Macrophage diversity in the pathogenesis of vasculitides:implications for diagnosis and treatment

Giant cell arteritis (GCA) and anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) are diseases marked by inflammation of blood vessels. Vascular damage occurring from GCA can lead to various severe complications such as blindness, stroke, and aortic dissection, while vascular damage from AAV often leads to organ malfunction such as kidney failure, leading to increased morbidity and mortality of the patients. Both GCA and AAV are marked by systemic inflammation and massive local leukocyte infiltration at the site of inflammation. In both diseases, macrophages account for the majority of infiltrating leukocytes – affirming their importance in the development of these diseases and making them attractive cellular markers for disease progression and treatment response. Macrophages can change their phenotypes depending on signals from the microenvironment. A deeper understanding of the macrophage phenotypes and their roles in the progression of vasculitides would allow us to exploit their heterogeneity therapeutically to specifically target proinflammatory macrophages. At the same time, this may aid the identification of markers suitable for diagnostic purposes and follow-up assessment. However, to date, our knowledge of macrophage heterogeneity in the pathogenesis of vasculitides is limited. This thesis identified distinct macrophage populations associated with distinct functions pertaining to the development of GCA and AAV. Additionally, soluble factors shed by these macrophages may aid in disease diagnosis and monitoring. Last but not least, we discussed the vast possibilities of specifically targeting these macrophages for the purpose of disease monitoring and treatment

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.</p

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.</p

Indication of activated senescence pathways in the temporal arteries of patients with giant cell arteritis

OBJECTIVES: 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. p21 WAF1/CIP1 and p16/INK4A play key roles in two distinct pathways leading to senescence. The proinflammatory molecules Interleukin (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 aim to investigate the presence of p21 and p16 positive 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: 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 three layers of the vessels. Expression of p16 and p21 was occasionally found in T cells but not B cells. p16+ and p21+ cells expressing GM-CSF/IL-6 were detected throughout the TABs.CONCLUSION: Our data suggests 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. This article is protected by copyright. All rights reserved.</p

A Distinct Macrophage Subset Mediating Tissue Destruction and Neovascularization in Giant Cell Arteritis:Implication of the YKL-40/Interleukin-13 Receptor alpha 2 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-1), previously linked to angiogenesis and tissue remodeling, remain elevated in GCA despite glucocorticoid treatment. Here, we aimed to investigate the contribution of YKL-40 to vasculopathy in GCA. METHODS: Immunohistochemistry was performed on GCA temporal artery biopsies (TABs; n=12) and aortas (n=10) for detection of YKL-40, its receptor IL-13Rα2, macrophage markers PU.1 and CD206, and the tissue-destructive protein MMP-9. Ten non-inflamed TABs served as controls. In vitro experiments with GM-CSF- or M-CSF-skewed monocyte-derived macrophages (GM-MØs or M-MØs) were conducted to study the dynamics of YKL-40 production. Next, silencing RNA (siRNA)-mediated knock-down of YKL-40 in GM-MØs 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 TABs and aortas. GM-MØs of GCA patients, but not of healthy controls, released significantly higher levels of YKL-40, compared to M-MØs. In inflamed TABs, IL-13Rα2 was expressed by macrophages and endothelial cells. Functionally, knock-down 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+MMP9+ 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 FRβ+ 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 FRβ 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 FRβ+ 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

Plasma Pyruvate Kinase M2 as a marker of vascular inflammation in Giant Cell Arteritis

OBJECTIVES: Giant Cell Arteritis (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]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 (HC, 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 immunofluorescence staining was performed on inflamed GCA TABs. [18F]FDG-PET scans of 23 GCA patients were analyzed and maximum standard uptake values (SUVmax) and target to background ratios (TBR) 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 GC-treatment. PKM2 was detected in both macrophages and T cells at the site of vascular inflammation. Circulating PKM2 levels correlated with average TBR PET scores. CONCLUSION: Elevated plasma PKM2 levels reflect active vessel inflammation in GCA and may assist in disease diagnosis and in disease monitoring

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, LTβ 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-1β, 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.</p