Bertus Eksteen

Generation of Gut-Homing IgA-Secreting B Cells by Intestinal Dendritic Cells

Normal intestinal mucosa contains abundant immunoglobulin A (IgA)–secreting cells, which are generated from B cells in gut-associated lymphoid tissues (GALT). We show that dendritic cells (DC) from GALT induce T cell–independent expression of IgA and gut-homing receptors on B cells. GALT-DC–derived retinoic acid (RA) alone conferred gut tropism but could not promote IgA secretion. However, RA potently synergized with GALT-DC–derived interleukin-6 (IL-6) or IL-5 to induce IgA secretion. Consequently, mice deficient in the RA precursor vitamin A lacked IgA-secreting cells in the small intestine. Thus, GALT-DC shape mucosal immunity by modulating B cell migration and effector activity through synergistically acting mediators.

Small intestinal CD103+ dendritic cells display unique functional properties that are conserved between mice and humans

A functionally distinct subset of CD103+ dendritic cells (DCs) has recently been identified in murine mesenteric lymph nodes (MLN) that induces enhanced FoxP3+ T cell differentiation, retinoic acid receptor signaling, and gut-homing receptor (CCR9 and α4β7) expression in responding T cells. We show that this function is specific to small intestinal lamina propria (SI-LP) and MLN CD103+ DCs. CD103+ SI-LP DCs appeared to derive from circulating DC precursors that continually seed the SI-LP. BrdU pulse-chase experiments suggested that most CD103+ DCs do not derive from a CD103− SI-LP DC intermediate. The majority of CD103+ MLN DCs appear to represent a tissue-derived migratory population that plays a central role in presenting orally derived soluble antigen to CD8+ and CD4+ T cells. In contrast, most CD103− MLN DCs appear to derive from blood precursors, and these cells could proliferate within the MLN and present systemic soluble antigen. Critically, CD103+ DCs with similar phenotype and functional properties were present in human MLN, and their selective ability to induce CCR9 was maintained by CD103+ MLN DCs isolated from SB Crohns patients. Thus, small intestinal CD103+ DCs represent a potential novel target for regulating human intestinal inflammatory responses.

Hepatic Endothelial CCL25 Mediates the Recruitment of CCR9+ Gut-homing Lymphocytes to the Liver in Primary Sclerosing Cholangitis

Primary sclerosing cholangitis (PSC), a chronic inflammatory liver disease characterized by progressive bile duct destruction, develops as an extra-intestinal complication of inflammatory bowel disease (IBD) (Chapman, R.W. 1991. Gut. 32:1433–1435). However, the liver and bowel inflammation are rarely concomitant, and PSC can develop in patients whose colons have been removed previously. We hypothesized that PSC is mediated by long-lived memory T cells originally activated in the gut, but able to mediate extra-intestinal inflammation in the absence of active IBD (Grant, A.J., P.F. Lalor, M. Salmi, S. Jalkanen, and D.H. Adams. 2002. Lancet. 359:150–157). In support of this, we show that liver-infiltrating lymphocytes in PSC include mucosal T cells recruited to the liver by aberrant expression of the gut-specific chemokine CCL25 that activates α4β7 binding to mucosal addressin cell adhesion molecule 1 on the hepatic endothelium. This is the first demonstration in humans that T cells activated in the gut can be recruited to an extra-intestinal site of disease and provides a paradigm to explain the pathogenesis of extra-intestinal complications of IBD.

Aberrant homing of mucosal T cells and extra-intestinal manifestations of inflammatory bowel disease.

Active inflammatory bowel disease (IBD) is often associated with simultaneous inflammation in the skin, eyes and joints. Inflammatory disease in the liver can also occur in patients with IBD but seems to be independent of inflammation in the bowel. In this Opinion article, we propose that the hepatic complications of IBD are mediated by long-lived mucosal T cells that are recruited to the liver in response to aberrantly expressed endothelial-cell adhesion molecules and chemokines that are normally restricted to the gut. Similar mechanisms might explain why certain diseases are associated with site-specific tissue distributions and might point to new therapeutic strategies that are based on modulating tissue-specific lymphocyte homing.

Distinct Roles for CCR4 and CXCR3 in the Recruitment and Positioning of Regulatory T Cells in the Inflamed Human Liver

Regulatory T cells (Tregs) are found at sites of chronic inflammation where they mediate bystander and Ag-specific suppression of local immune responses. However, little is known about the molecular control of Treg recruitment into inflamed human tissues. We report that up to 18% of T cells in areas of inflammation in human liver disease are forkhead family transcriptional regulator box P3 (FoxP3)+ Tregs. We isolated CD4+CD25+CD127lowFoxP3+ Tregs from chronically inflamed human liver removed at transplantation; compared with blood-derived Tregs, liver-derived Tregs express high levels of the chemokine receptors CXCR3 and CCR4. In flow-based adhesion assays using human hepatic sinusoidal endothelium, Tregs used CXCR3 and α4β1 to bind and transmigrate, whereas CCR4 played no role. The CCR4 ligands CCL17 and CCL22 were absent from healthy liver, but they were detected in chronically inflamed liver where their expression was restricted to dendritic cells (DCs) within inflammatory infiltrates. These DCs were closely associated with CD8 T cells and CCR4+ Tregs in the parenchyma and septal areas. Ex vivo, liver-derived Tregs migrated to CCR4 ligands secreted by intrahepatic DCs. We propose that CXCR3 mediates the recruitment of Tregs via hepatic sinusoidal endothelium and that CCR4 ligands secreted by DCs recruit Tregs to sites of inflammation in patients with chronic hepatitis. Thus, different chemokine receptors play distinct roles in the recruitment and positioning of Tregs at sites of hepatitis in chronic liver disease.

Epithelial Inflammation Is Associated with CCL28 Production and the Recruitment of Regulatory T Cells Expressing CCR10

Mucosal tissues require constant immune surveillance to clear harmful pathogens while maintaining tolerance to self Ags. Regulatory T cells (Tregs) play a central role in this process and expression of αEβ7 has been reported to define a subset of Tregs with tropism for inflamed tissues. However, the signals responsible for recruiting Tregs to epithelial surfaces are poorly understood. We have isolated a subset of CCR10-expressing CD25+CD4+Foxp3+ Tregs with potent anti-inflammatory properties from chronically inflamed human liver. The CCR10+ Tregs were detected around bile ducts that expressed increased levels of the CCR10 ligand CCL28. CCL28 was secreted by primary human cholangiocytes in vitro in response to LPS, IL-1β, or bile acids. Exposure of CCR10+ Tregs to CCL28 in vitro stimulated migration and adhesion to mucosal addressin cell adhesion molecule-1 and VCAM-1. Liver-derived CCR10+ Tregs expressed low levels of CCR7 but high levels of CXCR3, a chemokine receptor associated with infiltration into inflamed tissue and contained a subset of αEβ7+ cells. We propose that CXCR3 promotes the recruitment of Tregs to inflamed tissues and CCR10 allows them to respond to CCL28 secreted by epithelial cells resulting in the accumulation of CCR10+ Tregs at mucosal surfaces.

Gut Homing Receptors on CD8 T Cells Are Retinoic Acid Dependent and Not Maintained by Liver Dendritic or Stellate Cells

BACKGROUND & AIMS Lymphocytes primed by intestinal dendritic cells (DC) express the gut-homing receptors CCR9 and alpha4beta7, which recognize CCL25 and mucosal addressin cell-adhesion molecule-1 in the intestine promoting the development of regional immunity. In mice, imprinting of CCR9 and alpha4beta7 is dependent on retinoic acid during T-cell activation. Tissue specificity is lost in primary sclerosing cholangitis (PSC), an extraintestinal manifestation of inflammatory bowel disease, when ectopic expression of mucosal addressin cell-adhesion molecule-1 and CCL25 in the liver promotes recruitment of CCR9+alpha4beta7+ T cells to the liver. We investigated the processes that control enterohepatic T-cell migration and whether the ability to imprint CCR9 and alpha4beta7 is restricted to intestinal DCs or can under some circumstances be acquired by hepatic DCs in diseases such as PSC. METHODS Human and murine DCs from gut, liver, or portal lymph nodes and hepatic stellate cells were used to activate CD8 T cells. Imprinting of CCR9 and alpha4beta7 and functional migration responses were determined. Crossover activation protocols assessed plasticity of gut homing. RESULTS Activation by gut DCs imprinted high levels of functional CCR9 and alpha4beta7 on naïve CD8 T cells, whereas hepatic DCs and stellate cells proved inferior. Imprinting was RA dependent and demonstrated plasticity. CONCLUSIONS Imprinting and plasticity of gut-homing human CD8 T cells requires primary activation or reactivation by gut DCs and is retinoic acid dependent. The inability of liver DCs to imprint gut tropism implies that alpha4beta7+CCR9+ T cell that infiltrate the liver in PSC are primed in the gut.

CXCR3 Activation Promotes Lymphocyte Transendothelial Migration across Human Hepatic Endothelium under Fluid Flow

T cells infiltrating the inflamed liver express high levels of CXCR 3 and show enhanced migration to CXCR 3 ligands in chemotactic assays. Moreover, CXCR 3 ligands are up-regulated on hepatic endothelium at sites of T-cell infiltration in chronic hepatitis, and their presence correlates with outcome of inflammatory liver disease. We used a flow-based adhesion assay with human hepatic endothelium to investigate the function of CXCR 3 on lymphocyte adhesion to and transmigration through hepatic endothelium under physiological conditions of blood flow. To more accurately model the function of in vivo activated CXCR 3(high) lymphocytes, we isolated T cells from human liver tissue and studied their behavior in flow-based adhesion assays. We demonstrate that CXCR 3 not only promoted the adhesion of effector T cells to endothelium from flow but also drove transendothelial migration. Moreover, these responses could be stimulated either by endogenous CXCR 3 ligands secreted by the endothelium or by exogenous CXCR 3 ligands derived from other cell types and presented by the endothelium. This study thus demonstrates that activation of CXCR 3 promotes lymphocyte adhesion and transendothelial migration under flow and that human hepatic endothelium can present functionally active chemokines secreted by other cell types within the liver.

CXCR3-dependent recruitment and CCR6-mediated positioning of Th-17 cells in the inflamed liver

Background & Aims IL-17 secreting CD4 (Th17) and CD8 (Tc17) T cells have been implicated in immune-mediated liver diseases, but the molecular basis for their recruitment and positioning within the liver is unknown. Methods The phenotype and migratory behaviour of human liver-derived Th17 and Tc17 cells were investigated by flow cytometry and chemotaxis and flow-based adhesion assays. The recruitment of murine Th17 cells to the liver was studied in vivo using intra-vital microscopy. Results IL-17+ T cells comprised 1–3% of the T cell infiltrate in inflammatory liver diseases and included both CD4 (Th17) and CD8 (Tc17) cells. They expressed RORC and the IL-23 receptor and included subsets that secreted IL-22 and interferon-γ. Th17 and Tc17 cells expressed high levels of CXCR3 and CCR6, Tc17 cells also expressed CXCR6. Binding to human sinusoidal endothelium from flow was dependent on β1 and β2 integrins, CXCR3, and, in the case of Th17 cells, VAP-1. Th17 recruitment via sinusoids in mice with liver inflammation was reduced by treatment with antibodies against CXCR3 ligands, confirming the role of CXCR3 in Th17 recruitment in vivo. In human liver, IL-17+ cells were detected in portal infiltrates close to inflamed bile ducts expressing the CCR6 ligand CCL20. Cytokine-treated human cholangiocytes secreted CCL20 and induced CCR6-dependent migration of Th17 cells suggesting that local cholangiocyte chemokine secretion localises Th17 cells to bile ducts. Conclusions CXCR3 promotes recruitment of Th17 cells from the blood into the liver in both human and murine liver injury. Their subsequent positioning near bile ducts is dependent on cholangiocyte-secreted CCL20.

Immunology of the gut and liver: a love/hate relationship

The gut digests, absorbs and metabolises nutrients whilst also acting as a barrier to pathogens entering from the intestinal lumen. Not all intestinal microbes are harmful and the full development of the gut immune system is shaped by interactions with commensal bacteria in a complex interplay which allows responses to harmless bacteria and nutrients to be suppressed whilst developing protective immune response against pathogens(1;2). Most of these responses occur in the intestinal mucosa and underlying lamina propria however transport of nutrients or translocation of pathogens into the portal circulation means that a second level of protection is required in the liver to respond to antigens that evade the gut immune system(3). In addition pathogens can enter the liver directly via the systemic circulation or from the gut lumen via the biliary epithelium. Immune responses in the gut and to a lesser extent liver are well-described but the mechanisms that control immunological cross-talk between these two linked sites are only beginning to be elucidated(4). They provide insights into the pathogenesis of diseases that affect both sites including infections and inflammatory bowel disease (IBD).