SUGAR-FREE GLYCOBIOLOGY BASIC SCIENCE?sugar chain structure Unusual Sugar Chain It -- is a carboxylate Make lots of It Make an It antibody Inflammatory proteins?who binds to it? Who binds these inflammatory proteins RAGE INFLAMMATION
Expansion, homing and activation of CD4CD45RB hi naive T cells CD4CD45RB hi (naïve) Mesenteric lymph nodes Priming and clonal expansion Involves interaction with DCs IL12 CD8/86-CD28 CD4-CD154 CD134L-CD134 Acquire gut homing molecules α4β7, CCR9 Recognized by addressin molecules on vascular endothelium of intestine and home to lamina propria Th1 Lamina Propria CD4-CD154 CD8/86-CD28 CD134L-CD134 leukocyte recruitment from blood and Inflammation Proinflammatory Cytokines TNFα IFNγ IL-12, IL-23 NO MIF macrophages Apoptotic pathways
Expansion, homing and activation of CD4CD45RB hi naive T cells CD4CD45RB hi (naïve) Mesenteric lymph nodes Priming and clonal expansion Involves interaction with DCs IL12 CD8/86-CD28 CD4-CD154 CD134L-CD134 Acquire gut homing molecules α4β7, CCR9 Recognized by addressin molecules on vascular endothelium of intestine and home to lamina propria Th1 Lamina Propria CD4-CD154 CD8/86-CD28 CD134L-CD134 leukocyte recruitment from blood and Inflammation Proinflammatory Cytokines TNFα IFNγ IL-12, IL-23 NO MIF macrophages Apoptotic pathways
IL-1 pg/ml 16 12 IL-12 p4 Secretion of cytokines from LPS-activated RAW264.7 murine macrophages 8 4 TNFα ng/1 6 cells IL12p4 pg/ml 16 12 TNFα 8 4 2 16 12 8 4 IL-1 untreated LPS LPS+GB3.1 LPS+control Ab
mabgb3.1 inhibits LPS-induced TNFα and IL23p19 gene expression in murine macrophages.7.6.5.4.3.2.1 TNF α untreated LPS treated LPS + GB3.1 LPS + control Ab.6.5 IL-23p19.4.3.2.1. [-LPS] [+LPS] [+LPS/GB3] [+LPS/cAb]
% of total cells in unactivated cultures 12 1 8 6 4 2 GB3.1 augments cell death of activated macrophages 2 4 6 8 Time after LPS activation (h) -LPS LPS Activated macrophages +LPS +LPS/cAb +LPS/+GB3.1 12 % of total cells in untreated cultures 1 8 6 4 2 Unactivated macrophages 2 4 6 8 Time of treatment (h)
mabgb3.1 inhibits onset of colitis GB3.1 reduces CD4+ T cell accumulation specifically in colon tissues GB3.1 does not induce significant apoptosis of unactivated or activated T-cells GB3.1 inhibited recruitment of monocytes into inflammed areas or caused apoptosis of recruited macrophages No upregulation of MAdCAM expression Proinflammatory cytokine production is reduced Signaling pathways involved?
p65 [LPS activation] p65 [IFN/LPS] NF-kappaB p65.28.24.2.16.12.8.4 [unactivated] [activated] [activated/gb3.1] [activated/cab]
NF-kappaB p65 is enhanced in colon of Crohn s patients Did GB3.1 block NF-κB in treated mice cells? 1.4 NF-kB levels in colonic lamina propria cells cab Tx 1.2 1.8.6 GB3 Tx Normal p65 RelB.4.2 1 2 3 4 5 6 mouse #
WILL GB3.1 REVERSE COLITIS? early Day 9 Day21 Leithhauser et al Lab Invest 81, 1339, 21
Clinical Symptoms Reconstituted, Untreated/cAb treated Loss of weight, loss of hair, soft stools, ill Reconstituted, GB3.1 treated Minimal weight loss, no hair loss, normal stools, healthy
Weight loss curves 12 11 1 AJ944 (control B) AJ3524 (Control A) AJ2527 (GB3.1) 9 8 7 1 2 3 4 5 Days post transfer
mabgb3.1 treatment reverses colitis in the emerging phase of disease inflammation: proximal colon 7 6 5 4 3 2 1 no cell transfer cell transfer/untreated cell transfer/cab treated Treatment cell transfer/gb3.1 treated
mabgb3.1 treatment reverses colitis in the emerging phase of disease inflammation: distal colon 8 7 6 5 4 3 2 1 no cell transfer cell transfer/untreated cell transfer/cab treated Treatment cell transfer/gb3.1 treated
mabgb3.1 treatment reverses colitis in the emerging phase of disease No cell transfer Reconstituted/ untreated Reconstituted/ cab Treated Reconstituted/ mabgb3.1 treated
CONCLUSIONS ANTICARBOXYLATE ANTIBODY GB3.1 BLOCKS THE ONSET OF COLITIS THE ANTIBODY ALSO SEEMS TO REVERSES ESTABLISHED COLITIS EFFECTS ARE PROBABLY MEDIATED BY APOPTOSIS OF ACTIVATED MACROPHAGE GB3.1 MAY BE A POTENTIAL TREATMENT FOR IBD In this collaboration between the Freeze and Kronenberg labs Geetha Srikrishna pioneered these studies and contributed most of the data