BIOLOGY OF HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS G.E. Janka Hamburg Iran 2018

HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS Pathogenesis Which biological mechanisms lead to the disease? Pathophysiology Which physiological processes explain symptoms and laboratory findings? Biology of HLH

HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS Definition HLH is not an independent disease but a life-threatening clinical syndrome of severe uncontrolled hyperinflammation, due to activation of lymphocytes and macrophages secreting high levels of cytokines

HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS Pathogenesis There are similarities and differences in the pathogenesis of genetic and acquired HLH In both there is an inadequate immune reaction with high levels of cytokines In genetic HLH, in most cases this is due to permanently impaired function of natural killer cells and cytotoxic T cells, leading to the inability to kill the target and to terminate the immune response In acquired HLH mechanisms there are several possible mechanisms .

HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS Genetic defects Disease Chromosome Gene Gene function Familial HLH FLH-1 9q21.3-22 not known not known FLH-2 10q22 PFR1 induction of apoptosis FLH-3 17q25 UNC13D vesicle priming FLH-4 6q24 STX11 vesicle docking/fusion? FLH-5 19p13 UNC18B vesicle docking/fusion? (STXBP2) Griscelli syndr. 2 15q21 RAB27A vesicle docking Chédiak-Higashi 1q42.1 LYST vesicle maturation/ syndrome 1 - q42.2 fusion XLP (Purtilo Xq25 SH2D1A, signal transduction and syndrome) XIAP activation of lymphocytes

Uncontrolled and ineffective immune response in genetic HLH Normal immune response Trigger Activation Trigger Cytotoxic cell (NK cell, CTL) Activation ? Maturation Lyst Polarization Failure to kill (infected) target cell Failure to contract immune response Rab27a Docking Munc13-4 Priming Fusion Uncontrolled expansion of cytotoxic T-cells Syntaxin11 Munc18-2 Degranulation Immunological synapse Perforin Cytokines Target cell Uncontrolled activation of macrophages Apoptosis of target cell Granzymes Removal of antigen Clinical picture of HLH Termination of immune response

HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS Perforin Perforin is a key molecule for cytotoxic T-cells (CTLs) and NK cells Perforin leads to apoptosis of (infected) cells and down- regulation of the immune response (killing of iDCs, CTLs) It is important for immune homeostasis, protecting against autoimmune diseases. It plays a role in immune surveillance against malignancies.

HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS Pathogenesis Besides cytotoxic defects in T cells there are other pathogenetic mechanisms to induce HLH T cell activation is not always necessary for HLH: HLH can develop in patients with SCID and T cells <100/µl (Bode S 2015)

HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS Pathogenesis: Role of the innate immune system Activation of toll-like receptor (TLR)-9 induces HLH in mice (Behrens E 2011) Abrogating the function of the TLR-adaptor myd88 prevents HLH in UNC13D mice (Krebs P 2011) Myd88 is also an adaptor for signaling by IL-1 family cytokines In LCMV- infected Prf1-/- mice blockade of ST2, the receptor for the alarmin IL-33 reduced severity and mortality of HLH (Rood JE 2016) Mutations in human NLRC4, a component of the inflammasome, lead to recurrent HLH (Cannae SW 2014) In mice protection from excessive inflammasome activation is mediated by XIAP (Yabal M 2014)

HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS Other possible pathogenetic mechanisms in HLH

HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS Pathogenesis: possible factors Inhibition of cytotoxic function by viruses and cytokines Interference with apoptosis by viruses and tumor cells Secretion of cytokines from tumor cells Acquired immune dysfunction by drugs; HIV Genetic factors Imbalance between viral load and immune effector cells

HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS Inhibition of cytotoxic function Viral evasion strategies Avian influenza (H5N1) virus downregulates expression of perforin in CD8+ T cells (Hsieh SM 2006). EBV inhibits expression of SAP, impairing the cytolytic response of CTLs to B-cell mediated Ag presentation (Chuang HC 2005). Viruses can interfere with NK cell function by inhibiting activating receptors, stimulating inhibitory receptors, and by blocking chemokines and cytokines necessary for NK cell function (Orange JS 2002). Role of cytokines High levels of IL-6 (as present in sJIA) inhibit NK cell cytotoxicity (Cifaldi L 2015). High levels of pro-and anti-inflammatory cytokines in sepsis lead to decreased NK-cell activity and T-cell exhaustion (Hotchkiss RS 2013).

HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS Inhibiton of apoptosis Interference with apoptosis by viruses or tumor cells Viruses interfere both with the intrinsic and extrinsic pathway of apoptosis (Alcami A 2000, Tortorella D 2000). : - Expression of antiapoptotic molecules - Blocking of apoptotic signals via the extrinsic pathway - Inactivation of p53 - Inhibition of caspases Cancer cells interfere with all pathways of apoptosis (Hassan M 2014).

HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS Pathogenesis Secretion of cytokines by tumor cells (Nishiwaki U 2016) Acquired immune dysfunction Treatment with immunosuppressive agents HIV-infection

HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS Genetic factors Single nucleotide polymorphisms (SNPs) Severe malaria is associated with several SNPs (Sinha S 2008; Seaby E. 2016). Infectious mononucleosis or silent infection with EBV is associated with SNPs in HLA class I antigens (McAulay KA 2007). Severity of enterovirus-71 infection is associated with SNP in OAS3 encoding the IFN-induced oligoadenylate synthetase (Tan Y 2017). Risk of fatal outcome of avian or pandemic influenza is associated with SNPs in genes of innate immunity (Lee N 2017). Heterozygous mutations in FHL-related genes Mutations in other genes regulating the immune system

HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS Genetic factors Heterozygous mutations in HLH-related genes (Cheng X, Clin Genet 2018) Allele frequencies >1% excluded) 265 patients (243 children) Bi-allelic (19) or hemizygous (16) mutations (SAP, XIAP) 35/265 (13%) patients At least 1 mutation of uncertain significance 10/35 (29%) Heterozygous mutations 44/265 (17%) Of uncertain significance 24/44 (54%) Digenic mutations 7/265 (3%) At least one mutation of uncertain significance 4/7

HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS Genetic factors Mutations in other genes regulating the immune system (Chinn I Blood 2018) 101 children with genetic testing, fulfilling HLH-2004 criteria Whole-exome sequencing (WES) in 48 patients Biallelic mutations 19/101 (19%) 1 additional RAB27 case WES 14 potential primary immune deficiencies 28/48 (58%) patients with mutations 10 inflammasome genes + 1 NRAS 5 other candidate genes in pathways of cytotoxicity, regulation of immune activation, and cotrol of hematopoiesis

HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS Pathogenesis Adaptive immune system Cytotoxic defect - inherited biallelic mutations - acquired - immune evasion strategy by viruses - immune suppression, HIV - cytokines Loss of regulatory feedback loop CTL/NK cell Immunological synapse iDC Prolonged contact time Target cell Failure of apoptosis APCs Hypercytokinemia INFγ, IL-6, TNFα, IL-1ß, IL-10, IL-18 Hyperinflammation Viruses HLH

HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS Pathogenesis Adaptive immune system Cytotoxic defect - inherited biallelic mutations - acquired - immune evasion strategy by viruses - immune suppression, HIV - cytokines Innate immune system Loss of regulatory feedback loop CTL/NK cell Immunological synapse APC tissue damage Prolonged contact time Target cell necrosis Amplification of the immune response Failure of apoptosis APCs Hypercytokinemia INFγ, IL-6, TNFα, IL-1ß, IL-10, IL-18 Hyperinflammation Viruses alarmins, e.g. IL-33 INFγ T-cells HLH ST2 (IL-33 receptor)

HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS Pathogenesis Adaptive immune system Cytotoxic defect - inherited biallelic mutations - acquired - immune evasion strategy by viruses - immune suppression, HIV - cytokines Innate immune system Activation of TLRs Inflammasome disorders Loss of regulatory feedback loop CTL/NK cell Underlying inflammatory disorders, e.g. soJIA Immunological synapse APC tissue damage Prolonged contact time Target cell necrosis Amplification of the immune response Failure of apoptosis APCs Hypercytokinemia INFγ, IL-6, TNFα, IL-1ß, IL-10, IL-18 Hyperinflammation Viruses alarmins, e.g. IL-33 INFγ T-cells HLH ST2 (IL-33 receptor)

HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS Pathogenesis Adaptive immune system Cytotoxic defect - inherited biallelic mutations - acquired - immune evasion strategy by viruses - immune suppression, HIV - cytokines Innate immune system Activation of TLRs Inflammasome disorders Loss of regulatory feedback loop Underlying inflammatory disorders, e.g. soJIA CTL/NK cell Immunological synapse APC tissue damage Prolonged contact time Target cell necrosis Amplification of the immune response Failure of apoptosis APCs Hypercytokinemia INFγ, IL-6, TNFα, IL-1ß, IL-10, IL-18 Hyperinflammation Viruses alarmins, e.g. IL-33 INFγ antiapoptotic molecules T-cells HLH Malignant cells ST2 (IL-33 receptor) cytokines

HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS Pathogenesis Adaptive immune system Cytotoxic defect - inherited biallelic mutations - acquired - immune evasion strategy by viruses - immune suppression, HIV - cytokines Genetic factors Innate immune system - SNPs - monoallelic mutations Activation of TLRs Inflammasome disorders Loss of regulatory feedback loop CTL/NK cell Underlying inflammatory disorders, e.g. soJIA Immunological synapse APC tissue damage Prolonged contact time Target cell necrosis Amplification of the immune response Failure of apoptosis APCs Hypercytokinemia INFγ, IL-6, TNFα, IL-1ß, IL-10, IL-18 Hyperinflammation Viruses alarmins, e.g. IL-33 INFγ antiapoptotic molecules T-cells HLH Malignant cells ST2 (IL-33 receptor) cytokines

HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS Pathogenesis Adaptive immune system Cytotoxic defect - inherited biallelic mutations - acquired - immune evasion strategy by viruses - immune suppression, HIV - cytokines Genetic factors Innate immune system - SNPs - monoallelic mutations Activation of TLRs Inflammasome disorders Loss of regulatory feedback loop CTL/NK cell Underlying inflammatory disorders, e.g. soJIA Immunological synapse APC tissue damage Prolonged contact time Target cell necrosis Environment Amplification of the immune response Failure of apoptosis APCs Hypercytokinemia INFγ, IL-6, TNFα, IL-1ß, IL-10, IL-18 Hyperinflammation Viruses alarmins, e.g. IL-33 INFγ antiapoptotic molecules T-cells HLH Malignant cells ST2 (IL-33 receptor) cytokines

HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS PATHOPHYSIOLOGY Which physiological processes explain clinical and laboratory findings?

HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS Clinical symptoms and laboratory findings Fever, hepatosplenomegaly, cytopenias Neurological symptoms High ferritin, low fibrinogen, high triglycerides, transaminases, bilirubin, LDH CSF pleocytosis and/or elevated protein Hemophagocytosis

HLH: SYMPTOMS AND CYTOKINES Symptoms of HLH can be explained by hypercytokinemia and organ infiltration by histiocytes and lymphocytes • fever (interleukins) • pancytopenia (TNF-, INF-, heavy subunit of ferritin, phagocytosis) • high triglycerides (TNF-, INF- lipoprotein lipase ) • low fibrinogen (plasminogen activator hyperfibrinolysis) • high ferritin (activated macrophages), high levels of soluble interleukin-2 receptor (sCD25) (activated lymphocytes) • hepatosplenomegaly, elevated transaminases/bilirubin, neurological symptoms (infiltration by activated lymphocytes and histiocytes; cytokines and chemokines) All these symptoms and laboratory values can also be present in infections which take a normal course; however, in HLH these findings are abnormally out of proportion

HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS Physiological processes explaining laboratory findings Symptom Mechanisms Mediators References Cytopenias Selleri 1995 Papadaki 2002 Zoller 2011 Kuriyama 2012 Decreased production Increased apoptosis Hemophagocytosis (peripheral cells and stem cells) TNFα, INFγ Triglycerides Decreased lipoprotein lipase Increased synthesis TNFα, INFγ, IL-1 Henter 1991 Feingold 1989 Ferritin Increased synthesis Passive release from cells Increased Fe-absorption TNFα and oxidative stress HO-1 synthesis Otterbein 2003 Kirino 2005 Wu 2013 GDF15 (MIC-1) hepcidin Fibrinogen Fibrinolysis by plasmin plasminogen activator plasminogen plasmin various cytokines induce plasminogen activator plasmin increases influx of in- flammatory cells and cytokines Loscalzo 1996 Shimazu 2017 sCD25 Lymphocyte activation

HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS Pathophysiology HLH and bone marrow stem cells Kuriyama T, Blood 2012 Self-recognition to prevent phagocytosis is regulated by interaction of CD47 (expressed in hematopoietic cells) and SIRPA (expressed in macrophages). Inflammatory cytokines downregulate CD47 specifically in HSCs. CD47 is downregulated in stem cells of HLH patients with active disease, leading to increased phagocytosis. The number of HSCs in HLH patients was reduced to 23% of those in healthy adults. Pascutti MF, Frontiers Immunol 2016 Several viruses infect HSCs or supportive stromal cells in the bone marrow.

HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS Pathophysiology HLH and role of plasmin Shimazu H, Blood 2017 Activated macrophages produce plasminogen activator, leading to increased levels of plasmin. Plasmin can degrade fibrin clots. Plasmin regulates the influx of inflammatory cells and the inflammatory response by inducing transcription of cytokines. In a mouse model of HLH, established by repeated TLR-9 stimulation (CpG + DG), plasmin levels were excessively high. Genetic (plasminogen knock-out mice) and pharmacological (plasmin antibody) inhibition of plasmin reduced mortality and HLH symptoms.

HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS Summary Knowledge of HLH has increased considerably over the past years. Discovery of mutation of genes involved in the cytolytic granule pathway has given important insights into the pathogenesis of familial HLH and of the immune system in general. Pathogenesis of acquired HLH in children and adults is still poorly understood. HLH in these patients is probably due to a combination of host factors, pathogen-associated factors and other triggers. Better insight into the pathogenesis and pathophysiology of HLH may show new ways for treatment.

HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS Thank you for your attention! There is still much to learn about HLH!