we summarize and discuss the current knowledge on fibroblasts in LVV. Furthermore, we identify key questions that need to be addressed to fully understand the role of fibroblasts in the pathogenesis of LVV.

Evidence for increased interferon type I activity in CD8+ T cells in giant cell arteritis patients.

Introduction: Giant cell arteritis (GCA) is a vasculitis of the medium- and large-sized arteries. Interferon type I (IFN-I) is increasingly recognized as a key player in autoimmune diseases and might be involved in GCA pathogenesis, however evidence is limited. IFN-I activates Janus kinase/signal transducers and activators of transcription (JAK-STAT) pathways, leading to increased expression of interferon stimulated genes. In this study, IFN-I activity in GCA is explored, focusing on CD8+ T cells. Methods: Expression of phospho-STAT (pSTAT) 1, 3 and 5 was investigated in IFN-α-stimulated peripheral mononuclear cells (PBMCs) gated separately for CD8+ T cells of patients with GCA (n=18), healthy controls (HC, n=15) and infection controls (n=11) by Phosphoflow method combined with fluorescent cell barcoding technique. Furthermore, IFN-I induced myxovirus-resistance protein A (MxA) and CD8+ T cell expression was investigated by immunohistochemistry in temporal artery biopsies (TAB) of GCA patients (n=20) and mimics (n=20), and in aorta tissue of GCA (n=8) and atherosclerosis patients (n=14). Results: pSTAT1 expression was increased in IFN-α stimulated CD8+ T cells from GCA patients, whereas no difference was observed in pSTAT3 and pSTAT5 expression. MxA was present in TABs of 13/20 GCA patients compared to 2/20 mimics and in 8/8 GCA+ compared to 13/14 GCA- aorta tissues. MxA location partially co-localized with CD8+T cells. Conclusions: Our results provide evidence for increased IFN-I activity in CD8+ T cells of GCA patients, both systemically and locally. These findings warrant further investigation regarding IFN-I induced biomarkers and IFN-I related novel therapeutic options in GCA.

Indication of Activated Senescence Pathways in the Temporal Arteries of Patients With Giant Cell Arteritis.

OBJECTIVE: Giant cell arteritis (GCA) affects almost exclusively individuals above 50 years old, suggesting a role of aging-related changes such as cellular senescence in its pathobiology. The kinases p21(WAF1/CIP1) and p16/INK4A play key roles in 2 distinct pathways leading to senescence. The proinflammatory molecules interleukin-6 (IL-6) and granulocyte-macrophage colony-stimulating factor (GM-CSF), which are key components of the senescence-associated secretory phenotype (SASP), are effective targets of treatment in GCA. Here, we aimed to investigate the presence of p21+ and p16+ cells producing these SASP cytokines in temporal artery biopsies (TABs) of patients with GCA. METHODS: Eight patients with GCA and 14 age-matched, non-GCA individuals who underwent a TAB were included. Immunohistochemical staining of p21, p16, IL-6, and GM-CSF was performed. Multiplex immunofluorescent staining was performed to investigate the colocalization of p21 and p16 with IL-6, GM-CSF, and immune cell markers (CD68, CD3, CD20). RESULTS: We found that expression levels of p16, p21, IL-6, and GM-CSF were elevated in the TABs of patients with GCA. Both p16- and p21-expressing cells were mainly found near the internal lamina elastica, especially among giant cells and macrophages, although p21 and p16 expression could be found in all 3 layers of the vessels. Expression of p16 and p21 was occasionally found in T cells but not B cells. The p16+ and p21+ cells expressing GM-CSF/IL-6 were detected throughout the TABs. CONCLUSION: Our data suggest the presence of activated senescence pathways at the site of vascular inflammation in GCA and support further research into the role of senescence in the pathophysiology of GCA.

Positron Emission Tomography Imaging in Vasculitis.

Systemic vasculitides comprise a group of autoimmune diseases affecting blood vessels. [18F]-fluoro-2-deoxy-d-glucose positron emission tomography/computed tomography (FDG-PET/CT) plays an important role in the diagnosis and therapeutic monitoring of vasculitides affecting large-sized and medium-sized vessels. FDG-PET/CT also provides complementary information to other vascular imaging tools. The resolution and sensitivity of newer generation scanners continues to increase, hereby improving the ability of FDG-PET/CT to accurately assess the full disease extent in patients with vasculitis. Novel tracers targeting specific immune cells will allow for more detailed detection of vascular infiltrates.

Giant worm-shaped ESCRT scaffolds surround actin-independent integrin clusters.

Endosomal Sorting Complex Required for Transport (ESCRT) proteins can be transiently recruited to the plasma membrane for membrane repair and formation of extracellular vesicles. Here, we discovered micrometer-sized worm-shaped ESCRT structures that stably persist for multiple hours at the plasma membrane of macrophages, dendritic cells, and fibroblasts. These structures surround clusters of integrins and known cargoes of extracellular vesicles. The ESCRT structures are tightly connected to the cellular support and are left behind by the cells together with surrounding patches of membrane. The phospholipid composition is altered at the position of the ESCRT structures, and the actin cytoskeleton is locally degraded, which are hallmarks of membrane damage and extracellular vesicle formation. Disruption of actin polymerization increased the formation of the ESCRT structures and cell adhesion. The ESCRT structures were also present at plasma membrane contact sites with membrane-disrupting silica crystals. We propose that the ESCRT proteins are recruited to adhesion-induced membrane tears to induce extracellular shedding of the damaged membrane.

Novel PET Imaging of Inflammatory Targets and Cells for the Diagnosis and Monitoring of Giant Cell Arteritis and Polymyalgia Rheumatica.

Giant cell arteritis (GCA) and polymyalgia rheumatica (PMR) are two interrelated inflammatory diseases affecting patients above 50 years of age. Patients with GCA suffer from granulomatous inflammation of medium- to large-sized arteries. This inflammation can lead to severe ischemic complications (e.g., irreversible vision loss and stroke) and aneurysm-related complications (such as aortic dissection). On the other hand, patients suffering from PMR present with proximal stiffness and pain due to inflammation of the shoulder and pelvic girdles. PMR is observed in 40-60% of patients with GCA, while up to 21% of patients suffering from PMR are also affected by GCA. Due to the risk of ischemic complications, GCA has to be promptly treated upon clinical suspicion. The treatment of both GCA and PMR still heavily relies on glucocorticoids (GCs), although novel targeted therapies are emerging. Imaging has a central position in the diagnosis of GCA and PMR. While [18F]fluorodeoxyglucose (FDG)-positron emission tomography (PET) has proven to be a valuable tool for diagnosis of GCA and PMR, it possesses major drawbacks such as unspecific uptake in cells with high glucose metabolism, high background activity in several non-target organs and a decrease of diagnostic accuracy already after a short course of GC treatment. In recent years, our understanding of the immunopathogenesis of GCA and, to some extent, PMR has advanced. In this review, we summarize the current knowledge on the cellular heterogeneity in the immunopathology of GCA/PMR and discuss how recent advances in specific tissue infiltrating leukocyte and stromal cell profiles may be exploited as a source of novel targets for imaging. Finally, we discuss prospective novel PET radiotracers that may be useful for the diagnosis and treatment monitoring in GCA and PMR.

Contribution of pathogenic T helper 1 and 17 cells to bursitis and tenosynovitis in polymyalgia rheumatica.

Background: Although polymyalgia rheumatica (PMR) is a very common rheumatic inflammatory disease, current insight into the pathobiology of PMR is limited and largely based on studies in blood. We investigated T helper 1 (TH1) and T helper 17 (TH17) cell responses in blood, synovial fluid and bursa tissue of patients with PMR. Materials and methods: Blood samples were collected from 18 patients with new-onset PMR and 32 healthy controls. Synovial fluid was aspirated from the inflamed shoulder bursae or biceps tendon sheath of 13 patients. Ultrasound-guided biopsies of the subacromial-subdeltoid (SASD) bursa were obtained from 11 patients. T cells were examined by flow cytometry, immunohistochemistry and immunofluorescence staining. Results: Besides an increase of TH17 (CD4+IL-17+IFN-γ-) cells and T cytotoxic 17 (TC17; CD8+IL-17+IFN-γ-) cells, no other major changes were noted in the circulating T cell compartment of patients with PMR. Absolute numbers of CD4+ and CD8+ T cells were similar in blood and synovial fluid of patients with PMR. Synovial fluid T cells showed an effector-memory (CD45RO+CCR7-) phenotype. Percentages of TH1 (CD4+IFN-γ+IL-17-) cells and TH1/TH17 (CD4+IFN-γ+IL-17+) cells, but not TH17 or TC17 cells, were increased in the synovial fluid. Bursa tissue biopsies contained a small number of T cells, which were mostly CD8 negative. The majority of bursa tissue T cells produced IFN-γ but not IL-17. For comparison, B cells were scarcely detected in the bursa tissue. Conclusion: Although the circulating TH17 cell pool is expanded in patients with PMR, our findings indicate that TH1 cells are involved in the inflammation of bursae and tendon sheaths in this condition. Our study points towards the TH1 cell pathway as a potential target for therapy in PMR.

Functionally Heterogenous Macrophage Subsets in the Pathogenesis of Giant Cell Arteritis: Novel Targets for Disease Monitoring and Treatment.

Giant cell arteritis (GCA) is a granulomatous large-vessel vasculitis that affects adults above 50 years of age. In GCA, circulating monocytes are recruited to the inflamed arteries. With cues from the vascular microenvironment, they differentiate into macrophages and play important roles in the pathogenesis of GCA via pro-inflammatory cytokine production and vascular remodeling. However, a deeper understanding of macrophage heterogeneity in GCA pathogenesis is needed to assist the development of novel diagnostic tools and targeted therapies. Here, we review the current knowledge on macrophage heterogeneity and diverse functions of macrophage subsets in the pathogenesis of GCA. We next discuss the possibility to exploit their heterogeneity as a source of novel biomarkers and as targets for nuclear imaging. Finally, we discuss novel macrophage-targeted therapies and future directions for targeting these cells in GCA.

A Distinct Macrophage Subset Mediating Tissue Destruction and Neovascularization in Giant Cell Arteritis: Implication of the YKL-40/Interleukin-13 Receptor α2 Axis.

OBJECTIVE: Macrophages mediate inflammation, angiogenesis, and tissue destruction in giant cell arteritis (GCA). Serum levels of the macrophage-associated protein YKL-40 (chitinase 3-like protein 1), previously linked to angiogenesis and tissue remodeling, remain elevated in GCA despite glucocorticoid treatment. This study was undertaken to investigate the contribution of YKL-40 to vasculopathy in GCA. METHODS: Immunohistochemistry was performed on GCA temporal artery biopsy specimens (n = 12) and aortas (n = 10) for detection of YKL-40, its receptor interleukin-13 receptor α2 (IL-13Rα2), macrophage markers PU.1 and CD206, and the tissue-destructive protein matrix metalloproteinase 9 (MMP-9). Ten noninflamed temporal artery biopsy specimens served as controls. In vitro experiments with granulocyte-macrophage colony-stimulating factor (GM-CSF)- or macrophage colony-stimulating factor (M-CSF)-skewed monocyte-derived macrophages were conducted to study the dynamics of YKL-40 production. Next, small interfering RNA-mediated knockdown of YKL-40 in GM-CSF-skewed macrophages was performed to study its effect on MMP-9 production. Finally, the angiogenic potential of YKL-40 was investigated by tube formation experiments using human microvascular endothelial cells (HMVECs). RESULTS: YKL-40 was abundantly expressed by a CD206+MMP-9+ macrophage subset in inflamed temporal arteries and aortas. GM-CSF-skewed macrophages from GCA patients, but not healthy controls, released significantly higher levels of YKL-40 compared to M-CSF-skewed macrophages (P = 0.039). In inflamed temporal arteries, IL-13Rα2 was expressed by macrophages and endothelial cells. Functionally, knockdown of YKL-40 led to a 10-50% reduction in MMP-9 production by macrophages, whereas exposure of HMVECS to YKL-40 led to significantly increased tube formation. CONCLUSION: In GCA, a GM-CSF-skewed, CD206+MMP-9+ macrophage subset expresses high levels of YKL-40 which may stimulate tissue destruction and angiogenesis through IL-13Rα2 signaling. Targeting YKL-40 or GM-CSF may inhibit macrophages that are currently insufficiently suppressed by glucocorticoids.

Distinct macrophage phenotypes skewed by local granulocyte macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) are associated with tissue destruction and intimal hyperplasia in giant cell arteritis.

OBJECTIVE: To determine the presence and spatial distribution of different macrophage phenotypes, governed by granulocyte macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) skewing signals, in giant cell arteritis (GCA) lesions. METHODS: Temporal artery biopsies (TABs, n = 11) from treatment-naive GCA patients, aorta samples from GCA-related aneurysms (n = 10) and atherosclerosis (n = 10) were stained by immunohistochemistry targeting selected macrophage phenotypic markers, cytokines, matrix metalloproteinases (MMPs) and growth factors. In vitro macrophage differentiation (n = 10) followed by flow cytometry, Luminex assay and ELISA were performed to assess whether GM-CSF and M-CSF are drivers of macrophage phenotypic heterogeneity. RESULTS: A distinct spatial distribution pattern of macrophage phenotypes in TABs was identified. CD206+/MMP-9+ macrophages were located at the site of tissue destruction, whereas FRß+ macrophages were located in the inner intima of arteries with high degrees of intimal hyperplasia. Notably, this pattern was also observed in macrophage-rich areas in GCA aortas but not in atherosclerotic aortas. Flow cytometry showed that GM-CSF treatment highly upregulated CD206 expression, while FRß was expressed by M-CSF-skewed macrophages, only. Furthermore, localised expression of GM-CSF and M-CSF was detected, likely contributing to macrophage heterogeneity in the vascular wall. CONCLUSIONS: Our data document a distinct spatial distribution pattern of CD206+/MMP-9+ macrophages and FRß+ macrophages in GCA linked to tissue destruction and intimal proliferation, respectively. We suggest that these distinct macrophage phenotypes are skewed by sequential GM-CSF and M-CSF signals. Our study adds to a better understanding of the development and functional role of macrophage phenotypes in the pathogenesis of GCA and opens opportunities for the design of macrophage-targeted therapies.