OP0205 Gut Dysbiosis in Patients with HLA-B27+ Ankylosing Spondylitis is Associated with Ileitis, Down-Regulation of Tight Junction Proteins, Increased Serum Levels of LPS and Monocytes Anergy
Authors: F. Ciccia, M.M. Luchetti, G. Guggino, M. Rossini, S. Raimondo, A. Gabrielli, R. Alessandro, A. Rizzo, G. Triolo
Journal: Annals of the Rheumatic Diseases (2015)

<h3>Background</h3> Intestinal dysbiosis has been recently demonstrated in the inflamed ileum of AS patients. <h3>Objectives</h3> To study the ileal localization of bacteria in AS patients and their relationship with local and systemic immune responses. <h3>Methods</h3> Consecutive gut biopsies obtained from 30 HLA-B27<sup>+</sup> AS patients and 20 normal subjects were histologically classified in normal histology, acute inflammation and chronic inflammation. Giemsa and Silver stains were used to visualize bacteria and characterize their morphology. Intestinal bacteria were scored on the basis of the numbers of bacteria and their aggregation in clusters. The ileal expression and tissue distribution of claudin-2 and 4, Zonulin 1 and occludin were investigated by rt-PCR and immunohistochemistry. Serum levels of LPS and intestinal fatty acid binding protein (i-FABP) were also assayed by ELISA. The expression of HLA-DR, CD71 and CD14 on circulating monocytes and the monocyte immunologic behavior were studied by flow-cytometry. <h3>Results</h3> Cocci-like bacteria, but not segmented filamentous bacteria, were observed in the inflamed ileum of AS sometimes aggregated in clusters. Bacterial scores were significantly correlated with the percentages of infiltrating inflammatory cells (r<sup>2</sup>=0.56, p&lt;0.0001). The presence of invadent bacteria in AS was invariably associated with histologic changes characterized by i) the detachment of epithelial cell layer from the basement membrane; ii) an edematous lamina propria with extravasated red blood cells and iii) the down-regulation of occludin, claudin 2 and 4 and Zonulin 1 (∼10, ∼12, ∼8 and ∼ 6 fold decrease, respectively; p&lt;0.05) assessed by rt-PCR and confirmed by IHC. Higher serum levels of LPS and i-FABP significantly associated with the higher score of bacteria invasion and tissue inflammation were observed in AS patients. Circulating monocytes in AS displayed a reduced expression of CD14 (p&gt;0.0001), HLA-DR (p&lt;0.05) and CD71 (p&lt;0.001). Ex vivo stimulation of monocytes with LPS increased the expression of IL-23 in CD14<sup>+</sup> cells in both AS patients and controls that was, however significantly higher in AS patients. Production of IL-23 by CD14<sup>-</sup> monocytes in AS patients was however undetectable and not modified by LPS alone. Stimulation of monocytes with LPS+sCD14 increased IL-23 production in CD14<sup>+</sup> cells only in controls, strongly inducing the production of IL-23<sup>+</sup> in CD14<sup>-</sup> monocytes in AS. Neither LPS or LPS+CD14 modify the expression of UPR genes both in patients and controls. Conversely, LPS and more intensely, LPS+sCD14 stimulation significantly abrogated the expression of the autophagy genes ATG16L1 (∼5 fold decrease, p&lt;0.05); IRGM (∼ 3 fold decrease, p&lt;0.05) and MAP1LC3A (∼7 fold decrease, p&lt;0.05) only in AS monocytes. <h3>Conclusions</h3> Bacterial ileitis, accompanied by damaged intestinal mucosal barrier, characterizes AS patients. i-FABP and LPS are increased in the peripheral blood of AS patients and associated with the down-regulation of LPS co-receptor CD14 and with an anergic monocyte phenotype. Ex vivo stimulation with sCD14 may confer LPS-responsiveness to the cells not expressing CD14. Dysregulated autophagy response is present in the peripheral blood of AS patients. <h3>Disclosure of Interest</h3> None declared