Systemic Lupus Erythematosus
and DNA Methylation
Terrence Shin
MCB 5255
Dr. Lynes
Mar. 28, 2012
1
Systemic Lupus Erythematosus (SLE)

Chronic autoimmune disorder

Affects skin, joints, kidneys, and others






Joint pain and swelling
Lupus nephritis
Cause – Unknown
Ages: 10~50
More common in women
African Americans and Asians are affected
more often
2
Common Symptoms of SLE







Chest pain
Fatigue
Fever
Hair loss
Mouth sores
Swollen lymph nodes
Skin/Butterfly rash

Photosensitive
3
Possible Body Parts Targeted by SLE

Skin




Raynaud’s phenomenon
Brain & nervous system
Lungs
Heart

Arrhythmias



Tachycardia
Bradycardia
Digestive tract
4
Diagnosis of SLE





CBC
ANA
Chest x-ray
Urinalysis
Renal biopsy
5
Patients with SLE met at least four of
revised criteria of the American College of
Rheumatology.
6
Treatment for SLE


No cure
Control symptoms



Anti-inflammatory medication
Corticosteroids
Protection from sunlight
7
Ultraviolet B exposure of peripheral blood
mononuclear cells of patients with systemic
lupus erythematosus inhibits DNA
methylation
GS Wang, M Zhang, XP Li, H Zhang, W Chen, M Kan
and YM Wang
Lupus 2009
8
Background

Sunlight can induce exacerbation of SLE


SLE cells are hypomethylated


Ultraviolet B (UVB): 290~320 nm
Promoters of transcriptionally active gene
DNA methylation



Suppressive effects on gene expression
5th position of the cytosine ring
Catalyzed by DNMT1
9
Specific Aims

What are the effect of UVB on DNA
methylation in SLE?

What is its significance in the pathogenesis of
SLE ?
10
Materials & Methods






Study participants
Isolation of PBMCs
Irradiation protocol
DNA extraction and HPCE
RNA isolation and RT-PCR
Statistical analysis
11
Study Participants

45 patients with SLE




40 females, 5 males
Mean age: 36 years; range: 18~61 years
Active = SLEDAI ≥ 10 (21 out of 45 patients)
20 healthy volunteers (control)



Sex- and age-matched
18 females, 2 males
Mean age: 31 years, range: 22~46 years
12
Isolation of PBMCs

Total volume of 20 ml of
ethylenediaminetetraacetic acid (EDTA)-K2preserved venous peripheral blood drawn
from patients & controls

Ficoll gradient centrifugation


PBMCs
PBMCs cultured in RPMI 1640 medium

Supplemented with 10% heat-inactivated FBS, 2
mM sodium pyruvate, 100 IU/ml penicillin and 100
μg/ml streptomycin
13
Irradiation Protocol

UVB irradiation

Waldman UV109B lights with TL-12 lamps
(Waldman Lighting Ltd., Germany)


Emit within the UVB range (290–320 nm) with an
emission peak at 311 nm
PBMC were irradiated in PBS with different
doses of UVB (0, 50 and 100 mJ/cm2)

PBS removed  RPMI 1640 containing 10% FBS
added  cultured (24 h)  DNA & RNA
extractions
14
DNA Extraction and HPCE

Five deoxynucleoside standards purchased
from International Laboratory (CA, USA)






dA = 2’-deoxyadenosine
dT = 2’-deoxythymidine
dG = 2’-deoxyguanosine
dC = 2’-deoxycytidine
mdC = 5-methyl-2’-deoxycytidine
All nucleosides were dissolved in ddH2O
water
15
DNA Extraction and HPCE (cont.)



DNA extraction
 gDNA extraction kit (QIAmp, DNA mini kit; Qiagen, Hilden,
Germany)
RNA digestion
 Added 20 μg/μl RNase A (Sigma-Aldrich, St. Louis, MO)
DNA hydrolysis
 Heated 18 μl of DNA samples for 2 min in boiling water bath 
cooled rapidly in ice
 Added 4.5 μl of 10 mM ZnSO4 and 7.5 μl of nuclease P1 (SigmaAldrich, St. Louis, MO)
 Incubated at 37°C for 16 h
 Added 7.5 μl of Tris (0.5 M, pH 8.3), and 4.5 μl of alkaline
phosphatase (Sigma-Aldrich, St. Louis, MO) and 50 U/ml in 2.5 M
(NH4)2SO4
 Incubated at 37°C for 2 h
16
DNA Extraction and HPCE (cont.)

CE system (Beckman P/ACETM MDQ)






Uncoated fused-silica capillary (60 cm × 75 μm, effective length 57 cm)
Connected to Millennium data-processing station
Buffer: 48 mM NaHCO3 (pH 9.6) containing 60 mM SDS
Constant voltage (20 kV) and temperature (25°C)
Pressure injected for 5 s
Hydrolysed DNA or 0.1 mM free deoxynucleoside standard was injected into
a HPCE analysis system equipped with photodiode array (PDA) detection


Absorbance was monitored at 256 nm
Capillary conditioning

Washed with 1 M NaOH (2 min)  washed with 1 mM NaOH (1 min)  filled with
the running buffer (3 min)


Hydrolyzed samples


Buffers and washing solutions were filtered through 0.45 μm pores
Injected hydrostatically (30 s) from 9.8 cm above the cathode
Comparison of migration time & peak area of each sample with the
standards

Can calculate DNA methylation levels

Quantification of the relative methylation of each DNA sample

mdC/(dC + mdC) x 100%
17
RNA Isolation and RT-PCR

Total RNA

Isolated from PBMCs


Reverse transcription


Trizol reagent (Invitrogen, Carlsbad, CA, USA)
Reverse Transcription System (Promega, Madison, WI,
USA)
cDNA synthesis

1.0 μg total RNA—single round reverse transcriptase
reaction (TV = 20 μl) containing 0.5 μg oligo (dT) 15 primer,
2.0 μl dNTPs, 2.0 μl reverse transcriptase 10x buffer, 15 μl
AMV reverse transcriptase and 0.5 μl recombinant RNasin
ribonuclease inhibitor.
18
RNA Isolation and RT-PCR (cont.)

RT-PCR

ABI Prism 7500 Sequence Detection System




Using SYBR Premix Ex Taq Kit (TaKaRa Bio Inc.)
10 μl of 2x SYBR green Master Mix, 0.2 μM
primers, 5 μl 1:5 dilution of prepared cDNA, 20 μl
of water
40 cycles at 94°C (30 s), 60°C (40 s), and 72°C
(60 s)
Primers used:

β-actin (forward & reverse), DNMT1 (forward & reverse)

Purchased from TaKaRa Bio Inc
19
Statistical Analysis

Mann-Whitney U-test or one-way ANOVA


Equality of means—to compare values
Spearman’s rank correlation

Examine the relationship between two continuous
variables
20
Figure 1: Electrophoretogram of nucleosides
separated by HPCE
A. Electrophoretogram—standard
nucleosides
B. Electropherogram of hydrolyzed
gDNA from SLE patients
 Five deoxynucleosides
could be distinguished
under the given conditions
(running buffer of 48 mM
NaHCO3, at pH 9.6,
containing 60mM SDS, with
a separation voltage of 20
kV, injection pressure of 0.7
psi, at 25 °C and pressure
injection of 5 s)
21
Figure 2: Calibration curves
A. dC regression
•
B. mdC regression
Different quantities of dC and mdC were added to a mixture of dA, dT
and dG (0.1 mM) to obtain the concentrations of dC, and mdC
22
Figure 3: DNA methylation in SLE
patients and controls
•Significant decrease in DNA
methylation level in A and S
compared to C
•No difference between A & S
SLE patients
A=active; S=stable; C=control
23
All groups showed decreased DNA methylation levels
•Active SLE group showed significant decrease after 50 mJ/cm2
•Stabe SLE group showed significant decrease after 100 mJ/cm2
•Control group showed significant decrease after 100 mJ/cm2
24
Figure 4:
Electrophoretograms of
nucleosides from
irradiated PBMC
25
Figure 5: DNMT1 mRNA expression of
irradiated PBMC
• No significant differences
could be found in the
expression of DNMT1 before
and after UVB radiation
26
• Decrease in DNA methylation levels for all patients after UVB
irradiation irrespective of their clinical characteristics
• Patients with malar rashes have lower DNA methylation levels than
patients without rashes
• Patients with leucopenia had significantly lower DNA methylation
levels after UVB irradiation than that in patients with normal leucocyte
numbers
•No obvious differences were observed between patients with or
without thrombocytopenia
27
Discussion & Unknowns

SLE patients have significantly lower levels of
methylated DNA compared to controls (without UVB
irradiation)


Does down-regulation of DNA methylation play a role in the
pathogenesis of SLE?
DNA methylation levels decreased after UVB
exposure


Especially for patients with facial rash and leucopenia
Does UVB play a role in pathogenesis of SLE by
decreasing DNA methylation?
28
IL-6 Modulates CD5 Expression in B
Cells from Patients with Lupus by
Regulating DNA Methylation
Soizic Garaud, Christelle Le Dantec, Sandrine JousseJoulin, Catherine Hanrotel-Saliou, Alain Saraux, Rizgar
A. Mageed, Pierre Youinou and Yves Renaudineau
The Journal of Immunology, 2009
29
Background

Features of autoimmunity in SLE




B cell abnormalities in SLE




B cell hyperactivity
Spontaneous lymphocyte proliferation
Production of antibodies to self-antigens
Excess cytokine production
Autoantigen presentation to T cells
Modulation of the function of other immune cells
SLE is considered as a B cell disease

Therapies target B cells
30
Background (cont.)

CD5 isoforms


CD5-E1A
CD5-E1B

Demethylated in B cells of SLE patients

CD5-E1B mRNA is transcribed at the expense of CD5-E1A
mRNA transcription
31
Specific Aims

Does the engagement of the BCR with
constitutive IL-6 down-regulate the level of
membrane CD5, which negatively regulates
BCR signaling, in SLE B cells?

Does this signal promote the activation and
expansion of autoreactive B cells in SLE patients?
32
Materials & Methods










B lymphocyte isolation
FACS analysis
Cell culture
mRNA extraction and quantitative RT-PCR
RACE
Methylation-specific PCR
Bisulfite sequencing
Chromatin immunoprecipitation (ChIP)
Computational promoter analysis
Statistics
33
B Lymphocyte Isolation

PBMCs from the
blood of 25 SLE
patients and
HCs


Centrifugation on
Ficoll-Hypaque
Active = SLEDAI
≥5
34
B Lymphocyte Isolation (cont.)

Cells permeabilized with 70% methanol 
intracellular staining

FITC-anti-CD19 (clone J4-119) and PE-anti-CD5 (clone
BL1a)


Anti-DNMT1 and anti-p27kip1


Obtained from Abcam
CD5-CD19+ B cells



Obtained from Beckman-Coulter
Sorted on an Epics Elite FACS (Beckman-Coulter)
98% CD19+
Daudi human B cell line

Purchased from American Type Culture Collection
35
FACS Analysis

Number of CD5 molecules per cell

Quantum Simply Cellular kit


Amount of Ab binding to the cells (ABC) at saturating
concentrations
FACS analysis

50μl calibrated microspheres stained with 20 μl of
the same anti-CD5 Ab

Mean fluorescence intensity  standard ABC curve 
arbitrary ABC value
36
Cell Culture

B cells (sorted via FACS)

Suspended in RPMI 1640


Supplemented with 10% heat-inactivated FCS, 2 mM Lglutamine, 200 U/ml penicillin and 100 μg/ml streptomycin.
B lymphocytes were seeded at 2 x 105 cells per well

Incubated with 1 μg/ml anti-IgM Ab-coated Sepharose beads
(BioRad) and 10 U/ml IL-2


In the presence or absence of 10–40 ng/ml anti-IL-6RAb (R&D
Systems), or 100 ng/ml rhIL-6 (Immuno Tools).
Repression of DNMTs

Incubated with 50 μM of the ras signal blocker PD98059


Detected IL-6 and IFN-ɣ in sera
Detected IL-6 in the supernatant of cultured cells using ELISA kits
(Beckman Coulter).
37
mRNA Extraction and qRT-PCR


Total mRNA
 Extracted using the RNAble method (Eurobio)
cDNA synthesis
 Reverse transcription


Quantitative RT-PCR
 Conducted in 20 μl mixtures





20 μl volume with Superscript II RNase H-RT (Invitrogen
Corporation).
50 ng template cDNA
1X Sybr Green PCR Master mix (Applied Biosystems)
500 nM of each primer (Table II)
All assays included a negative and positive controls
CT method
 Comparison of cycle thresholds
 18S as an internal control
38
mRNA Extraction and qRT-PCR (cont.)
39
RACE

mRNA 5’ transcript ends


cDNA first strand synthesis


Sense UPM primer and the gene-specific antisense primer CD5 E5 (Table II)
PCR protocol


Amplified with SMART-RACE kit (Clontech)
Initial denaturation at 94°C for 5 min  5 touchdown-PCR cycles of
denaturation at 94°C for 30 s  Annealing at 72°C for 3 min  Another 5
cycles at 94°C for 30 s, 70°C for 30 s, 72°C for 3 min  decreasing
temperature for 35 cycles of 94°C or 30 s, 68°C for 30 s, 72°C for 3 min.
Nested PCR


Sense NUP primer and the gene-specific antisense primer CD5 E3
Second PCR round:

40 cycles at 94°C for 30 s, 56°C for 1 min, and 72°C for 1 min with a final extension
at 72°C for 10 min.
40
RACE (cont.)
UPM Primer
NUP Primer
41
Methylation-specific PCR

Genomic DNA


Purification – QIAmp 96 DNA blood kit (Qiagen)
Digestion – 20 U of the methylation-sensitive REs (HpaII, HaeII, FauI, HgaI)
or the methylation insensitive restriction enzyme (MspI) – 37°C for 3 h

Undigested Genomic DNA


PCR primers

Positioned upstream and downstream of E1A and E1B promoter recognition
sites


Positive control
E1A and E1B of CD5, CD19 CD70, Pax5, Syk, and HRES-1 genes (Table II)
PCR protocol


Initial denaturation at 94°C for 5 min  35 cycles of denaturation at 94°C for
30 s, annealing at 56°C for 1 min, and primer extension at 72°C for 1 min 
final extension at 72°C for 10 min.
Products separated on agarose gel

Visualized with 0.5 g/ml ethidium bromide.
42
Methylation-specific PCR (cont.)
43
Bisulfite Sequencing

Methylation status of DNA

Non-methylated cytosines were converted to uridines by bisulfite treatment


Unmodified DNA (100 ng)



Amplified 40 times at 56°C using specific primers
Sequenced using specific primers
Bisulfite-converted DNA


EZ-DNA methylation-Gold kit (Zymo Research)
Amplified by nested PCR using two rounds of 40 cycles each at 56°C with
primers specific for methylated cytosines (Table II)
PCR products


Purification – high pure PCR product purification kit (Roche)
Sequencing – Internal primers


BigDye Terminator Cycle Sequencing kit using an automated ABI-310 genetic
analyzer (Applied Biosystems)
Electrophoregram


Quantification of T and C peaks
Determination of methylation status = [peak (C)/peak (T) + peak (C)] x 100
44
Bisulfite Sequencing (cont.)
45
ChIP



EpiQuik kit (Epigentek Group)
Evaluation of the activation of the CD5-E1B promoter
Sonicated DNA (200–1000 bp)

Transferred into strip wells



Incubated for 90 min at room temperature, then washed
Precipitated DNA-protein complexes



Treated with 250 μg/ml proteinase K in the DNA release buffer for 15 min
Left in the same buffer for 90 min at 65°C
Collection of DNA samples


Precoated with mouse anti-RNA polymerase II, or with a nonspecific mouse IgG, used as a
negative control
P-spin columns, washed with ethanol, and eluted
PCR




Template – purified DNA
GAPDH & CD5-E1B specific primers (Table II)
40 cycles at 56°C
Products were separated on agarose gel

Visualized with 0.5 g/ml ethidium bromide.
46
ChIP (cont.)
47
Computational Promoter Analysis

Identification of putative transcription factor
binding sites



Alibaba (v2.1)
TESS (transcription element search system)
Genomatix
48
Statistical Analysis


Results as arithmetic means with SD
Mann-Whitney U test


Wilcoxon test


Unpaired data
Paired data
Spearman’s rank correlation
49
Figure 1: CD5 expression in B cells
Scattergrams
A. Percentage of
CD5-expressing B
cells
B. Anti-CD5 Ab
bound to the cell
membrane
C. FACS profile
of one SLE
patient and one
HC: cell surface
expression of
CD5
50
Figure 1: CD5 expression in B cells (cont.)
D. Histograms from qRT-PCR; Bcells were stimulated with antiIgM (24h)  increase in CD5E1B transcription
Incubating HC IgM with PD98059
 upregulation of CD5-E1B
Expression of CD5-E1A also
increased in both
51
Figure 1: CD5 expression in B cells (cont.)
E. CD5-E1B = 639 bp
CD5-E1A = 259 bp
• B cells from HCs
• BCR engagement induce
CD5-E1A, but not CD5-E1B
transcripts
• Stimulated with anti-IgM in the
presence of PD98059
 both are induced
52
Figure 2: Amplification of methylation sensitive, endonucleasedigested genomic DNA reveals methylation status of the
alternative promoters of CD5 in resting B cells
LTR
A. Affected promoters and corresponding restriction enzymes
•
•
•
CD5-E1B promoter arises from LTR
1177-bp CD5-E1B amplicon contains six HpaII/MspI motifs and one
HaeII motif
783-bp CD5-E1A amplicon contains one HgaI motif, one FauI motif
and two HpaII/MspI motifs
53
Figure 2: Amplification of methylation sensitive, endonucleasedigested genomic DNA reveals methylation status of the
alternative promoters of CD5 in resting B cells (cont.)
B. Amplification of gDNA digested with
methylation sensitive HaeII, HpaII, or
methylation insensitive MspI enzymes
HaeII – demethylated CD5-E1B
promoter only in SLE patients
HpaII – demethylation in two of six
SLE patients
MspI (HpaII isoschizomer) – negative
control; cut the CpG sequences  no
PCR products
Daudi – positive control
54
Figure 2: Amplification of methylation sensitive, endonucleasedigested genomic DNA reveals methylation status of the
alternative promoters of CD5 in resting B cells (cont.)
C. Analysis of the CD5-E1A promoter
region
CpG motifs – demethylated or
hemimethylated in both SLE and HCs
55
Figure 3: The U3-LTR HERV-CD5 region is demethylated in B cells from SLE patients
A. Circles = CpG; boxes = U3/R/U5
regions
HaeII site (4), HpaII sites (1, 7, 13)
 HERV U3-R-U5 regulatory
elements may be demethylated in B cells from
SLE patients
56
Figure 3: The U3-LTR HERV-CD5 region is demethylated in B cells from SLE patients (cont.)
B. Bisulfite C  T transition
• White = SLE patients; black = HCs
• Bisulfite sequencing of gDNA  level
of CpG methylation
• Five CpG sites (2~6) in the U3 region
are hemi- or de-methylated.
• C residues near the E2/Rb binding
sites (3, 4), and the CpG site (5), were
significantly less affected by the bisulfite
treatment in the SLE patients compared
to HCs
*, p < 0.05; **, p < 0.001
57
Figure 3: The U3-LTR HERV-CD5 region is demethylated in B cells from SLE patients (cont.)
C. SLE patients have an inverse correlation between CD5 cell surface
expression and methylation status of CpG sites (3~5), but not site 6
White circles = SLE patients; black circles = HCs
58
Figure 4: Effect of BCR engagement on CD5
gene methylation
A, B. Effect of BCR engagement on methylation of the CD5 locus—FACSsorted CD5-negative B cells stimulated with anti-IgM (24 h)
• BCR engagement did not alter SLE B cell methylation status
• HCs stimulated with anti-IgM in the presence of PD98059 were
demethylated
• Not much change in the CD5-E1A for both
59
Figure 4: Effect of BCR engagement on
CD5 gene methylation (cont.)
C. The level of CpG methylation
(in SLE patients) measured by
bisulfite sequencing
• White = with; black = without
BCR engagement
• BCR engagement did not alter
the methylation status
60
Figure 4: Effect of BCR engagement on
CD5 gene methylation (cont.)
D. CpG methylation in B cells of
HCs—stimulated with anti IgM
• Gray = with PD98059; white =
without PD98059
• Methylation of the CD5-E1B
promoter at sites 3 & 4 with BCR
engagement
*, p < 0.05
• HCs stimulated with anti-IgM in
the presence of PD98059
 CpG sites 3, 4, and 5 were
demethylated (similar to SLE
B cells)
61
DNMT1, DNMT3a, DNMT3b, MBD2, and MBD4
levels in B cells were similar
62
Figure 5: Involvement of DNMTs on CD5-E1B expression
A. DNMT1 expression in B cells with BCR engagement
 Increased by 2.3 ± 0.2-fold in SLE patients
 Increased by 16.6 ± 13.4-fold in the controls
MBD expressions were not affected by BCR engagement or by the
addition of PD98059
63
Figure 5: Involvement of DNMTs on CD5-E1B expression (cont.)
B. FACS analyses—BCR engagement modified DNMT1 expression
• Resting B cells vs. B cells with anti-IgM (% DNMT1bright)
 Patients: 23.8 ± 7.9%  46.1 ± 7.8%
 HCs: 25.3 ± 12.3%  86.1 ± 12.2%
 Induction of DNMT1 following BCR engagement is reduced in
patients with SLE
64
Figure 6: The effect of IL-6 on CD5-E1B
expression and promoter methylation
A. FACS-sorted B cells from six HCs were
stimulated for 48 h with rhIL-6 in the
presence or absence of anti-IgM
•
Test if IL-6 requires BCR engagement
•
CD5-E1B expression increased by 4.1
± 3.13-fold in B cells cultured with rhIL6, and by 54.8 ± 11.3-fold in B cells
cultured with rhIL-6 and anti-IgM
•
rhIL-6 negated DNMT1 mRNA induction
by anti-IgM
65
Figure 6: The effect of IL-6 on CD5-E1B
expression and promoter methylation (cont.)
B. CD5-E1B induction upon anti-IgM/rhIL-6
stimulation
 RNA pol II was recruited to the CD5-E1B
promoter upon stimulation
c- = nonspecific mouse IgG as
negative control
c+ = mouse anti-RNA polymerase as
positive control
66
Figure 6: The effect of IL-6 on CD5-E1B expression
and promoter methylation (cont.)
C. DNMT1 and p27kip1 from the HCs incubated with IL-6 in the presence or
absence of anti-IgM
• Number of DNMT1bright cells was reduced after anti-IgM stimulation in the
presence of rhIL-6
 Due to cell cycle blockade?
 Over-expression of p27kip1 in anti-IgM/rhIL-6-stimulated B cells
 Control of CpG methylation by IL-6 (in SLE B cells) may be resulting from its
effect on arresting cells at the late G1 phase of the cell cycle.
67
Figure 7: Anti-IgM-induced methylation of promoters for
CD19, CD70, Pax5, Syk, and HRES-1
A. HC B cells incubated with anti-IgM in
the presence or absence of anti-IL-6R
Ab
•
HpaII = methylation-sensitive;
MspI = methylation-insensitive
Methylation of five promoters
(determined by PCR)
 Known to be regulated by
methylation
 Determine the effect of IL-6 on
methylation
• Hypomethylated in resting B cells
• Stimulation with anti-IgM only
increased the methylation of the
HRES-1
 Reversed when IL-6 was
present
68
Figure 7: Anti-IgM-induced methylation of promoters for
CD19, CD70, Pax5, Syk, and HRES-1 (cont.)
*, p < 0.05
B.
Methylation of the
HRES-1 promoter
=
HpaII-digested bands (in six HCs)
undigested bands
69
Figure 8: IL-6-dependent modulation of methylation in
SLE B cells
A. CD5 cell surface expression in
relation to the detection level of IL-6
B. CD5 cell surface expression in
relation to the detection level of
IFN-ɣ—not significant
IL-6  decreased CD5 expression
70
Figure 8: IL-6-dependent modulation of methylation in
SLE B cells (cont.)
C. FACS-sorted B cells cultured
with anti-IgM in the presence or
absence of up to 40 ng/ml anti-IL6R Ab
 Blocking IL-6 increased DNMT1,
and decreased CD5-E1B
expression as the dose of anti-IL6R Ab went up
71
Figure 8: IL-6-dependent modulation of methylation in
SLE B cells (cont.)
D. Effect of anti-IgM/anti-IL-6R
(gray) on methylation status of
CD5-E1B promoter
 Blocking of IL-6 contributed to
the methylation of the U3-LTR sites
3 ~ 5.
72
Discussion



B cells from SLE patients have reduced membrane CD5 levels
 Due to increased expression of CD5-E1B (retained in the
cytoplasm)
CD5-E1B promoter (U3-LTR) is demethylated in SLE B cells
 Differential methylation is more pronounced with BCR
engagement
 CD5-E1B mRNA is transcribed at the expense of CD5-E1A
mRNA transcription
B cells (of SLE patients) cannot induce DNMT1 due to production
of IL-6
 Cannot methylate DNA
 Similar demethylation patterns of CpG islands when compared to
HCs stimulated with IL-6 or PD98059
73
Therapeutic Treatment?

IL-6 activates CD5-E1B transcription

SLE patients with anti-IL-6R mAb could inhibit
autoreactive B cell expansion by restoring DNA
methylation and cell cycle progression
74