Gabriela M a 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  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: Variable domains Constant domain Fc Diversity Specificity Cellular effector pathways Fc immune complex

IVIG mechanism of action  Neutralization ??? Target: harmful antibodies  Idiotypic Network Theory A B C A e B = IDIOTYPE A e C = ISOTYPE Vaz & Pordeus. Visita à imunologia. Arq. Bras. Cardiol. vol.85 no.5 São Paulo Nov OK

 Antibody Feedback: Cross-linking between BCR and Fc  IIR  B cell blocked Fc mechanism of IVIG action

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)? FcγR FcαR FcεR FcγRI FcγRIIA FcγRIIB1 FcγRIIB2 FcγRIIIA FcγRIIIB FcεRIFcεRII FcαRI Fcα/μR FcRn

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 property  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? Defined defects in specific immune cell populations IVIG 1 hour after Arthritis inducing sera (K/BxN) Clinical score analysis Specific macrophage populations in the splenic marginal zone might be required for the anti- inflammatory effect of the 2,6 sialylated Fc found in IVIG

Results Which receptor expressed in macrophages is required for IVIG protection? Interacting with glycopeptides: Scavenger receptor (MARCO) – bacterias Sialoadhesin receptor (CD169) – sialic acid C-type lectin receptor (SIGN-R1) – polysaccharide dextran Blocking antibodies 1 hour after Arthritis inducing sera (K/BxN) 1 hour after IVIG 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 (2,6 Fc) 1 hour after Arthritis inducing sera (K/BxN) Clinical score analysis The c-type lectin, SIGN-R1, is required for IVIG protection Ankle bones

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? Resident peritoneal m  were harvested 1.C57BL/6 mice 2.Lack all IgG Fc receptors 3.SIGN-R1 -/- 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 CRD - carbohydrate recognition domains Yellow – Identical amino acids Green – Similar amino acids Human DC-SIGN expressed on dendritic cells

Results Did DC-SIGN able to bind to the 2,6-sialyted Fc? CHO cells expressing SIGN-R1, hDC-SIGN or hFc  RIIb Pulsed with 2,6-Fcs Mannan = ligand for DC-SIGN Fibrinogen = similar to Fc linked glycan Human DC-SIGN, binds 2,6-sialylated Fc

Results 2,6-sialylation Fc antiinflammatory response Fc  RIIb FcR binding mSIGN-R1, hDC- SIGN binding 1 hour after Arthritis inducing sera (K/BxN) 1 hour after IVIG C57Bl/6 SIGN-R1 -/- FcγRIIb -/-

Results Did Fc  RIIb involve in the mechanism by which the 2,6-Fc mediates an anti-inflammatory response? The absence of FcRIIb in the recipient prevented the protection afforded by these splenocytes K/BxN

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? hDC-SIGN + /SIGN- R1 -/- WT 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

BMMФ Were hDC-SIGN + macrophages sufficient to induce an anti-inflammatory response? hDC- SIGN + WT 30min sFc or asyaloFc + WT Transfered to WT mice hDC-SIGN + Macrophages treated with sFC showed reduced joint inflammation Challenged with K/BxN Clinical score assessemen t

Is FcγRIIB required to the anti-inflammatory property induced hDC-SIGN + macrophages? hDC-SIGN + - BMMФ hDC- SIGN + 30min sFc or PBS + SIGN-R1 - /- Transfered to FcγRIIB -/- The anti-inflammatory property induced by hDC-SIGN + macrophages depends on FcγRIIB Challenged with K/BxN Clinical score assesseme nt

Was IL-4 responsable for mediating IVIG anti- inflammatory activity? BMMФ hDC- SIGN + 30min sFc or PBS + WT Transfered to IL-4 -/- IL-4 is crucial for mediating IVIG anti-inflammatory activity Challenged with K/BxN Clinical score assessemen t

Could Th2 cytokines supress K/BxN-induced inflammation? WT FcγRIIB -/- Treated with IL- 4, IL-13 or IL-3 K/BxN Clinical score evaluation Inflammation was attenuated after Th2 cytokines administration

Did sFc administration increase Th2 cytokines production? SIGN-R1 - /- WT 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

WT Treated with PBS, IL-33, IL-25 or TSLP K/BxN Clinical score evaluation and analyses of IL-4 levels Can IL-33 induce IL-4 production? IL-33 reverts K/BxN-induced inflammation by increasing IL-4 levels

hDC-SIGN + /SIGN- R1 -/- Treated with sFc or sFc+anti-IL-33Rα K/BxN Clinical score evaluation Does Anti-IL-33Rα ablate the sFc protection? 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? hDC-SIGN + /SIGN- R1 -/- Treated with sFc or sFc+anti-FcεRI K/BxN Clinical score evaluation Basophils contribute for IVIG anti-inflammatory activity

IL-4-GFP mice Treated with PBS or sFc K/BxN Clinical score evaluation and quantification of IL- 4-producing basophils Are basophils the main source of IL-4 production during sFc treatment? Increased IL-4-producing basophils were induced during sFC treatment

Were basophils associated with anti- inflammatory activity induced by sFc? WT or FcγRIIB -/- Basophils (DX5 + FcεRI + c-Kit - ) PBS, IVIG or IL Transfered to WT Challenged with K/BxN IL-33-treated basophils also increased anti-inflammatory activity in a FcγRIIB- dependent manner

CONCLUSION