Tumor Necrosis Factor Inhibitors Exacerbate Whipple's Disease by Reprogramming Macrophage and Inducing Apoptosis

Abstract

Tropheryma whipplei is the agent of Whipple's disease, a rare systemic disease characterized by macrophage infiltration of the intestinal mucosa. The disease first manifests as arthralgia and/or arthropathy that usually precede the diagnosis by years, and which may push clinicians to prescribe Tumor necrosis factor inhibitors (TNFI) to treat unexplained arthralgia. However, such therapies have been associated with exacerbation of subclinical undiagnosed Whipple's disease. The objective of this study was to delineate the biological basis of disease exacerbation. We found that etanercept, adalimumab or certolizumab treatment of monocyte-derived macrophages from healthy subjects significantly increased bacterial replication in vitro without affecting uptake. Interestingly, this effect was associated with macrophage repolarization and increased rate of apoptosis. Further analysis revealed that in patients for whom Whipple's disease diagnosis was made while under TNFI therapy, apoptosis was increased in duodenal tissue specimens as compared with control Whipple's disease patients who never received TNFI prior diagnosis. In addition, IFN-γ expression was increased in duodenal biopsy specimen and circulating levels of IFN-γ were higher in patients for whom Whipple's disease diagnosis was made while under TNFI therapy. Taken together, our findings establish that TNFI aggravate/exacerbate latent or subclinical undiagnosed Whipple's disease by promoting a strong inflammatory response and apoptosis and confirm that patients may be screened for T. whipplei prior to introduction of TNFI therapy.

Keywords: IFNγ; TNF inhibitor; Tropheryma whipplei; Whipple’s disease; macrophages.

Figure 1

TNFI increase T. whipplei replication in macrophages. (A) Macrophages were infected with T. whipplei (50 bacteria per cell) alone (black bars) or in the presence of etanercept (white bars), certolizumab (grey bars) or adalimumab (hashed bars) and washed after 2, 4 or 8 h. (B) Macrophages were infected with T. whipplei (50 bacteria per cell) alone (black bars) or in the presence of etanercept (white bars), certolizumab (grey bars) or adalimumab (hashed bars) for 4 h, washed to remove free bacteria and incubated for the indicated periods with etanercept (white bars), certolizumab (grey bars) or adalimumab (hashed bars) or left untreated (black bars). Bacterial DNA copy number was determined by qPCR. (C) Representative pictures of infected macrophages incubated for 12 days under the indicated treatment and stained with an anti-T. whipplei antibody (red), phalloidin (green) and DAPI (blue), scale bar = 20 μm). The experiment was performed using three different donors (N = 3), and the values represent the mean ± standard error of the mean. * and ***P < 0.05 and 0.001, respectively by two-way ANOVA and the Dunnett’s test for post-hoc comparisons.

Figure 2

TNFI interfere with macrophage polarization. (A, B) Macrophages were stimulated for 6 h with T. whipplei (A) or LPS (B) in the presence or not of etanercept, certolizumab or adalimumab. The expression of macrophage M1 (purple) and M2 (green) polarization genes was investigated by qRT-PCR after normalization to the actin endogenous control and expressed as log2-transformed-foldchanges relative to the appropriate unstimulated condition. The experiment was performed using three (N = 3) or six different donors (N = 6) for T. whipplei or LPS stimulation, respectively. The mean log2-transformed-foldchange value was used in the ClustVis webtool to generate the heat-maps. (C, D) Macrophages were stimulated for 24 h with T. whipplei (C) or LPS (D) in the presence of etanercept (white bars), certolizumab (grey bars) or adalimumab (hashed bars) or left untreated (black bars) and TNF, IL-6, IL-1β and IL-10 release in the culture supernatants was assessed by ELISA (N = 3). The experiment was performed using three different donors (N = 3), and the values represent the mean ± standard error of the mean. * and ***P < 0.05 and 0.001, respectively by two-way ANOVA and the Tukey’s test for post-hoc comparisons.

Figure 3

TNFI increase T. whipplei-induced macrophage apoptosis. (A) Macrophages were infected or not with T. whipplei (50 bacteria per cell) alone (black bars) or in the presence of etanercept (white bars), certolizumab (grey bars) or adalimumab (hashed bars) for 24 h. Cell metabolic activity was assessed by MTT assay and expressed as % of uninfected and untreated cells (N = 3). (B) THP-1 macrophages were infected or not with T. whipplei (50 bacteria per cell) alone (black bars) or in the presence of etanercept (white bars), certolizumab (grey bars) or adalimumab (hashed bars) for 24 h. Apoptosis was assessed by flow cytometry after annexin V staining (N = 3). Representative plots are shown (red line: untreated; blue line: etanercept; orange line: certolizumab and green line: adalimumab). (C) Macrophages were infected or not with T. whipplei (50 bacteria per cell) alone or in the presence of etanercept (eta.), certolizumab (certo.) or adalimumab (ada.) for 24 h. As a positive control, cells were treated with staurosporine for 4 h. Representative pictures of macrophages stained with anti-active caspase-3 antibody (green), phalloidin (purple) and DAPI (blue) are shown (scale bar = 20 μm). Values represent mean ± standard error of the mean. * and **P < 0.05 and 0.01, respectively by two-way ANOVA and the Tukey’s test for post-hoc comparisons.

Figure 4

TNFI increase apoptosis in duodenal tissue and local and systemic IFN-γ expression. (A) Duodenal biopsy specimen from patients diagnosed under TNFI treatment (left) or not (right) were stained with PAS. Arrows indicate PAS-positive cells (original magnification × 250). (B) Cell apoptosis on duodenal biopsy specimen was assessed by TUNEL assay and observed by confocal microscopy. Representative images are shown. Nuclei are visualized in blue after DAPI staining and TUNEL-positive cells (arrows) appear in red, scale bar = 20 μm. (C) IFN-γ expression was evaluated by immunohistofluorescence staining and observed by confocal microscopy. Representative images are shown. Nuclei are visualized in blue after DAPI staining and IFN-γ -positive cells (arrows) appear in green, scale bar = 20 μm. IFN-γ (D), IL-10 (E) and TNF (F) were measured by ELISA in the sera from patients diagnosed under TNFI treatment (square) or not (circle). **P < 0.01 by Mann-Whitney U test.