Introduction Results Conclusions Materials and ...

Absence of LPS in the lamina propria of UC identifies patients in remission who share immunological and structural features with active disease and may be at risk of relapse Louise Greathead1, Robert Goldin2, Peter Kelleher1,Alan Steel3 & Vaccinology, Imperial College London, 2Department of Cellular Pathology, Imperial College London 3Gastroenterology Unit, Chelsea and Westminster NHS Trust

Ethical approval was obtained from South West London REC2 (10/H0706/26) and local site approval was obtained from Chelsea Westminster Hospital NHS Trust (CW 10/090). Paired blood and colonic samples were collected from 22 healthy controls and 30 UC patients. LPS, claudin 1, claudin 4 and occludin staining was performed on formalin fixed colonic tissue sections by indirect immunohistochemistry. Quantitative image analysis was performed using high-resolution scans of whole tissue sections. Microbial translocation was assessed using quantitative real time PCR. Soluble CD14 and LPS-binding protein were measured by ELISA. Mucosal mononuclear cells (MMCs) were obtained from biopsies following collagenase digestion. Cytokine staining was performed on PBMCs and MMCs following a 6hr SEB + Brefeldin A stimulation. Median values and interquartile ranges are reported. Mann-Whitney or Kruskal Wallis with Dunn’s correction were used to look for differences between groups and correlations were performed using Spearman’s Rho.

Breach/Intact Ratio

15 10 5 0

30

HC

UC

60 40 20 HC

UC

HC

4 2 0

UC

HC

IL-17F

0.5

HC

UC PBMC

HC

MMC

6

1.0

0.0

UC

IL-22 0.0259

1.5

HC

PBMC

MMC

PBMC

UC

UC

0.0226

4

UC patients were significantly younger than controls (p=0.0009) and this group also contained more females (X2=11.33, p=0.0008). However blood and mucosal T cell proportions were similar. Mucosal CD4+CD161+ TH17 precursor cells were reduced in UC and IL-17f and IL-22 elevated (Figure 1). Mucosal TH17 expression in UC was heterogeneous and correlated with UCSS (rs=0.43, p=0.0271). UC was associated with significant increases in disruption to tight junction proteins and there was evidence of elevated immune response to LPS (raised LBP) but we found reduced microbial translocation in plasma compared to controls (Figure 2). Translocation of LPS into the lamina propria was also seen in more controls (59%) than UC patients (37%) (Figure 4). UC patients with LPS in the lamina propria had significantly reduced levels of pro inflammatory cytokine production compared to UC patients with complete LPS clearance (Figure 3). We divided our patients into those with active disease (C) and those in remission (Montreal severity score of 0 or 1) with LPS (A) or without LPS (B) in the lamina propria. We found that group B shared the ultrastructural abnormalities seen in relapsed disease but tight junctions in group A were similar to controls (Figure 5,6). LBP and mucosal IL-17a in Group A were also significantly lower than in the relapsed Group C but this was not seen in Group B (Figure 6). We obtained follow up data on our UC patients in remission. Median follow up within Group A was available for 4 patients and was 973 days. Two patients had a disease flare within this time frame. In Group B median follow up time was 644 days and 5/8 patients (63%) experienced disease flare by this time point. The calculated median time to disease escalation was 1054 days in Group A and 761 days in Group B (Figure 6).

Conclusions

2 0

Results

HC

MMC

UC PBMC

HC

UC MMC

UC is associated with elevated mucosal TH17 cytokines and reduced CD4+CD161+ TH17 precursor cells

UC patients with inactive disease who share immunological and mucosal ultrastructural features with active UC can be identified by the absence of LPS in the lamina propria. Measurement of LPS by IHC may have a role in identifying cases at greater risk of clinical relapse.

Figure 6

Claudin 4

20 15 10 5 0

HC

p= 0.0191

40

UC Serum LBP

30

20 10

20 10

0

HC

p=

Example LPS staining in the lamina propria of patients with UC (x200)

0

UC

HC

SCD14 ug/ml

UC

Figure 5

Plasma 16s rDNA 100 p= 0.0002

Copies/ml

3

μg/ml

p= 0.0013

25

Occludin

p= 0.0231

μg/ml

Breach/Intact Ratio Breach/Intact Ratio

20

6

0.0374

80

0

TH17 cells

Figure 4

Figure 2 Claudin 1 p= < 0.0001

Precursor TH17 cells 100

UC (n=30) 34 (27-45) 18:12 64.85 (53.25-75.90) 62.85 (58.64-69.31) 25.55 (18.25-35.25) 22.13 (18.73-25.00) 6.3 (1.4 – 11.8) 5 10 7 8 4 15 11 14 16 23 7 7

Figure 1 %CD4 Memory

Materials and Methods

%CD4 Memory

The pathology of ulcerative colitis (UC) is thought to arise from an aberrant or excessive immune response to physiologically normal levels of gut bacteria in a genetically susceptible host. We measured levels of lipopolysaccharide (LPS), a component of the outer membrane of gramnegative bacteria, in the periphery and colonic lamina propria of patients to assess the extent of microbial translocation in UC. We further examined ultra structural changes in the distribution of tight junction proteins in the colon to assess mucosal healing in remission. As mutations in the TH17/IL-23 axis are associated with increased susceptibility to IBD we also measured peripheral blood and mucosal levels of TH17 cells and inflammatory cytokines.

Controls (n=22) Age in years* 47 (40-55) Gender F:M* 3:19 PBMC 64.10 (53.00-73.45) %CD4 MMC 64.78 (56.66-70.59) PBMC 27.95 (20.50-36.61) %CD8 MMC 22.20 (16.99-27.95) Disease Duration in years 0 Montreal 1 Severity 2 3 1 Disease Score Montreal Extent 2 3 5 5-ASA Treatment AZA/E Steroids

%CD4 Memory

Introduction

% CD4 Memory

1Immunology

2 1

80 60 40 20

0

HC

0

UC

HC

UC

UC patients have increased disruption to the tight junction network and elevated response to LPS (LBP) but peripheral microbial translocation is reduced UC patients in remission who lack LPS staining in the lamina propria (Group B) share features with relapsed disease and have shorter event free days compared to LPS+ve UC patients in remission (Group A)

Figure 3

2

0

10

LPS- UC

LPS+ UC

30

p= 0.0104

8

%CD4 Memory

4

TNF MMC

IFNγ MMC %CD4 Memory

%CD4 Memory

6

IL-17a MMC p= 0.0003

6 4 2 0 LPS+ UC

Acknowledgements

20

10

0 LPS- UC

p= 0.0190

LPS- UC

LPS+ UC

UC patients with LPS present in the lamina propria have lower pro inflammatory cytokine production by mucosal CD4 T cells

Example claudin 1 staining in the lamina propria of a control and patients with UC in remission (A, B) and with relapsed disease (C) (x200)

We would like to thank the Blizzard Institute for the use of their Hamamatsu NanoZoomer scanning microscope. Funding for this project was provided by Westminster Medical School Research Trust, St Stephens AIDS Trust and Pfizer. The authors report no conflicts of interest.