Memorias Conferencia Científica Anual sobre Síndrome Metabólico 2015 - Programa Científico - Dra. Julia Kzhyshkowska - Jefa del Departamento de Inmunidad Innata y Tolerancia, Facultad de Medicina, Universidad de Heidelberg, Alemania. Miembro del Editorial Board, Revista Immunobiology
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Programming of innate immune cells in diabetic conditions: Emerging epigenetic mechanisms
1. PROGRAMMING OF INNATE IMMUNE CELLS IN
DIABETIC CONDITIONS:
EMERGING EPIGENETIC MECHANISMS
Prof. Dr. Julia Kzhyshkowska
Institute of Transfusion Medicine and Immunology,
Medical Faculty Mannheim, University of Heidelberg
Mexico 14.08.2015
2. Innate and adaptive immune system cooperate
Innate system:
Non-specific
“non-self” and
“unwanted-self” sensing,
local inflammation
Resolution of inflammation
Wound healing
Adaptive system:
Antigen specific
amplification of inflammation
Immunological memory
Antigen recognition
Activation, Recruitment,
Inhibition
Antigen presentation
Activation, Recruitment,
Inhibition
•Macrophages
•Mast cells
•Neutrophils
•Basophils & Eosinophils
•NK cells
Bone Osteoclasts
Brain Microglia
Liver Kupffer cells
Lung Alveolar macrophages
Skin Langerhans cells
8. MACROPHAGES AS DIAGNOSTIC
BIOMARKERS AND CELL THERAPY TOOLS
Analysis of macrophage polarisation
Re-programming of macrophage
Immune cell therapy
Status of innate immune system activation in circulation and tissues
DiagnosticsDiagnostics
TherapyTherapy
Monocyte Macrophages
9. Phagocyte
“Whenever the organism enjoys immunity, the
introduction of infectious microbes is followed by the
accumulation of mobile cells, of white corpuscles of the
blood in particular which absorb the microbes and
destroy them. The white corpuscles and the other cells
capable of doing this have been designated
"phagocytes", i.e. devouring cells, and the whole
function that ensures immunity has been given the
name of "phagocytosis".
“It has been established as a general rule that in all
cases of immunity, natural or acquired, either by
preventive vaccination or following an attack of
infectious illness, phagocytosis takes place to a
marked degree, whereas in fatal or very dangerous
diseases, this phenomenon does not exist at all or is
attenuated.”
Nobel Lecture, December 11, 1908
Ilya Metchnikov
10. Activated macrophage
The mouse was found to be natively susceptible to Listeria
monocytogenes. Its susceptibility was attributed to the capacity of the
organism to survive and multiplying in host macrophages. During the first
3 days of a primary infection the bacterial populations of spleen and liver
were found to increase at a constant rate. On the 4th day of infection the
host became hypersensitive to Listeria antigens and at the same time
bacterial growth ceased. A rapid inactivation of the organism ensued.
Convalescent mice were resistant to challenge, but no protective factor
could be found in their serum. Histological evidence suggested that
acquired resistance was the result of a change occurring in the host's
mononuclear phagocytes. When challenged in vitro, the macrophages of
convalescent mice were found to resist infection with Listeria
monocytogenes. Listeria-resistant cells appeared during the course of
infection at a time which corresponded with the development of the
antibacterial mechanism in the spleen. They persisted for as long as the
antibacterial mechanism remained intact in this organ.
This period of absolute resistance to Listeria lasted about 3 weeks.
Thereafter, the host remained hypersensitive but unable to inactivate a
challenge inoculum of Listeria. However, it remained capable of
producing an accelerated response to reinfection. This was thought to
depend upon an ability to generate a new population of resistant cells
from a residuum of specifically sensitized macrophages or macrophage
precursors still surviving in the tissues as a result of the immunological
activation which occurred during the primary infection.
JEM 1962
George Mackaness
11. IFNgamma is the key factor activating macrophages
“IFN gamma activates human macrophage oxidative
metabolism and antimicrobial activity, and appeared to
be the only factor consistently capable of doing so in
the diverse lymphokine preparations tested”
JEM 1983
Carl Nathan
17. Signal transduction and vesicular transport define macrophage
function: example TGFbetaRII and stabilin-1
Inflammation Tolerance
Exogene effectors
Endogene effectors
Exogene effectors
Endogene effectors
Original papers:
Kzhyshkowska et al, J Leuk Biol, 2004
Kzhyshkowska et al J Immun. 2006
Kzhyshkowska et al J Immun. 2008
Gratchev; Kzhyshkowska et al, J Immun. 2008
Mosig et al, FASEB 2009
Zhang et al, Kzhyshkowska, MCB 2009
Schledzewski et al, Kzhyshkowska J Clin Invest 2011
Popova A et al, Immunobiology 2012
Ovsiy et al, manuscript in preparation
Nurgazieva te al, J Immun, 2015
Reviews:
Kzhyshkowska et al, J Cell Mol Med 2006
Kzhyshkowska et al, Immunobiology 2007;
Kzhyshkowska and Krussel, Immunobiology 2009
Kzhyshkowska, ScientificWorldJournal 2010
Kzhyshkowska et al, Immunobiology 2012
Gratchev et al, Kzhyshkowska, Immunobiology 2012
Gratchev et al, Kzhyshkowska, Curr Pharm Des. 2013
19. DIABETIC COMPLICATIONS
AND INFLAMMATION
microvascular complications
diabetic retinopathy
diabetic nephropathy
diabetic neuropathy
macrovascular complications
atherosclerosis
cardio-vascular diseases
other chronic complications
depression, dementia, sexual disfunction
doubled over all risk of dying
inflammatory processes involved
not everybody suffer from complications
http://www.idf.org/sites/default/files/pictures/Complications-graphic.jpg
20. ATHEROSCLEROSIS
Major Risk factors: Hypercholesterolemia/ dyslipedimia, hypertension, tabacco
consumption, diabetes mellitus
Inflamed atherosclerotic plaques had a higher amount of macrophages and are thought to be at
higher risk to rupture [Puig 2011]
LDL indices stabilin-1 and CD36 expression on monocytes promoting their adhesion to endothelium
[Mosig S 2009]
TLR2 and 4 activation by modified LDL, Tenascin C or HMGB promotes foam cell formation [Howell
2011; Liu 2012; Tsung 2014]
21. Open question:
what are the mechanisms of long-term
pathological programming of innate
immune cells
by metabolic factors?
24. DNA METHYLATION
In mammals only in CpG context
Blocking agent
for DNA methyl-transferase
For investogation or therapy
25. CpG ISLANDS
CpG dinucleotides occur in clusters known as ‘CpG islands’.
CpG islands are often associated with sites where the transcription of DNA
into RNA begins - the promoter regions.
CpG islands are normally unmethylated, consistent with the ability of genes
to be transcribed in the presence of necessary transcription factors.
Major function: regulation of gene expression and genome organization
26. DNA METHYLATION IN CANCER
DNA hypomethylation contributes to cell transformation by 3 mechanisms:
generation of chromosomal instability,
reactivation of transposable elements,
loss of imprinting.
I: Hypomethylation
II: Hypermethylation
Hypermethylation of the CpG islands in the promoter regions of tumor-
suppressor genes (DNA-repair genes hMLH1, BRCA1, MGMT, p15, p16,
APC etc) is a major event in the origin of many cancers.
28. REGULATION OF DNA METHYLATION
There are 2 major types of DNA methylation activity – maintenance
methylation and de novo methylation
A critical step in DNA methylation involves DNMT (DNA methyl-
transferase), the enzymes that catalyze the methylation process (DNMT1,
DNMT3a, and DNMT3b).
Loss of methylation requires active cell division
29. from Horn and Peterson Science, 2002
CHROMATIN AND HISTONES
30. Euchromatin
Transcriptionally active, less compacted
Heterochromatin
Less transcriptionally active, very compacted
a) constitutive heterochromatin
centromeres, telomeres
b) facultative heterochromatin
transposons, inactive X chromosome
33. “Histone Code” hypothesis
Modifications of the Histone tails act as marks that can
be read by other proteins to control the expression or
replication of chromosomal regions. The coding in the
histones may be heritable.
Generally, histone acetylation is associated with
transcriptionally active genes
Deacetylation is associated with inactive genes
(= gene silencing)
Role of methylation is context-specific
34. POI – protein of interest, may be histone or modified histone
CHIP-ON-CHIP
36. EPIGENETIC REGULATION OF INFLAMMATION
Gaps of knowledge:
How metabolic
conditions affect histone
code?
37. EPIGENETICS IN DIABETIC
ENDOTHELIAL CELLS
Reddy and Natarajan, Cardiovascular research, 2010c
Hyperglycemia and AGEs result in production of
pro-inflammatory mediators (cytokines, GF)
Pro-inflammatory mediators activate oxidant stress,
signal transduction (TK, PKC, MAPK) leading to
activation of transcription factors (NFkappaB) and
dysregulation of epigenetic mechanisms including
DNA methylation, histone modifications and miRNA
Outcome: loss of repressive chromatin marks and
gain of activation marks leading to formation of open
chromatin sate at the promoters of pathological
genes
Long-term fixation of open chromatin state works as
“metabolic memory”
Open question: why open state of chromatin is
fixed? Molecular mechanisms?
HMTs: histone methyltransferases
HDM: histone demethylases
HDAC: histone deacetylases
DNMTs: DNA methyltransferases
DeMet: DNA demethylases
38. Gaps of knowledge:
What are the key differences in
epigenetic changes in
subpopulations of macrophages in
health and disease Saeed S et al,
Science, 2014
EPIGENETICS CONTROLS
MONOCYTE TO MACROPHAGE DIFFERENTIATION
39. Our question:
which genes are controlled by
epigenetic mechanisms by metabolic conditions
in macrophages?
40. MODEL SYSTEM, MET
Monocyte isolation from buffycoats by Biocoll and Ficoll centrifugation and
magnetic seperation of CD14+
monocytes with MACS beats
CD14+
monocytes
low glucose conditions (5 mM) high glucose conditions (25 mM)
IL-4 IL-4IFN-γ IFN-γ
Macrophage differentitation by culuturing and stimulation for
6d
RNA isolation and cDNA synthesis
PCR and quantitative realtime PCR analysis
41. TLR AND DIABETES
TLR2 knockout in diabetic mice attenuates developing chronic
inflammation or incipient diabetic nephropathy [Devaraj, 2011]
TLR4 knockout attenuates the proinflammatory state of diabetes
[Devaraj, 2011]
increased expression of macrophage TLR4 is reported in obesity [Olefsky,
2010]
NF-κB signaling is more active under obese conditions or in insulin
resistance [Osborn, 2012]
application of TNF-α has been shown to increase blood sugar levels
and insulin resistance in rodents [Könner, 2011]
42. TLR EXPRESSION
TLR2 + 4 mRNA and protein expression in THP-1 human monocytic cell
line are increased under hyperglycemic conditions (3d) [Dasu, 2008]
An increased TLR2 and TLR4 expression in mRNA and protein in T1D
and T2D patients’ monocytes, compared with controls, correlate with
theire HbA1C levels [Devaraj, 2008; Dasu, 2010]
TLR2 + 4 surface expression (FACS) increased in monocytes from T1D
patients with microvascular complications compared with NC and
"healthy" T1D [Devaraj, 2011]
Does glucose alter TLR expression in macrophage
subpopulation?
43. HYPOTHESIS
Hyperglycemia leads to an altered TLR expression in
macrophage subtypes and drive pro-inflammatory responses to
endogenous unwanted-self ligands
49. EDOGENOUS TLR LIGANDS
AND DIABETES
Toll-like receptor ligand
TLR2
necrotic cell products
apolipoprotein CIII
serum amyloid A
vesican
human cardiac myosin
snapin
TLR2/ CD36
oxidised phospholipids
saturated fatty acids
lipoprotein A
TLR2 or TLR4
hyaluron fragments
biglycan
oxLDL
HSP60, 70
endoplasmin
HMGB1
TLR4
surfactanc protein A
tenascin C
fibrinogen
fibronectin EDA
heparan sulphate
ß-defensin 2
amyloid ß peptid
HSP22, 72
minimally modified LDL
AGE-LDL
Shalhoub J. Inflamm. 2011
Yu J. Cell. Mol. Med. 2010
damage-associated
molecular patterns
endogenous activation of
TLR
50. TLR EXPRESSION IN FAT TISSUE OF
METABOLIC SYNDROME PATIENTS
qPCR results
51. CONCLUSIONS
Conclusions
higher levels of TLR2, 1,6, 4 and TLR8 expression in M1compared to M2
no increased expression of TLR4, but rather tendency to lower expression
levels of TLR4 in high glucose conditions
individual TLR expression pattern in high glucose conditions
Induction by glucose of TLR2, 1,6 in macrophage correlates with their
increase in fat tissue in metabolic syndrome patient
Next steps:
Analysis of individual TLR responses to stimulation by unwanted –self
ligands related to metabolic conditions
52. High glucose induces donor-specific
cytokine responses in human macrophages
Cytokines RT-PCR ELISA
TNFa Increased in 5 out of 6
donors after 6h in M1
Increased in 4 out of 6
donors after 6h in M1
CCL18 Decreased in 8 out of 8
donors on day 6 in M2
Decreased 8 out of 8 donors
on day 6 in M2
IL1beta Increased in 5 out of 8
donors on day 6 in M1
Increased 6 out of 8 donors
on days 1, 3 and 6 in M0,
M1 and M2
IL1ra Increased in 2 out of 8
donors on day 6 in M2
Increased in 6 out of 8
donors on day 6 in M0
Green = High glucose induces
Red = High glucose supresses
53. Sig Index for Estimate of IFNg:
High glucose vs Low Glucose
M1
M2
M0
Sig Index for Estimate of NS:
High Glucose vs Low Glucose
Sig Index for Estimate of IFNg:
High Glucose vs Low Glucose
Sig Index for Estimate of IL4:
High Glucose vs Low Glucose
M1 M0
M2
Searching for glucose regulated genes in
macrophages: Affymetrix Microarray analysis
54. EPIGENETICS IS A SENSOR TRANSLATED TO MACROPHAGES
DIFFERENTIATION AND ACTIVATION
IN METABOLIC CONDITIONS
metabolic, life style factors,
diabetic conditions
Epigenetic changes
Epigenetic memory
High glucose, ,
endogenous unwanted-self
products
Pathologic response
Pro-inflammatory
monocytes in the circulation
Changed M1/M2 balance in
the tissues
Modifications in
macrophages plasticity
Pathological
profile of cytokines
and receptors
Chronic
inflammation
Vascular
complications
Gaps of knowledge: exact
mechanisms, specific for
macrophage subtypes
55. Take home messages
Metabolic conditions induce epigenetic changes leading to the
inflammatory responses in endothelial cell
Histone code is major epigenetic mechanism of inflammatory
programming of macrophages in response to infection
High glucose induces gene expression for TLRs, cytokines and other
regulatory genes not only in M1, but also and M2
Next horizons in research: to identify epigenetic mechanism of long-
term metabolic memory in macrophages
Translational perspective for clinic: drugs that block pathological
metabolic memory in immune cells and decrease risk of diabetic
complications.
56. Institute of Transfusion Medicine
and Immunology
Kondaiah Moganti
Bin Song
Alexandru Gudima
Shuiping Yin
Feng Li
Michael Balduff
Maria Verdiell-Lopes
Vladimir Ryabov,
Christna Schmuttermaier
Sandu Gudima
Amanda Mickley
Harald Klüter
Karen Bieback
Peter Buggert
Thank you
Financial support
DFG SFB „Immune tolerance“ Project B12
DFG GRK880 “Vascular Medicine”
Ministry of Science, Research
and Art of Baden-Württemberg
Margarete von Wrangell
Habilitationsprogramm 2003-2008
BMBF 2010-2012
BMBF BIOIN 2013-2015
FP7-ERANET
GRK1874 DIAMICOM
FP7-IMMODGEL
FP7 EULAMDIMA
Helmholtz-Zentrum, München
Elisabeth Kremmer
University of Heidelberg,
Medical Faculty
Mannheim
Oxford University
Siamon Gordon
DKFZ, Heidelberg
Berndt Arnold
University of Jena,
S. Mosig, K Rennert, Prof. H. Funke
Centre for Medical Research,
Lab for Microarray analysis
Xaolei Yu
M. Saile
C. Sticht
Department of Dermatology
Sergij Goerdt
Kai Schledzewski
Alexei Gratchev
I. Ovsij
N. Wang
S. Mamidi
L. Krusell
H. Brundiers
A. Dittmann
E. Usselmann
J. Dvoracek
H. Ahmed
A. D. Nurgazieva
A. Popova
All DIAMICOM members
UMC Groningen
Martin Harmsen
Jan-Luuk Hillebrands
EULAMDIMA consortium
University of Mexico,
Galileo Escobedo
57. OUR PROGRESS
Model system to examine
the high glucose effects on
human M0, M1 and M2
is established
Effect of high glucose on
M1 and M2 cytokines is identified
Kondaiah Moganti: glucose
affects cytokine expression
on transcriptional and post-
transcriptional levels
Affymetrix profiling: high glucose
affect transcription in M0, M1 and
M2, with strongest effect in M1
Effect of high glucose on TLR
expression profile in M1 and M2
is identified
Michael Balduff: glucose
affects TLR expression on
transcriptional level
ChIP-on-chip using
activation and repression
histone marks: effect of HG
on M1, M2, plasticity
58. Green color represents significantly upregulated
Red color represents significantly
Epigenetic regulatory factors
59. EPIGENETICS IN DIABETES
Conclusions
It is established that epigenetics plays a key role in
glucose memory and diabetic complications
development
It involves both DNA methylation and histone code
Identification of particular mechanisms requires large
clinical studies
60. EPIGENETICS IN DIABETES
Keating ST, El-Osta A. Epigenetic changes in diabetes. Clin Genet 2013
Gap of knowledge:
for how long
glucose memory
persists in vivo?
62. ENHANCERS ARE CRITICAL FOR
REGULATION OF EPIGENETIC IN MACROPHAGES
Jan Van den Bossche, Curr
Opin Lipidol, 2014
Gaps of knowledge:
What are the key
differences in
epigenetic changes in
subtypes of
macrophages
63. EPIGENETIC REGULATION OF INFLAMMATION IN
HYPOXIC CONDITIONS
HIF-1 binds to a specific site of the
promoter of histone 3 lysine 9
demethylases (JMJD1A, JMJD2A)
and activate these genes.
Brigati et al, 2010 Mediators of Inflammation
64. Jan Van den Bossche, Curr
Opin Lipidol, 2014
JMJD3: HISTONE DEMETHYLASE WITH THE MOST
COMPLEX ROLE IN MACROPHAGES POLARISATION
67. Enzymes providing histone modifications
Acetylation: HATs - CBP,p300, GCN5, ATF2, Tip 60…
Deacethylation: HDACs- class I and II
Methyaltion: Lysine: SET-domain HMTase and non-SET domain
HMTase (Dot1)
Arginine: PRMT family, CARM1
Demethylation: LSD1, JMJD3
Ubiquitination: ubiquitin conjugase Rad6/ligase Bre1 for H2B
De-Ubiquitination: SAGA-associated Ubp10
68. Macrophage activation dichotomy
(1985-1992, S.Gordon)
Th1
IFNγ
LPS
Th2IL-4,
Mannose
receptor
TNFα, IL-6,
IL-1
NO, H2O2
Classically activated macrophage
Alternatively activated macrophage
Initially alternative activation of
macrophages was defined on the
basis of IL-4- dependent induction
of macrophage mannose receptor
(MMR)
Editor's Notes
During the next decade the concept of alternative activation became so popular that several different cytokines and also hormones were classified as “alternative activators” of macrophages. At the same time the only thing they have in common is the inability to induce inflammatory activation of macrophages.
Here the results of our 10 years long studies are presented that demonstrate that every stimulus induces specific molecular markers and functions in macrophages. I will illustrate these work with experimental data.
IL-4 can induce cytokine response on any stage and type of macrophage differentiation. IFN-gamma induces cytokine response only in monocytes, but not in macrophages, however it can induce bactericidal activity independent on differentiation type and stage. LPS induces cytokine response in all macrophage populations.
From our mechanistic studies of receptors we see that vesicular trafficking can define macrophage activation without activation of transcription.
Thus transcriptional profiling does not reveal all changes in macrophage phenotype. Thus we decided to use proteomic approach and profile of secreted cytokines, growth factros and other soluble mediators of macrophage functions
IL-4 can induce cytokine response on any stage and type of macrophage differentiation. IFN-gamma induces cytokine response only in monocytes, but not in macrophages, however it can induce bactericidal activity independent on differentiation type and stage. LPS induces cytokine response in all macrophage populations.
Here, I summarised all the donors responded to high glucose by realtime and also by ELISA
In this table 15 donors are responded out of 26