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

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

3. 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

4. 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 .

5. HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS
Genetic defects
Disease Chromosome Gene Gene function
Familial HLH
FLH q not known not known
FLH q PFR induction of apoptosis
FLH q UNC13D vesicle priming
FLH q STX vesicle docking/fusion?
FLH p UNC18B vesicle docking/fusion?
(STXBP2)
Griscelli syndr q RAB27A vesicle docking
Chédiak-Higashi 1q LYST vesicle maturation/
syndrome q fusion
XLP (Purtilo Xq SH2D1A, signal transduction and
syndrome) XIAP activation of lymphocytes

6. 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

7. 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.

8. 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)

9. 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)

10. HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS
Other possible pathogenetic mechanisms
in HLH

11. 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

12. 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).

13. 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).

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

15. 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

16. 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

17. 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

18. 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

19. 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)

20. 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)

21. 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

22. 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

23. 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

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

25. 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

26. 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

27. 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

28. 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.

29. 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.

30. 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.

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