Volume 138, Issue 1, Pages 210-220 (January 2010) Impaired Immune Functions of Monocytes and Macrophages in Whipple's Disease  Verena Moos, Carsten Schmidt, Anika Geelhaar, Désirée Kunkel, Kristina Allers, Katina Schinnerling, Christoph Loddenkemper, Florence Fenollar, Annette Moter, Didier Raoult, Ralf Ignatius, Thomas Schneider  Gastroenterology  Volume 138, Issue 1, Pages 210-220 (January 2010) DOI: 10.1053/j.gastro.2009.07.066 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 Similar expression of CD68, enhanced expression of stabilin-1, and reduced expression of iNOS in the duodenum of Whipple's disease. Positive cells are stained red by specific immunohistochemistry. (A and B) CD68 in a treated Whipple's disease patient (A) and in a healthy control (B). (C and D) Stabilin-1 in a treated Whipple's disease patient (C) and a healthy control (D). (E and F) iNOS in a treated Whipple's disease patient (E) and a healthy control (F). Scale bars, 100 μm. Representative examples of stainings of the duodenum of healthy subjects (n = 27), treated Whipple's disease patients (n = 21), untreated Whipple's disease patients (n = 19), and disease controls (n = 9) are shown. Gastroenterology 2010 138, 210-220DOI: (10.1053/j.gastro.2009.07.066) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 Immunohistochemical double staining of CD163 (brown) and T whipplei (blue) in duodenal biopsy samples from untreated and treated Whipple's disease patients and healthy and disease controls. CD163 and T whipplei double positive cells were marked with black arrowheads, CD163 single positive macrophages with open arrowheads, and T whipplei single positive macrophages with grey arrowheads. (A) CD163 and T whipplei double positive macrophages in untreated Whipple's disease. (C) CD163 and T whipplei double positive, single CD163, and single T whipplei positive macrophages in Whipple's disease treated for 6 months. (E) Faintly T whipplei and CD163 double positive and single CD163 positive macrophages in Whipple's disease treated for 18 months. (B and D) CD163 single positive cells in healthy controls. (F) CD163 single positive cells in a biopsy sample of a patient with duodenal tuberculosis. Scale bars, 100 μm. Representative examples of stainings of the duodenum of healthy subjects (n = 27), treated Whipple's disease patients (n = 39), untreated Whipple's disease patients (n = 20), and disease controls (n = 9) are shown. Gastroenterology 2010 138, 210-220DOI: (10.1053/j.gastro.2009.07.066) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 Increased numbers of AAM in the duodenum and peripheral blood of Whipple's disease patients. (A–D) Numbers of CD68+, CD163+, stabilin-1+, and iNOS+ cells in the duodenum of healthy and disease controls, treated and untreated Whipple's disease patients determined by immunohistochemistry. (A) CD68+ macrophages; (B) CD163+ macrophages; (C) Stabilin-1+ macrophages; (D) iNOS+ macrophages. (E and F) The percentages of CD68+ and CD163+ cells within CD14+ monocytes in the peripheral blood of healthy controls and treated and untreated Whipple's disease patients were determined by flow cytometry. (E) CD68+ of CD14+ monocytes; (F) CD163+ of CD14+ monocytes. *P value < .05, **P value < .01, ***P value < .001; WD, Whipple's disease. Gastroenterology 2010 138, 210-220DOI: (10.1053/j.gastro.2009.07.066) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 Up-regulation of CD163 on CD14+ monocytes of Whipple's disease patients by T whipplei. Whole blood was incubated for 6 hours either with T whipplei or without stimulus followed by the analysis of the percentage of CD163+ cells of CD14+ monocytes by flow cytometry; Whipple's disease patients (A) and healthy subjects (B). ***P value < .001. Gastroenterology 2010 138, 210-220DOI: (10.1053/j.gastro.2009.07.066) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 Elevated expression of mRNA of CCL2 and IL-10 in the duodenum of Whipple's disease patients. Analysis of mRNA for CCL2 (A), IL-10 (B), IL-1β (C), and TNF-α (D). Data are presented as adjusted transcripts/microliter cDNA. WD, Whipple's disease. *P value < .05, **P value < .01, ***P value < .001. Gastroenterology 2010 138, 210-220DOI: (10.1053/j.gastro.2009.07.066) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 Elevated expression of CCL2 and IL-10 in serum samples from patients with Whipple's disease. Serum concentrations of CCL2 (A), IL-10 (B), IL-1β (C), and TNF-α (D) were determined by cytometric bead array. WD, Whipple's disease. *P value < .05, ***P value < .001. Gastroenterology 2010 138, 210-220DOI: (10.1053/j.gastro.2009.07.066) Copyright © 2010 AGA Institute Terms and Conditions

Figure 7 Functional impairment of macrophages and monocytes from Whipple's disease patients. (A) Reduced nitrite production in supernatants of duodenal biopsy samples from Whipple's disease patients after stimulation with IFN-γ, TNF-α, and LPS. (B–D) Normal phagocytosis (B) and reduced oxidative burst (C and D) of monocytes from Whipple's disease patients following incubation with T whipplei. (B) The mean fluorescence intensity of monocytes after phagocytosis of FITC-labeled T whipplei, C cellulans, and E coli. (C and D) The mean fluorescence intensity of monocytes after induction of oxidative burst by opsonized T whipplei and C cellulans (C) and E coli (D). Clear dots: healthy subjects; light grey dots: treated Whipple's disease patients; dark grey dots: untreated Whipple's disease patients. *P value < .05, **P value < .01. Gastroenterology 2010 138, 210-220DOI: (10.1053/j.gastro.2009.07.066) Copyright © 2010 AGA Institute Terms and Conditions