Transcript 2,6-sialylated Fc

Gabriela
Ma Claudia
Tiago
 IVIG is a blood product administered intravenously
 It contains the pooled IgG extracted from the plasma of over one thousand blood
donors
 Immunoglobulin products from human plasma were first used in 1952 to treat immune
deficiency. It was initially shown to be effective in ITP in 1981.
 Treatment for: Immune deficiencies (plasma protein replacement therapy)
Autoimmune Diseases (anti-inflammatory at a high dose ≈1-2 g/Kg)
Acute Infections
 IVIG cost is climbing and well over $50/g. ($8,000 for a 80 kg person at 2g/kg)
 IVIG's effects last between 2 weeks and 3 months
 primary immune dysfunction: 100 to 400 mg/kg of body weight every 3 to 4 weeks.
 autoimmune diseases: 2 grams per kilogram of body weight for three to six months over a five day
course once a month. Then maintenance therapy of 100 to 400 mg/kg of body weight every 3 to 4
weeks follows.
Rational basis
 IVIG: pool of IgG
Immunoglobulins: effector molecules of immune defense
Igs properties:
immune complex
Variable domains
Constant domain
Fc
Fc
Diversity
Specificity
Cellular
effector
pathways
IVIG mechanism of action
 Neutralization ??? OK
Target: harmful antibodies
 Idiotypic Network Theory
AeB=
IDIOTYPE
A e C = ISOTYPE
A
B
C
Vaz & Pordeus. Visita à imunologia. Arq. Bras. Cardiol. vol.85 no.5 São
Fc mechanism of IVIG action
 Antibody Feedback: Cross-linking between BCR and FcIIR  B cell blocked
IVIG mechanism of action
The precise mechanism by which IVIG suppresses harmful inflammation has not been
definitively established
BUT….
is believed to involve the Fc receptor…
How? Which one(s)?
FcαR
FcαRI
FcεR
FcεRI
FcγR
Fcα/μR
FcεRII
FcγRI
FcγRIIIA
FcγRIIA
FcγRIIB1
FcγRIIB2
FcRn
FcγRIIIB
IVIG mechanism action
 Fc Receptors (FcyR)
a protein found on the surface of NK cells, macrophages, neutrophils, mast
cells and others
FcγRs are the most important Fc receptors for inducing phagocytosis of
opsonized microbes
Glycoforms of IgG (Asn297)
Carbohydrate whit terminal sugar residues such as galactose, sialic acid,
N-acetylglucosamine, and fucose
 more than 30 different antibody glycovariants have been detected in human
serum, with about 25%–30% of them in the IgG glycoform.
 Thus, these variants, multiplied by the four different IgG subclasses, result in
more than 120 different glycoproteins in the IVIG preparation that could contain
the active anti-inflammatory component
 IVIG has anti-inflammatory effect at a high dose ≈1-2 g/Kg
120 different glycoproteins in the IVIG preparation
 terminal sugar residues of sialic acid confers anti-inflammatory p
 1-3% of IgGs in IVIG have sFc (sialylation)
 recombinant sFc: enhanced 35 fold of action in vivo
Carbohydrate
Carbohydrate-Binding Proteins
C-Type Lectins
Siglecs
Galectins
CD1
DC-SIGN is a C-type lectin receptor
 binds to mannose type carbohydrates.
Phagocytosis
Cell rolling interactions (ICAM) and activation of CD4+ T cells
• Binds sFc  anti-inflammatory responses
- Population of regulatory macrophage
- Splenic Marginal Zone
Maria Claudia
Tiago
Objective:
• To define the mechanism by which the 2,6-sialylated Fc mediates an anti- inflammatory
response
• To identify the properties of the regulatory macrophage population
• To identify the receptor required for initiating this pathway in response to 2,6-sialylated Fc.
Results
Are the splenic marginal zone macrophage necessary for IVIG-mediated immune
suppression?
1 hour after
IVIG
Arthritis inducing sera
(K/BxN)
Clinical score
analysis
Defined defects in specific
immune cell populations
Specific macrophage populations in the splenic marginal zone might be required for the antiinflammatory effect of the 2,6 sialylated Fc found in IVIG
Results
Which receptor expressed in macrophages is required for IVIG protection?
1 hour after
IVIG
Blocking
antibodies
Interacting with glycopeptides:
Scavenger receptor (MARCO) – bacterias
Sialoadhesin receptor (CD169) – sialic acid
C-type lectin receptor (SIGN-R1) – polysaccharide dextran
1 hour after
Arthritis inducing sera
(K/BxN)
TKO-SIGNR1 - antibody that results in the transient
down-regulation of SIGN-R1 expression
Results
Which receptor expressed in macrophages is required for IVIG protection?
C57BL/6 and
SIGN-R1-/-
IVIG 1 hour after
(2,6 Fc)
Clinical score
analysis
Arthritis inducing sera
(K/BxN)
Ankle bones
The c-type lectin, SIGN-R1, is required for IVIG protection
Results
Did SIGN-R1 able to bind to the 2,6-sialyted Fc?
Transfected
macrophage
(RAW-SIGN-R1)
Pulsed with
flourochrome-labeded
2,6-Fcs (red)
SIGN-R1 binds 2,6-sialylated Fc.
Results
Did SIGN-R1 able to bind to the 2,6-sialyted Fc and asialylated Fcs?
1.
2.
3.
C57BL/6 mice
Lack all IgG Fc receptors
SIGN-R1-/-
Resident peritoneal
m were harvested
Pulsed with 2,6-Fcs
or asialylated Fcs
The amount of
bound Fcs were
determined
The 2,6-sialylation of the IgG Fc converts the molecule to a species that acquires the ability
to engage a mSIGN-R1 and mediate an antiinflammatory response.
Results
Human DC-SIGN expressed on dendritic cells
Yellow – Identical amino acids
Green – Similar amino acids
CRD - carbohydrate recognition domains
Results
Did DC-SIGN able to bind to the 2,6-sialyted Fc?
CHO cells expressing
SIGN-R1, hDC-SIGN or
hFcRIIb
Mannan = ligand for DC-SIGN
Fibrinogen = similar to Fc linked glycanHuman
Pulsed with
2,6-Fcs
DC-SIGN, binds 2,6-sialylated Fc
Results
2,6-sialylation Fc
1 hour after
C57Bl/6
SIGN-R1-/FcγRIIb-/-
FcR
binding
mSIGN-R1, hDCSIGN binding
antiinflammatory
response
FcRIIb
IVIG
1 hour after
Arthritis inducing sera
(K/BxN)
Results
Did FcRIIb involve in the mechanism by which the 2,6-Fc mediates an
anti-inflammatory response?
K/BxN
The absence of FcRIIb in the recipient prevented the protection afforded by these splenocytes
Conclusion
Objective: To study hDC-SIGN in the context of IVIG
anti-inflammatory activity in expressing-hDC-SIGN
mice.
Could hDC-SIGN mediate anti-inflammatory
protection by IVIG?
WT
SIGN-R1- hDC-SIGN+/SIGN/R1-/-
Treated with
sFc
Challenged
with
arthritogenic
K/BxN serum
Clinical score
assessement
hDC-SIGN substitutes for SIGN-R1 in mediating IVIG
anti-inflammatory protection
Were hDC-SIGN+ macrophages sufficient to
induce an anti-inflammatory response?
WT
hDCSIGN+
BMMФ
+
sFc or
asyaloFc
30min
Transfered
to WT mice
WT
Challenged
with K/BxN
Clinical
score
assessemen
t
hDC-SIGN+ Macrophages treated with sFC showed reduced
joint inflammation
Is FcγRIIB required to the anti-inflammatory property
induced hDC-SIGN+ macrophages?
hDC-SIGN+BMMФ
+
hDCSIGN+
30min
sFc or
PBS
Transfered
to
SIGN-R1/-
FcγRIIB-/-
Challenged
with K/BxN
Clinical
score
assesseme
nt
The anti-inflammatory property induced by hDC-SIGN+
macrophages depends on FcγRIIB
Was IL-4 responsable for mediating IVIG antiinflammatory activity?
hDCSIGN+
BMMФ
+
30min
sFc or
PBS
Transfered
to
WT
IL-4-/-
Challenged
with K/BxN
Clinical
score
assessemen
t
IL-4 is crucial for mediating IVIG anti-inflammatory
Could Th2 cytokines supress K/BxN-induced
inflammation?
Treated with IL4, IL-13 or IL-3
FcγRIIB-/-
WT
K/BxN
Clinical score
evaluation
Inflammation was attenuated after Th2 cytokines
administration
Did sFc administration increase Th2 cytokines
production?
WT
SIGN-R1/-
Treated with
sFc (1h)
Splenic cells
were
removed
Quantification of IL-4,
IL-33 and IL-25
mRNA expression
(qPCR)
IL-33 mRNA was upregulated in WT mice after sFc
administration
Can IL-33 induce IL-4 production?
Treated with
PBS, IL-33, IL-25
or TSLP
WT
K/BxN
Clinical score
evaluation and
analyses of IL-4 levels
IL-33 reverts K/BxN-induced inflammation by
increasing IL-4 levels
Does Anti-IL-33Rα ablate the sFc
protection?
Treated with sFc or
sFc+anti-IL-33Rα
hDC-SIGN+/SIGNR1-/-
K/BxN
Clinical score
evaluation
The IL-33Rα blocking increases joint inflammation
Did IL-33 and IL-4 increase FcγRIIB expression
on monocytes?
hDC-SIGN+Monocytes
(CD11b+Ly6G+)
+
PBS or IL-4 or
IL-33 or IL-25
24h
FcγRIIB
expression by
FACS
FcγRIIB expression on monocytes was increased
after IL-33 and IL-4 treatment
Are basophils involved with reduced joint
inflammation?
Treated with sFc or
sFc+anti-FcεRI
hDC-SIGN+/SIGNR1-/-
K/BxN
Clinical score
evaluation
Basophils contribute for IVIG anti-inflammatory
activity
Are basophils the main source of IL-4
production during sFc treatment?
Treated with
PBS or sFc
IL-4-GFP
mice
K/BxN
Clinical score
evaluation
and quantification of IL4-producing basophils
Increased IL-4-producing basophils were induced
during sFC treatment
Were basophils associated with antiinflammatory activity induced by sFc?
WT or FcγRIIB-/Basophils
+
(DX5+FcεRI
+ c-Kit )
PBS, IVIG or IL33
Transfered to
WT
Challenged
with K/BxN
IL-33-treated basophils also increased anti-inflammatory activity in a FcγRIIBdependent manner
CONCLUSION