Cheng T. Tee

Skin‐ and gut‐homing molecules on human circulating γδ T cells and their dysregulation in inflammatory bowel disease

Changes in phenotype and function of γδ T cells have been reported in inflammatory bowel disease (IBD), including Crohns disease (CD) and ulcerative colitis (UC). Dysregulation of lymphocyte migration plays a key role in IBD pathogenesis; however, data on migratory properties of γδ T cells are scarce. Human circulating γδ T cells from healthy controls (n = 27), patients with active CD (n = 15), active UC (n = 14) or cutaneous manifestations of IBD (n = 2) were characterized by flow cytometry. Circulating γδ T cells in healthy controls were CD3hi and expressed CD45RO. They expressed gut‐homing molecule β7 but not gut‐homing molecule corresponding chemokine receptors (CCR)9, or skin‐homing molecules cutaneous lymphocyte‐associated antigen (CLA) and CCR4, despite conventional T cells containing populations expressing these molecules. CCR9 expression was increased on γδ T cells in CD and UC, while skin‐homing CLA was expressed aberrantly on γδ T cells in patients with cutaneous manifestations of IBD. Lower levels of CD3 expression were found on γδ T cells in CD but not in UC, and a lower proportion of γδ T cells expressed CD45RO in CD and UC. Enhanced expression of gut‐homing molecules on circulating γδ T cells in IBD and skin‐homing molecules in cutaneous manifestations of IBD may be of clinical relevance.

T-cell proliferation and forkhead box P3 expression in human T cells are dependent on T-cell density: physics of a confined space?

T-cell proliferation rates in vitro depend on factors including initial T-cell number, dose of stimulus, culture time, and available physical space. The role of forkhead box P3 (FoxP3) in the identification of T cells with a regulatory phenotype remains controversial in humans. Through 5-carboxyfluorescein diacetate succinimidyl ester labeling of human T cells and subsequent culture of different numbers of T cells and antigen-presenting cells (APC), we studied proliferative T-cell responses and FoxP3 expression in divided T cells. T-cell proliferation rates depended on initial T-cell/APC numbers. Proliferation rates decreased when high initial T-cell numbers were increased. FoxP3 expression was expressed exclusively in virtually all divided T cells cultured at high T-cell densities, irrespective of their CD4 nature or cytokine content, and was coexpressed with T-bet. However, when T cells were cultured on larger surfaces or at lower initial numbers, FoxP3 expression was not induced in divided T cells, even when most of the cells had undergone cell division. FoxP3(+) T cells generated at high cell densities did not elicit a suppressive phenotype and FoxP3 expression was subsequently lost in time when the stimulus was removed. Therefore, caution should be observed in the use of FoxP3 expression to identify regulatory T cells in humans because its expression may be only a consequence of activation status in a restricted environment.

Dysregulated Circulating Dendritic Cell Function in Ulcerative Colitis Is Partially Restored by Probiotic Strain Lactobacillus casei Shirota

Background. Dendritic cells regulate immune responses to microbial products and play a key role in ulcerative colitis (UC) pathology. We determined the immunomodulatory effects of probiotic strain Lactobacillus casei Shirota (LcS) on human DC from healthy controls and active UC patients. Methods. Human blood DC from healthy controls (control-DC) and UC patients (UC-DC) were conditioned with heat-killed LcS and used to stimulate allogeneic T cells in a 5-day mixed leucocyte reaction. Results. UC-DC displayed a reduced stimulatory capacity for T cells (P < 0.05) and enhanced expression of skin-homing markers CLA and CCR4 on stimulated T cells (P < 0.05) that were negative for gut-homing marker β7. LcS treatment restored the stimulatory capacity of UC-DC, reflecting that of control-DC. LcS treatment conditioned control-DC to induce CLA on T cells in conjunction with β7, generating a multihoming profile, but had no effects on UC-DC. Finally, LcS treatment enhanced DC ability to induce TGFβ production by T cells in controls but not UC patients. Conclusions. We demonstrate a systemic, dysregulated DC function in UC that may account for the propensity of UC patients to develop cutaneous manifestations. LcS has multifunctional immunoregulatory activities depending on the inflammatory state; therapeutic effects reported in UC may be due to promotion of homeostasis.

PMO-231 Ileal and colonic mucosal dendritic cell cytokine profiles differ at rest and after in vitro bacteria and pro-biotic challenge in postoperative Crohn's disease patients

Introduction Postoperative Crohns disease (CD) recurrence predominantly affects the ileal mucosa at the ileo-colonic anastomosis with the colonic side often spared. Altered immune responses to bacterial flora are thought to be a driving force in the pathogenesis of CD recurrence. Gut dendritic cells (DC) are key in the initiation of immune response, through cytokine production, when stimulated with bacterial antigens. We postulate that differences between ileal and colonic DC resting characteristics and functional responses may be responsible for the propensity of recurrence to occur at the ileal aspect of the anastomosis. We aimed to assess ongoing intracellular cytokine production in DC from ileal and colonic postoperative CD mucosa and assess their functional response to bacterial stimulation and modulation with probiotics. Methods Paired ileal and colonic biopsies were taken from postoperative CD patients at colonoscopy (n=11). Lamina propria mononuclear cells were collected after collagenase digestion. DC intracellular cytokine responses (IL-2, IL-6, IL-17a, TGFβ and INFγ) were assessed in basal conditions and after culture with LPS and two probiotic bacterial strains Bifidobacterium Longum; Lactobacillus Casei (B longum and L casei) using multi-colour flow cytometry. Results Unstimulated ileal DC showed higher levels of ongoing intracellular production of pro-inflammatory cytokines than unstimulated colonic DC: IL6 (34.21±12.80 vs 10.47±3.574 cells/υl [mean±SEM], p=0.037), IL17a (24.62±12.38 vs 14.94±9.865 cells/υl, p=0.05, n=5) and TGFβ (74.12±17.96 vs 32±16.27 cells/υl, p=0.031). Incubation with LPS resulted in higher DC intracellular cytokine levels of INFγ in ileal derived DC with a borderline p value (27.49±12.61 vs 0.39±0.391 cells/υl p=0.06) but not colonic derived DCs (19.55±10.12 vs 12.40±7.039, p=0.6). L casei incubation, however, led to a larger decrease in ongoing TGFβ (−42.35±16.02 vs 4.42±11.46 cells/υl p=0.023) and INFγ (−14.76 ±7.196 vs 20.33±10.16 cells/υl-, p=0.05) DC cytokine production in colonic tissue compared with ileal. Conclusion Ileal mucosa DC demonstrate a cytokine profile implicating a Th17 response compared with colonic mucosa. Upon bacterial stimulation with LPS ileal mucosa demonstrate increased INFγ DC production compared with unstimulated DC. These results suggest a role of for a Th1/Th17 response in driving post-operative CD recurrence. The probiotics L casei and B longum failed to show significant effects in modulation of intracellular cytokine production in ileal DC. Competing interests None declared.

Unravelling the immunomodulatory functions of glucagon like peptide-2 through dendritic cells

Introduction Animal studies have shown that glucagon like peptide-2 (GLP-2) may reduce mucosal inflammation; it decreases proinflammatory cytokines and ameliorates chronic colitis1. However, we do not yet know whether this anti-inflammatory effect occurs in humans. If so, it potentially opens the door for use of GLP-2 as therapy in conditions like inflammatory bowel disease. Therefore we studied the immunomodulatory functions of GLP-2 in humans. Methods Dendritic cells (DC) enriched from human blood of healthy volunteers were cultured in-vitro for 24 h with GLP-2 at concentrations of 1 pM, 1 nM and 1 mM. The effect of GLP-2 on DC survival was determined using apoptosis experiments. Phenotype and functions of DC were then assessed by flow cytometry and mixed leucocyte reaction (MLR), respectively. Each experiment was performed independently at least 3 times and analysed for statistically significant effects. Results Apoptosis experiments showed that GLP-2 at all concentrations did not have a toxic effect on DC; their survival after in-vitro culture with GLP-2 was similar to that in basal control culture (p=NS). GLP-2 conditioning changed the phenotype of DC with reduction in HLA-DR intensity (p=0.0243) and increase in CD14 expression (p=0.0237), compared with basal control culture. However, the down-regulation of HLA-DR intensity and up-regulation of CD14 expression did not correlate with an increase in the phagocytic capacity (p=NS). Other markers of immature DC, ILT3 and DC SIGN, were not affected. TLR2/4 expression was also not affected by the treatment (p=NS). Finally, MLR experiments showed that GLP-2 treatment on DC did not have an effect on their stimulation of T cell proliferation (p=NS). Conclusion GLP-2 reduced HLA-DR and increased CD14 on DCs, though this effect does not correlate with T-cell stimulation in-vitro. More studies are needed to detect any functional significance to the changes GLP-2 induced on dendritic cells.

TH1/TH17 profiles in crohn's disease: a cross sectional single centre study in postoperative crohn's disease

Introduction Th1 and Th17 pathways are implicated in Crohn9s disease (CD). In operative resection samples healthy ileum shows high TGFβ levels in patients who develop recurrence, with TGFβ being a known activator of the Th17 response. Other studies in CD show a dominant Th1 cytokine profile, with high levels of IFNγ, which reduce Th17 response and augment Th1 response. The relationship of Th1/Th17 cytokine profiles in postoperative CD has not been examined. The authors aimed to study tissue Th1/Th17 cytokine secretion after in vitro biopsy culture in postoperative CD. Methods Colonoscopy was undertaken in postoperative CD patients. Recurrence graded as no/minimal inflammation (Rutgeert Score (RS) ≥1) or progressive inflammation (RS≥2). Ileal biopsies were cultured overnight and cell free supernatants obtained. Supernatant cytokines (IL-2, IL-4, IL-10, IL-17 TNFα, INFg and IL-6) were assessed by flow cytometry using cytometric bead array (Becton Dickinson). Statistical analysis was via unpaired t tests. Results Consecutive patients attending endoscopy (n=24, 9M/15F) were identified. Mean age 45.0 years and time from I to C resection was 5.8 years; 5 patients were smokers. Drugs were thiopurines 13, Infliximab 1 and nil 10. Endoscopic severity was i0 n=5, i1 n=6, i2 n=5, i3 n=3, i4 n=5. Mean cytokine concentrations from supernatants are shown in the table 1. Comparison between RS≥1 and ≥2 showed that pro-inflammatory cytokines IL-17a (p Conclusion Cytokine profiles in those with RS≥2, show higher levels of IL-17a and IFNγ and reduced IL-10 compared to RS≥1. This profile supports a Th17 and Th1 mediated response as one of the early instigators of endoscopic progression in postoperative CD. The authors9 observation is consistent with recent findings of a T cell subset able to produce cytokines involved in both Th1 and Th17 responses. Previous therapies directed at Th1 pathway, for example, anti-IL-12p40 antibody ustekinumab and anti-IFNγ Fontolizumab failed to show significant clinical benefit in CD. Given our findings targeting the Th17 response, for example, with anti-IL-23 antibodies and anti-IL-17 may deliver improved therapeutic outcome.

Increase in Dendritic Cell Migration Markers CCR7 and CCR9 in the Neo-Terminal Ileum of Postoperative Crohn's Disease: An Adaptive Response to Bacterial Exposure?

Introduction Gut dendritic cells (DCs) are crucial in bacterial recognition, T cell signalling and inflammatory regulation. DC TLR expression is altered in CD: increased on myeloid DC (MDC) in colonic CD and reduced on plasmacytoid DCs (PDC) in postoperative CD (POCD) ileum. In active CD, peripheral CD4 and CD8 T cells showed increased intestinal homing with high CCR9 levels. In Crohn9s colonic tissues, CCR7, a homing marker crucial for DC trafficking to mesenteric lymph nodes, was elevated compared with controls. Additionally CCR7 expression on MDC is higher in ileal compared with colonic tissue in CD. CCR7 and CCR9 expression on DC in POCD is unknown. After ileo-caecal resection the neo-terminal ileum is exposed to the bacteria rich contents of the colon. The authors hypothesise that alteration in gut microflora after surgery may modulate expression of homing markers on DC from POCD patients. The authors aimed to examine homing marker expression on MDC and PDC from the ileum and the colon in healthy controls (HC) and POCD patients. Methods HC and POCD patients were identified at colonoscopy. Intestinal lamina propria mononuclear cells were collected using collagenase digestion and labelled with directly conjugated monoclonal antibodies to CCR7 and 9. PDC and MDC were characterised as CD11c+ve and –ve respectively and expression of CCR7 and 9 by multicolour flow cytometry measured. Statistical analysis was via unpaired t tests. In experiments with paired colonic and ileal samples paired t tests were performed. Results In paired samples, HC ileal CCR9+ve PDC concentrations were lower than colonic PDC (26.46±10.43/ml SEM vs 53.76±20.16/ml SEM, p There were significantly higher concentrations of CCR9+ve and CCR7+ve PDCs within ileal POCD compared with ileum normal controls (103.8±22.64/ml vs 37.68±7.434/ml, p=0.039 and 117.4±26.97/ml SEM vs 40.47±3.97/ml SEM, p=0.03). No differences in MDC concentrations of both homing markers in all types of tissue existed. Conclusion POCD neo-terminal ileum showed higher CCR7+ve and CCR9+ve PDC than normal ileum. This novel finding indicates a potential role for PDC in CD pathogenesis. The loss of the ileocaecal valve in POCD may alter microbiota flora exposure in the ileum with an adaptive response of CCR9+ve DC to a level seen in normal colonic tissue. Additionally, this may induce migration of PDC by upregulation of CCR7 expression. Further studies to examine the changes with disease progression may unravel the function of PDC in ileal POCD tissues.

W1816 Loss of TLR 2 and 4 on Ileal Plasmacytoid Dendritic Cells in Post-Operative Crohn's Disease Patients

Introduction Aberrant responses to bacterial flora are implicated in post-operative recurrence of Crohn9s disease (CD) after ileo-ceacal resection. Response to gut flora is conducted by dendritic cells (DC) which can induce a tolerogenic or active immune response. DC express Toll like receptors (TLR) which recognise bacterial products and are important in immune tolerance in health and highly expressed in IBD colonic tissue. Two subsets of DC are plasmacytoid (PDC) and myeloid cells (MDC). These DC show functional differences in their response to bacteria and are able to cross-talk. Expression of TLR on DC in the small intestine of post-operative CD patients is still largely unknown. We aimed to assess expression of TLR on DC from lamina propria mononuclear cells (LPMC) in post-operative CD patients as compared with normal controls. Methods Small intestine biopsies were obtained from 18 post-operative CD patients and 5 controls by colonoscopy. LPMC were collected using collagenase digestion, labelled with monoclonal antibodies to TLR2 and 4 and assessed by multicolour flow cytometry. Result A high percentage of DC from normal ileum expressed TLR2 and 4 with no significant differences in expression between PDC and MDC. However, expression of TLR 2 and 4 was significantly lower in PDC from post op CD patients compared with PDC in normal controls (mean 22.30% vs 60%, p=0.0003 and 38.55% vs 65.16%, p=0.02), respectively. Furthermore, PDC and MDC differed in expression of TLR2 and 4 in tissues from post-op CD patients; PDC expressed significantly less TLR2 and 4 compared with MDC (22.30% vs 38.11%, p=0.002 and 38.55% vs 50.89%, p=0.01), respectively. Conclusion Reduction of TLR2 and 4 expression in PDC compared with their counterparts in normal controls is likely to be the reason for the reduced expression of both TLRs on PDC compared with MDC in CD tissues. The lack of TLR2 and 4 expression on PDC maybe related to the disease activity as the normal controls did not show any statistical differences in expression of either TLR between the DC subsets. Other groups have suggested that functional differences exist between PDC and MDC in their activation by bacterial products and ability to produce innate and acquired inflammatory responses. In particular, blood PDC require the presence of MDC to respond to bacterial products to activate T cells rather than driving innate immunity. Our results indicate that PDC response to intestinal microflora may differ from that in MDC and provide initial background to the DC function and diversity in post-operative CD recurrence.