Age-related macular degeneration and myeloproliferative neoplasms - A common pathway

Abstract

DANSK RESUMÉ (DANISH SUMMARY): Aldersrelateret makuladegeneration (AMD) er den hyppigste årsag til uopretteligt synstab og blindhed i højindkomstlande. Det er en progredierende nethindesygdom som gradvist fører til ødelaeggelse af de celler som er ansvarlige for vores centralsyn. De tidlige stadier er ofte asymptomatiske, imens senstadie AMD, som opdeles i to former, neovaskulaer AMD (nAMD) og geografisk atrofi (GA), begge udviser gradvist synstab, dog generelt med forskellig hastighed. Tidlig AMD er karakteriseret ved tilstedevaerelsen af druser og pigmentforandringer i nethinden mens nAMD og GA udviser henholdsvis karnydannelse i og atrofi af nethinden. AEtiologien er multifaktoriel og udover alder omfatter patogenesen miljø- og genetiske risikofaktorer. Forskning har specielt fokuseret på lokale forandringer i øjet hvor man har fundet at inflammation spiller en betydelig rolle for udviklingen af sygdommen, men flere studier tyder også på at systemiske forandringer og specielt systemisk inflammation spiller en vaesentlig rolle i patogenesen. De Philadelphia-negative myeloproliferative neoplasier (MPNs) er en gruppe af haematologiske kraeftsygdomme med en erhvervet genetisk defekt i den tidlige pluripotente stamcelle som medfører en overproduktion af en eller flere af blodets modne celler. Sygdommene er fundet at udvikle sig i et biologisk kontinuum fra tidligt cancerstadie, essentiel trombocytose (ET) over polycytaemi vera (PV) og endelig til det sene myelofibrose stadie (PMF). Symptomer hos disse patienter skyldes isaer den aendrede sammensaetning af blodet, hyperviskositet, kompromitteret mikrocirkulation og nedsat vaevsgennemblødning. Den øgede morbiditet og mortalitet beror i høj grad på tromboembolier, blødninger og leukemisk transformation. En raekke mutationer som driver MPN sygdommene er identificeret, bl.a. JAK2V617F-mutationen som medfører en deregulering JAK/STAT signalvejen, der bl.a. har betydning for cellers vaekst og overlevelse. Et tidligere stort registerstudie har vist at patienter med MPNs har en øget risiko for neovaskulaer AMD og et pilotstudie har vist øget forekomst af intermediaer AMD. Dette ønsker vi at undersøge naermere i et større studie i dette Ph.d.- projekt. Flere studier har også vist at kronisk inflammation spiller en vigtig rolle for både initiering og udvikling af den maligne celleklon hos MPNs og herfra er en "Human Inflammationsmodel" blevet udviklet. Siden er MPN sygdommene blevet anvendt som "model sygdomme" for en tilsvarende inflammationsmodel for udvikling af Alzheimers sygdom. I dette Ph.d.-projekt vil vi tilsvarende forsøge at undersøge systemisk inflammation i forhold til forekomst af druser. Det vil vi gøre ved at sammenligne systemiske immunologiske markører som tidligere er undersøgt hos patienter med AMD og sammenligne med MPN. Specielt er vi interesseret i systemiske immunologiske forskelle på patienter med MPN og druser (MPNd) og MPN med normale nethinder (MPNn). Denne afhandling består af to overordnede studier. I Studie I, undersøgte vi forekomsten af retinale forandringer associeret med AMD hos 200 patienter med MPN (artikel I). Studie II, omhandlede immunologiske ligheder ved AMD og MPN, og var opdelt i yderligere tre delstudier hvor vi undersøgte hhv. systemiske markører for inflammation, aldring og angiogenese (artikel II, III og IV). Vi undersøgte markørerne i fire typer af patienter: nAMD, intermediaer AMD (iAMD), MPNd og MPNn. Undersøgelsen af forskelle mellem MPNd og MPNn, vil gøre det muligt at identificere forandringer i immunsystemet som kunne vaere relevante for AMD-patogenesen. Vi vil endvidere sammenholde resultaterne for patienter med MPN med patienter som har iAMD og nAMD. I studie I (Artikel I) fandt vi at patienter med MPN har en signifikant højere praevalens af store druser og AMD tidligere i livet sammenlignet med estimater fra tre store befolkningsundersøgelser. Vi fandt også at forekomst af druser var associeret med højere neutrofil-lymfocyt ratio, hvilket indikerer et højere niveau af kronisk inflammation i patienterne med druser sammenlignet med dem uden druser. I studie II (Artikel II, III og IV) fandt vi flere immunologiske forskelle mellem patienter med MPNd og MPNn. Da vi undersøgte markører for inflammation, fandt vi en højere grad af systemisk inflammation i MPNd end MPNn. Dette blev vist ved en højere inflammationsscore (udregnet på baggrund af niveauer af pro-inflammatoriske markører), en højere neutrofil-lymfocyt ratio, samt indikationer på et dereguleret komplementsystem. Ved undersøgelse af aldringsmarkører fandt vi tegn på accelereret immunaldring hos MPNd i forhold til MPNn, hvilket kommer til udtryk ved en større procentdel af "effector memory T celler". Endelig fandt vi en vaesentlig lavere ekspression af CXCR3 på T celler og monocytter hos patienter med nAMD sammenlignet med iAMD, MPNd og MPNn. Dette er i overensstemmelse med tidligere studier hvor CXCR3 ekspression er fundet lavere end hos raske kontroller. Derudover fandt vi en faldende CXCR3 ekspression på monocytter over det biologiske MPN-kontinuum. Disse studier indikerer en involvering af CXCR3 i både nAMD og PMF, begge sygdomsstadier som er karakteriseret ved angiogenese og fibrose. Ud fra resultaterne af denne afhandling kan vi konkludere at forekomsten af druser og AMD hos MPN er øget i forhold til baggrundsbefolkningen. Endvidere viser vores resultater at systemisk inflammation muligvis spiller en vaesentlig større rolle i udviklingen af AMD end tidligere antaget. Vi foreslår derfor en AMD-model (Figur 18) hvor inflammation kan initiere og accelerere den normale aldersafhaengige akkumulation af affaldsstoffer i nethinden, som senere udvikler sig til druser, medførende øget lokal inflammation og med tiden tidlig og intermediaer AMD. Dette resulterer i den øgede risiko for udvikling til de invaliderende senstadier af AMD. ENGLISH SUMMARY: Age-related macular degeneration (AMD) is the most common cause of irreversible vision loss and blindness in high-income countries. It is a progressive retinal disease leading to damage of the cells responsible for central vision. The early stages of the disease are often asymptomatic, while late-stage AMD, which is divided into two entities, neovascular AMD and geographic atrophy (GA), both show vision loss, though generally with different progression rates. Drusen and pigmentary abnormalities in the retina characterise early AMD, while nAMD and GA show angiogenesis in and atrophy of the retina, respectively. The aetiology is multifactorial and, in addition to ageing, which is the most significant risk factor for developing AMD, environmental- and genetic risk factors are implicated in the pathogenesis. Research has focused on local changes in the eye where inflammation has been found to play an essential role, but studies also point to systemic alterations and especially systemic inflammation to be involved in the pathogenesis. The Philadelphia-negative myeloproliferative neoplasms (MPN) are a group of haematological cancers with an acquired genetic defect of the pluripotent haematopoietic stem cell, characterised by excess haematopoiesis of the myeloid cell lineage. The diseases have been found to evolve in a biological continuum from early cancer state, essential thrombocythemia, over polycythaemia vera (PV), to the advanced myelofibrosis stage (PMF). The symptoms in these patients are often a result of the changes in the blood composition, hyperviscosity, microvascular disturbances, and reduced tissue perfusion. The major causes of morbidity and mortality are thromboembolic- and haemorrhagic events, and leukemic transformation. A group of mutations that drive the MPNs has been identified, e.g., the JAK2V617F mutation, which results in deregulation of the JAK/STAT signal transduction pathway important, for instance, in cell differentiation and survival. A previous large register study has shown that patients with MPNs have an increased risk of neovascular AMD, and a pilot study has shown an increased prevalence of intermediate AMD. We wish to study this further in a larger scale study. Several studies have also shown that systemic inflammation plays an essential role in both the initiation and progression of the malignant cell clone in MPNs. From this knowledge, a "Human inflammation model" has been developed. Since then, the MPNs has been used as model diseases for a similar inflammation model for the development of Alzheimer's disease. In this PhD project, we would like to investigate systemic inflammation in relation to drusen presence. We will do this by comparing systemic immunological markers previously investigated in patients with AMD and compare with MPN. We are primarily interested in systemic immunological differences between patients with MPN and drusen (MPNd) and MPN with normal retinas (MPNn). This thesis consists of two main studies. Study I investigated the prevalence of retinal changes associated with AMD and the prevalence of different AMD stages in 200 patients with MPN (paper I). Study II examined immunological similarities between AMD and MPNs. This study was divided into three substudies exploring systemic markers of inflammation, ageing and angiogenesis, respectively. This was done in four types of patients: nAMD, intermediate AMD (iAMD), MPNd and MPNn. Investigating, differences between MPNd and MPNn, will make it possible to identify changes in the immune system, relevant for AMD pathogenesis. Additionally, we will compare patients with MPNs with patients with iAMD and nAMD. In study I (Paper I), we found that patients with MPNs have a significantly higher prevalence of large drusen and consequently AMD from an earlier age compared to the estimates from three large population-based studies. We also found that drusen prevalence was associated with a higher neutrophil-to-lymphocyte ratio indicating a higher level of chronic low-grade inflammation in patients with drusen compared to those without drusen. In study II (papers II, III and IV), we found immunological differences between patients with MPNd and MPNn. When we investigated markers of inflammation, we found a higher level of systemic inflammation in MPNd than MPNn. This was indicated by a higher inflammation score (based on levels of pro-inflammatory markers), a higher neutrophil-to-lymphocyte ratio, and indications of a deregulated complement system. When examining markers of ageing, we found signs of accelerated immune ageing in MPNd compared to MPNn, shown by more senescent effector memory T cells. Finally, when exploring a marker of angiogenesis, we found a lower CXCR3 expression on monocytes and T cells in nAMD compared to iAMD, MPNd and MPNn, in line with previous studies of nAMD compared to healthy controls. Further, we found decreasing CXCR3 expression over the MPN biological continuum. These studies indicate CXCR3 involvement in both nAMD and PMF, two disease stages characterised by angiogenesis and fibrosis. From the results of this PhD project, we can conclude that the prevalence of drusen and AMD is increased in patients with MPN compared to the general population. Further, our results show that systemic inflammation may play a far more essential role in AMD pathogenesis than previously anticipated. We, therefore, propose an AMD model (Figure 18) where inflammation can initiate and accelerate the normal age-dependent accumulation of debris in the retina, which later evolve into drusen, resulting in increased local inflammation, and over time early- and intermediate AMD. This results in the increased risk of developing the late debilitating stages of AMD.

FIGURE 1

Anatomy of the human eye and the layers of the retina. Left: Eyeball anatomy – sagittal view. Right: Enlargement and anatomy of the retina, showing the ten layers – the neuroretina in blue text and the RPE in red. Created with BioRender.

FIGURE 2

visual distortions seen in patients with age‐related macular degeneration. Created with Adobe Illustrator. Royalty free photos from dreamstime.com.

FIGURE 3

Simplified diagram of haematopoiesis and simplified characteristics view of the MPNs. The JAK2V617 mutation (or other relevant mutations) result in an abnormal haematopoietic stem cell, leading to excess haematopoiesis of the myeloid lineage. In ET, thrombocytosis is the dominating sign, but leukocytosis is also seen. The classical characteristic of PV is erythrocytosis, but nearly all patients develop leukocytosis and thrombocytosis if not already present at diagnosis. The classic PMF patient characteristics are anaemia, variable leukocyte and platelet count changes, bone marrow fibrosis and splenomegaly. Created with BioRender.

FIGURE 4

Symptoms and complications in patients with MPNs. Patients with MPNs have a huge symptom and associated disease burden/comorbidity burden, and some of these are presented in this figure. For ocular manifestations in these patients please see review by Liisborg et al. (2020).Created with BioRender.

FIGURE 5

A simplified drawing of the complement system. The three different complement activation pathways culminate in a common terminal pathway. The classical pathway is activated by antigen/antibody complexes (C1q interact with C1r and C1s to form the C1 complex that interacts with antigen/antibodies on the pathogen surface). The proteases C1r and C1s cleave C2 and C4, generating the classical pathway convertase C4b2a (not shown). The lectin pathway is initiated by mannose‐binding lectin (MBL) or ficolin binding to carbohydrates on the pathogen or target membrane. The MBL‐associated serine proteases (MASPs) then cleave C2 and C4, generating the C4b2a convertase (not shown). In the alternative pathway, constant low‐level hydrolysis of C3 to C3H₂O is seen, augmented by factors B, D and Properdin. The pathway functions as an amplification loop when C3b bind to a target membrane (pathogen, damaged tissue, foreign material) and interact with the C3H₂0, factors B and D to form the alternative pathway convertase C3bBb stabilised by Properdin. All three pathways converge in the final pathway where C3 convertases cleave C3 forming C3a and C3b. C3b bind to C3 convertases generating C5 convertases. The C5 convertase cleaves C5 into C5a and C5b. The anaphylatoxins C3a and C5a activate and attract inflammatory cells and lead to vasodilation, inflammation, and phagocytosis. C5b bind to C6, C7, C8 and multiple C9, forming the membrane attack complex (MAC). The MAC forms pores on target membranes and can cause cell lysis. Figure created with BioRender.

FIGURE 6

Redrawing of figure “MPN Human Inflammation Model” (Modified and simplified) from Hasselbalch HC, Bjørn ME. MPNs as Inflammatory Diseases: The Evidence, Consequences, and Perspectives. Mediators Inflamm 2015; 2015: 1–16.⁶⁷ Chronic inflammation initiate and drives clonal expansion and is represented by an increased level of inflammatory cytokines and reactive oxygen species (ROS). This creates a dangerous microenvironment with genetic instability – DNA damage. When the JAK2 mutation happens, it increases the cytokine and ROS levels further and raises platelet and or leukocyte levels giving rise to even more inflammatory products. This creates a self‐fuelling positive feedback loop – a vicious cycle of increasing inflammation and clonal expansion. The rounded arrows outside the large circle represents both the mutational burden (allele burden), the inflammation and comorbidity burden in the different stages in the biological continuum of the MPNs from early cancer state ET/PV to the late burned‐out phase of myelofibrosis with increased risk of leukemic transformation. The coloured smaller circles represent the increasing levels of inflammatory mediators (cytokines) also following the biological continuum. JAK2 46/1: the JAK2 46/1 haplotype. ET: Essential thrombocythemia, PV: Polycythaemia vera, PMF: primary myelofibrosis. Created with BioRender.

FIGURE 7

Colour fundus photography of (a) an individual with a healthy retina (b) a patient showing drusen associated with AMD. From IMAGEnet iBase.

FIGURE 8

Optical Coherence Tomography (OCT) image showing the layers of the macula of a healthy individual. BM, Bruch's membrane; ELM, external limiting membrane; GCL, ganglion cell layer; ILM, inner limiting membrane; INL, inner nuclear layer; IPL, inner plexiform layer; OPL, outer plexiform layer; PR1/2, photoreceptor inner and outer layer; RNFL, retinal nerve fibre layer; RPE, retinal pigment epithelium. Arrows show choroidea, CC, choriocapillaris; CS, choroidal stroma. The neuroretina is defined as the part of the retina from the INL to the PR. From Heidelberg Eye Explorer. Reprinted from The Lancet, EClinicalMedicine 2020; 26: 100526, Liisborg C, Nielsen MK, Hasselbalch HC, Sørensen TL, Patients with myeloproliferative neoplasms and high levels of systemic inflammation develop age‐related macular degeneration. (supplemental material), Copyright (2020), with permission from Elsevier. https://www.thelancet.com/journals/eclinm/article/PIIS2589‐5370(20)30270‐4/fulltext#articleInformation.

FIGURE 9

Fundus autofluorescence (FAF) image of a patient eye with geographic atrophy (GA) The image shows a large central atrophic area. From Heidelberg Eye Explorer.

FIGURE 10

Basic components of a flow cytometer – simplified. The cell suspension contains cells with fluorochrome labelled antibodies. The suspension is run on a flow cytometer which contains the above five basic components marked with blue boxes. The flow chamber/fluidics system funnels the suspension through a nozzle that forges a stream of single cells. The cells flow past a set of focused lasers lasers (light source). When the light hits the cells, it is scattered. The scattered light is detected by two types of optical detectors (the optical system). One detects light along the light path of the laser, referred to as forward scatter (FSC) and the other measures scatter at other angles, the side scatter (SSC). The FSC provides information about cell size, while the side scatter provides information about the granularity of the cells (the internal complexity). This gives the opportunity to discriminate between cells when the information is sent to a computer. The lights from the optical system are recorded by light detectors and converted by an electronic system and can hereafter be presented on a computer as either single parameter histograms or as two‐parameter plots called cytograms. Created with BioRender.

FIGURE 11

Prevalence of large drusen in patients with MPNs compared to estimates from three large population‐based studies. Comparison of the prevalence of drusen >125μm as the largest drusen present within a 3000μm radius of the fovea between patients with myeloproliferative neoplasms and three large population‐based studies (Beaver Dam Eye Study, Blue Mountains Eye Study and Rotterdam Study). Reprinted from The Lancet, EClinicalMedicine 2020; 26: 100526, Liisborg C, Nielsen MK, Hasselbalch HC, Sørensen TL, Patients with myeloproliferative neoplasms and high levels of systemic inflammation develop age‐related macular degeneration., Copyright (2020), with permission from Elsevier. https://www.thelancet.com/journals/eclinm/article/PIIS2589‐5370(20)30270‐4/fulltext#articleInformation.

FIGURE 12

Summary inflammation score in patients with AMD and MPN Plot of summary inflammation score = ([z score (IL6) + z score (IL8) + z score (TNF‐R2) + z score (TNF‐a) + z score (IL‐1β)]) ‐ with 95% CIbars. Comparisons between patients with nAMD (n=29), iAMD (n=28), MPNd (n=35), MPNn (n=27). Reprinted from The Lancet, EClinicalMedicine 2022; 43: 201248, Liisborg C, Skov V, Kjær L, Hasselbalch HC, Sørensen TL, Patients with MPNs and retinal drusen show signs of complement system dysregulation and a high degree of chronic low‐grade inflammation, Copyright (2022), with permission from Elsevier. https://www.thelancet.com/journals/eclinm/article/PIIS2589‐5370(21)00529‐0/fulltext. iAMD, intermediate AMD; MPNd, myeloproliferative neoplasms with drusen; MPNn, myeloproliferative neoplasms with normal retinas; nAMD, neovascular AMD.

FIGURE 13

Expression of CD59 in patients with PV. Expression of complement regulatory protein CD59 in patients with polycythemia vera with (n=26) and without drusen (n=13). Reprinted from The Lancet, EClinicalMedicine 2022; 43: 201248, Liisborg C, Skov V, Kjær L, Hasselbalch HC, Sørensen TL, Patients with MPNs and retinal drusen show signs of complement system dysregulation and a high degree of chronic low‐grade inflammation, Copyright (2022), with permission from Elsevier. https://www.thelancet.com/journals/eclinm/article/PIIS2589‐5370(21)00529‐0/fulltext. PV, Polycytheamia vera.

FIGURE 14

Effector memory cells in patients with AMD and MPNs. MPNn patients stand out with lower percentage of effector memory T cells. Reprinted from Impact Journals, LLC, Aging 2021; 13: 25763–25777, Liisborg C, Skov V, Kjær L, Hasselbalch HC, Sørensen TL, Retinal drusen in patients with chronic myeloproliferative blood cancers are associated with an increased proportion of senescent T cells and signs of an ageing immune system, Copyright (2021), with permission from Impact Journals, LC. https://www.aging‐us.com/full/13/25763. iAMD, intermediate AMD; MPNd, patients having myeloproliferative neoplasms with drusen; MPNn, patients having myeloproliferative neoplasms having normal retinas; nAMD, neovascular AMD.

FIGURE 15

CXCR3 expression T cells in patients with AMD and MPNs. A significantly lower CXCR3 expression in CD4+ and CD8+ t cells is found in the nAMD group compared to the other groups. Reprinted from Impact Journals, LLC, Aging 2021; 13: 25763‐25777, Liisborg C, Skov V, Kjær L, Hasselbalch HC, Sørensen TL, Retinal drusen in patients with chronic myeloproliferative blood cancers are associated with an increased proportion of senescent T cells and signs of an ageing immune system, Copyright (2021), with permission from Impact Journals https://www.aging‐us.com/full/13/25763. iAMD, intermediate AMD; MPNd, patients having myeloproliferative neoplasms with drusen; MPNn, patients having myeloproliferative neoplasms having normal retinas; nAMD, neovascular AMD.

FIGURE 16

Expression of CXCR3 in subtypes of MPNs and neovascular AMD. A decreased CXCR3 expression is observed over the MPN biological continuum. Reprinted from Impact Journals, LLC, Aging 2021; 13: 25763‐25777, Liisborg C, Skov V, Kjær L, Hasselbalch HC, Sørensen TL, Retinal drusen in patients with chronic myeloproliferative blood cancers are associated with an increased proportion of senescent T cells and signs of an ageing immune system, Copyright (2021), with permission from Impact Journals, LCC. https://www.aging‐us.com/full/13/25763. ET, essential thrombocythemia; nAMD, neovascular AMD; PMF, primary myelofibrosis; PV, polycythemia vera.

FIGURE 17

Heatmap of selected significant results from study II. Heatmap showing levels of selected markers from study II. Colour coding is done from lowest to highest measured level in the four groups for each marker. Dark colours represent high levels, while light colours represent low levels.

FIGURE 18

AMD models The current understanding of how drusen develop are the accumulation of oxidative insults and debris that increase with age. Oxidative damage is believed to be the initial trigger of AMD. The highly metabolically active retinal tissue are exposed to a large amount of light and have a high content of polyunsaturated fatty acids, resulting in a high sensitivity to oxidative stress. The accumulating damage exhausts the local autonomous cell response, with decreased autophagy, and the stressed cells may undergo senescence or die. Senescent cells may secrete inflammatory cytokines and chemokines activating the local immune regulating system, and if the damage/ stress again exceeds the capacity, this will eventually lead to decreased phagocytosis resulting loss of clearance and lipid accumulation. The microglia and macrophages of the local regulatory system may release additional cytokines and chemokines which reach the systemic circulation and activate the systemic immune system. The earliest pathological changes are the appearance of basal deposits called basal laminar deposits (BLamD) and basal linear deposits (BLinD) and thickening of BM, with reduced permeability. (a) Our group have previously proposed a model for AMD development ‐ a two‐level model hypothesis”. The accumulation of age‐related ocular damage comprise the first step in the development and the subsequent inflammatory host response the second step. Depending on the host response, the progress rate varies. Adding to the complexity, a “collection” of risk factors (genetic, environmental and health behaviours) is involved in the pathogenesis and can either halt or speed up the process (not shown). (b) Based on the findings in this PhD project, we now propose that the systemic inflammation can trigger and drive the accumulation of debris that leads to drusen formation. All steps in the accumulation process can be affected by and initiated by systemic inflammation and accelerate the ageing process and the following decline in different functions and mechanisms. The systemic inflammation is capable of damaging retinal tissue and triggering the local response.