Whipple’s disease (WD) is a rare systemic disease caused by Tropheryma whipplei. We showed that T. whipplei was eliminated by human monocytes but replicated in monocyte-derived macrophages (Mφ) by inducing an original activation program. Two different host molecules were found to be key elements for this specific pattern. Thioredoxin, through its overexpression in infected monocytes, was involved in bacterial killing because adding thioredoxin to infected Mφ inhibited bacterial replication. IL-16, which was up-regulated in Mφ, enabled T. whipplei to replicate in monocytes and increased bacterial replication in Mφ. In addition, anti-IL-16 Abs abolished T. whipplei replication in Mφ. IL-16 down-modulated the expression of thioredoxin and up-regulated that of IL-16 and proapoptotic genes. In patients with WD, T. whipplei replication was higher than in healthy subjects and was related to high levels of circulating IL-16. Both events were corrected in patients who successfully responded to antibiotics treatment. This role of IL-16 was not reported previously and gives an insight into the understanding of WD pathophysiology.

Whipple’s disease (WD),3 a rare systemic disease, is associated with an initial phase of migratory polyarthritis, fatigue, weight loss, and anemia followed by a progressive syndrome of abdominal pain, steatorrhea, and cachexia with lymphadenopathy and hyperpigmentation (1, 2). Although a bacterial etiology had been suggested since the original description (3), the identification of the causative agent of WD as a novel bacterium was very recently assessed by PCR and sequence analysis (4). In 2000, we successfully cultivated the WD agent from the valve of a patient with endocarditis (5). Subsequently, the name of Tropheryma whipplei was officially ascribed to the WD agent (6). The complete sequence of the genome has been reported recently for two strains of T. whipplei (7, 8). The diagnosis of WD was usually based on the presence of large, foamy macrophages (Mφ) containing periodic acid-Schiff-positive inclusions in the lamina propria of duodenal biopsy specimens (9, 10), but these periodic acid-Schiff-positive cells may be detected in other organs (2).

The presence of bacterial material in tissue Mφ suggested that T. whipplei had a tropism for myeloid cells. It is well known that the microbicidal activity of monocytes (Mo) and Mφ can be disarmed by immunoregulatory cytokines such as IL-4 or IL-10 and is stimulated by T cell-mediated signals such as IFN-γ (11). T. whipplei multiplies after cytokine-mediated deactivation of Mo (12). The chronic evolution of WD evokes long-term persistence of T. whipplei in Mφ, which requires the inhibition of Mφ microbicidal competence. Although impaired microbicidal activity in Mφ (13) and cell-mediated immunity, including defective production of IL-12 and IFN-γ (14), have been reported, the nature of immune impairment leading to T. whipplei persistence is still not known. In this study, we showed that T. whipplei organisms replicated in Mφ but were killed by Mo from healthy individuals. T. whipplei survival and killing were associated with specific and unusual transcriptional patterns. The expression of thioredoxin was, indeed, found to be up-regulated in Mo but not in Mφ, where its expression partly reduced T. whipplei replication. Conversely, IL-16 was found to be produced only by T. whipplei-infected Mφ and was able to stimulate T. whipplei replication in Mo. In addition, our data showed that high amounts of circulating IL-16 were associated with WD. These findings offer new insights on the role of IL-16 in microbicidal potency of Mφ and the understanding of the WD pathophysiology.

We included WD patients in the study after informed consent and approbation by the Ethics Committee of the Université de la Méditerranée. The diagnosis was based on clinical features, histological findings, and PCR studies of tissue samples (15). The patients, consisting of 10 men and 3 women (mean age, 55 years), presented neurological (n = 6) or digestive (n = 4) manifestations or endocarditis (n = 3). Among them, four were successfully treated by antibiotherapy. Two presented endocarditis and were treated with doxycycline and hydroxychloroquine, one presented digestive symptoms and was treated with trimethropim-sulfamethoxazole, and the last one presented neurological manifestations and was treated with doxycycline, hydroxychloroquine, and trimethropim-sulfamethoxazole. Ten healthy controls (6 men, 4 women) with mean age of 45 years were also included in the study.

The Twist-Marseille strain of T. whipplei (CNCM I-2202) was cocultured with HEL cells (CCL-37; American Type Culture Collection) and purified as described previously (5). Bacteria were counted by Gimenez staining and indirect immunofluorescence, and their viability was assessed using the LIVE/DEAD BacLight bacterial viability kit (Molecular Probes) (16). We cultured axenic T. whipplei, as described recently (17). Briefly, a cell-culture medium providing amino acids for which biosynthetic pathways are missing or impaired in T. whipplei (DMEM:Ham’s F-12; Invitrogen Life Technologies) was supplemented with 10% FCS, 2 mM l-glutamine, and 1% nonessential amino acids (Invitrogen Life Technologies).

Mo from circulating PBMC were isolated by Ficoll gradient (MSL; Eurobio) and adherence in flat-bottom 24-well plates (Nunc; PolyLabo). Mφ were derived from Mo by a 7-day culture, as described previously (18). Mo and Mφ (105 cells/assay) were infected with T. whipplei, washed to remove free bacteria, and incubated for additional period of time in RPMI 1640 containing 10% FCS. In some experiments, thioredoxin (Sigma-Aldrich) was added to infected cells every day. Negative control was obtained by thioredoxin using a maleimide treatment. Briefly, thioredoxin was incubated with maleimide (1:2, molar reaction; Sigma-Aldrich) for 2 h to alkylate thiol groups. Inactivated thioredoxin was then added to T. whipplei-infected cells as described above. Human rIL-16 (R&D Systems) was added to Mo and Mφ 18 h prior infection, as described by Ferland et al. (19). To neutralize endogenous IL-16, monoclonal anti-IL-16 Abs (mouse IgG1; R&D Systems) were added at 1 μg/ml to cell culture every other day. Human rIL-1β (R&D Systems) was added to Mo 18 h prior infection. In microarray experiments, 106 cells were seeded in 3.5-cm dishes and stimulated with T. whipplei (50:1 bacterium-to-cell ratio) or 2 μg/ml Escherichia coli LPS (Sigma-Aldrich) for 6 h.

We lysed Mo and Mφ, and extracted DNA by using the QIAamp DNA MiniKit (Qiagen). PCR was performed using the LightCycler-FastStart DNA Master SYBR Green system (Roche), and conducted with primers specific for T. whipplei 16S-23S ribosomal intergenic spacer region (tws3f and tws4r), as described previously (20). In each PCR run, a standard curve was generated using serial dilutions ranging from 10 to 108 copies of the intergenic spacer region, and established by the LightCycler 5.32 software (LC-Run version 5.32; Roche).

Mo and Mφ total RNA were purified with TRIzol (Invitrogen Life Technologies) and digested using DNA-free kit (Ambion) for 1 h to remove contaminating genomic DNA. Quality of the RNA was assessed using the 2100 Bioanalyzer and the RNA 6000 Nano LabChip kit (Agilent). Labeled cDNA was synthesized using cyanine 3- or 5-dCTP from 10 μg of RNA with an oligo(dT) primer according to the manufacturer’s protocol (Agilent). Labeled cDNA were hybridized to Human 1 cDNA Microarray (Agilent) containing 12,840 sequenced human genes and expressed sequence tags (ESTs) for 17 h at 65°C. The hybridized slides were scanned with a ScanArray Express HT using the ScanArray software (PerkinElmer). We analyzed the dataset with the QuantArray software (PerkinElmer) and Microsoft Excel. We normalized the fluorescence intensities with the LocFit algorithm of MIDAS from the TM4 The Institute for Genomic Research microarray suite available at 〈www.tigr.org/software/tm4/〉. We calculated fold changes relative to uninfected controls, and we considered as regulated only gene expression with fold change >2.5. We then analyzed the dataset following hierarchical clustering using the MeV software from the TM4 The Institute for Genomic Research microarray suite.

Reverse transcription (Superscript II; Invitrogen Life Technologies) of 10 ng of RNA was performed with an oligo(dT) primer. We conducted PCR for IL-12p35, thioredoxin, glutaredoxin, IL-16, and IL-1β encoded by IL12A, TXN, GLRX, IL16, and IL1B, respectively, using the following primers: IL-12f, 5′-TCAGCAACATGCTCCAGAAGGC-3′, and IL-12r, 5′-TGCATTCATGGTCTTGAACTCCACC-3′; TXNf, 5′-CCATTTCCATCGGTCCTTAC-3′, and TXNr, 5′-CACTCTGAAGCAACATCCTTGAC-3′; GLRXf, 5′-GGGAAGGTGGTTGTGTTCAT-3′, and GLRXr, 5′-GT TAGTGTGGTTGGTGGCGT-3′; IL-16f, 5′-AAGGGGCATCTCCAACATCATCAT-3′, and IL16r, 5′-CTCCTGCCAAGCTGAACCCAAGAC-3′; IL-1Bf, 5′-AGCACCTCTCAAGCAGAAAACAT-3′, and IL-1Br, 5′-AGACAACAGGAAAGTCCAGGCTA-3′; IL-10f, 5′-GCT CCAAGAGAAAGGCATCTACA-3′, and IL-10r, 5′-GGGGGTTGAGGTATCAGAG GTAA-3′; and TGFB1f, 5′-TCTATGACAAGTTCAAGCAGA-3′, and TGFB1r, 5′-GACATCAAAAGATAACCACTC-3′. Reverse transcriptase was omitted in negative control. The fold change in target gene cDNA relative to the β-actin endogenous control (ACTBf, 5′-GGAAATCGTGCGTGACATTA-3′, and ACTBr, 5′-AGGAAGGAAGGCTGGAAGAG-3′) was determined as follows: fold change = 2−ΔΔCt, where ΔΔCt = (CtTarget − CtActin)stimulated − (CtTarget − CtActin)unstimulated (21). Ct values were defined as the number of cycles for which the fluorescence signals were detected.

Mo and Mφ were incubated with or without T. whipplei (50:1 bacterium-to-cell ratio) in RPMI 1640 supplemented with 10% FCS for 6 h. Cells were washed to remove free bacteria and were then cultured for 48 h. We determined apoptosis as follows: cells were stained with 20 μl of annexin V-FITC (Roche) diluted in 1 ml of a medium containing 10 mM HEPES (pH 7.4), 135 mM NaCl, and 5 mM CaCl2 for 10 min. The percentage of annexin V-positive cells was determined by fluorescence microscopy (Axioskop; Zeiss).

Mo and Mφ (105/assay) were incubated with T. whipplei (50:1 bacterium-to-cell ratio) for 16 h. Cell supernatants and plasma from controls and WD patients were assessed for the presence of IL-16 and IL-1β. IL-16 and IL-1β detection kits were provided by R&D Systems (detection limit, 6.2 pg/ml) and Beckman Coulter (detection limit, 1.5 pg/ml), respectively. The intra- and interspecific coefficients of variation of what ranged from 5 to 10%. We measured thioredoxin with a TRX immunoassay kit (Redox Bioscience) according to the manufacturer’s protocol. The detection limit of this assay was 2.0 ng/ml. The intra- and interassay coefficients of variation were 4 and 7%, respectively.

Infected Mo and Mφ were scrapped and fixed in 2.5% glutaraldehyde diluted in 0.1 M cacodylate buffer (pH 7.2) containing 0.1 M sucrose for 1 h at 4°C. After washing, they were incubated with 1% osmium tetroxide diluted in 0.1 M cacodylate buffer for 1 h. Dehydration was performed through washes of graded concentrations of acetone (25–100%), and cells were then embedded in Araldite (Sigma-Aldrich). Sections from embedded blocks were poststained with saturated solution of methanol/uranyl acetate and lead nitrate with sodium citrate in water before examination using a JEOL 1220 electron microscope.

To evaluate the role of IL-16 in Mo maturation, Mo (106 cells) were incubated with and without rIL-16 (10 ng/ml) for 18 h before washing. At 2 and 6 days of incubation, cells were harvested, washed, and reincubated with PE-CD16 and FITC-CD68 (Beckman Coulter) or irrelevant mAbs of the same isotypes. Cells were then washed, fixed in 1% paraformaldehyde, and analyzed by flow cytometry on a Coulter EPICS-XL cytometer. On average 25,000 cells were counted, and the results are expressed therefore as fluorescence mean.

Results were expressed as mean ± SD or median for WD patients. Quantitative datasets were compared using a two-tailed nonparametric Mann-Whitney U test. Differences were considered significant at p < 0.05. Statistical analyses were performed using Prism 4.0 software (GraphPad Software).

First, we assessed the uptake of T. whipplei by human circulating Mo and Mo-derived Mφ from healthy controls by using qPCR for bacterial DNA quantification. T. whipplei DNA was detected after 2 h, and the number of DNA copies reached a plateau between 4 and 8 h (Fig. 1,A), although the number of T. whipplei DNA copies was significantly higher in Mφ than in Mo at 2, 4, and 8 h. T. whipplei was found inside vacuoles in Mo (data not shown) and Mφ (Fig. 1,B) after 4 h of incubation, emphasizing results obtained with HeLa cells (16). Second, the intracellular fate of T. whipplei was assessed by qPCR. In Mo, T. whipplei DNA became undetectable after 3 days and remained undetectable during 12 days. In contrast, the DNA copy number increased from day 6 to day 12 after the infection in Mφ (Fig. 1,C). As T. whipplei uptake was distinct in Mo and Mφ (Fig. 1,A), we wondered whether uptake differences would account for changes in survival pattern. Bacterial uptake by Mo was enhanced to levels observed in Mφ by increasing bacterium-to-cell ratio to 200:1. This did not enable T. whipplei survival in Mo. In opposition, decreasing bacterial uptake in Mφ (bacterium-to-cell ratio of 25:1) to the levels found in Mo did not impair T. whipplei replication (Fig. 1 D). Hence, our results clearly showed that T. whipplei organisms replicate in Mφ and are killed by Mo.

FIGURE 1.

T. whipplei replicates in Mφ, not in Mo. A, Mo (□) and Mφ (▪) were incubated for different times with T. whipplei and washed to remove free bacteria. Bacterial DNA copy number was determined by qPCR after DNA extraction (n = 6). B, Mφ were incubated with T. whipplei for 4 h. The representative electron micrograph showed that T. whipplei was found inside vacuoles (arrows, bar: 1.5 μm). C, Mφ were infected with T. whipplei for 4 h and cultured for different times. T. whipplei replication was assessed by determining the bacterial DNA copy number by qPCR. (n = 6). D, Mφ were infected with T. whipplei at different bacteria-to-cell ratios (□, ▪, and ▦: 25, 50, and 200 bacteria/cell, respectively) for 4 h and cultured for different times. T. whipplei uptake (insets) and replication were assessed by determining the bacterial DNA copy number by qPCR (n = 6). ∗, p = 0.002.

FIGURE 1.

T. whipplei replicates in Mφ, not in Mo. A, Mo (□) and Mφ (▪) were incubated for different times with T. whipplei and washed to remove free bacteria. Bacterial DNA copy number was determined by qPCR after DNA extraction (n = 6). B, Mφ were incubated with T. whipplei for 4 h. The representative electron micrograph showed that T. whipplei was found inside vacuoles (arrows, bar: 1.5 μm). C, Mφ were infected with T. whipplei for 4 h and cultured for different times. T. whipplei replication was assessed by determining the bacterial DNA copy number by qPCR. (n = 6). D, Mφ were infected with T. whipplei at different bacteria-to-cell ratios (□, ▪, and ▦: 25, 50, and 200 bacteria/cell, respectively) for 4 h and cultured for different times. T. whipplei uptake (insets) and replication were assessed by determining the bacterial DNA copy number by qPCR (n = 6). ∗, p = 0.002.

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To determine whether T. whipplei survival or killing are associated with different gene expression programs, we monitored transcriptional responses of Mo and Mφ stimulated by T. whipplei. Among the 12,840 arrayed sequences from which 97% map to named human genes, only those whose expression decreased or increased >2.5-fold compared with uninfected cells were considered as down- or up-regulated, respectively. As a control for non-WD-specific host gene regulation, we used LPS, a canonical agonist of myeloid cells. The number of genes whose expression was modulated in response to LPS was similar in Mo (764 genes or ESTs) and Mφ (1009 genes or ESTs), whereas T. whipplei infection induced a modulation in the expression of only 327 genes or ESTs in Mo and 923 in Mφ. Hence, T. whipplei induced a higher transcriptional activity in Mφ than in Mo. In addition, the transcriptional programs stimulated by T. whipplei were clearly distinct in Mo and Mφ, whereas LPS induced a similar transcriptional pattern. Indeed, hierarchical clustering analysis revealed that Mo and Mφ coclustered in response to LPS stimulation. Clustering algorithm placed the program of T. whipplei-stimulated Mφ on a separate branch of the dendrogram (Fig. 2 A). The transcriptional pattern of T. whipplei-stimulated Mo also differed from that elicited by LPS. Taken together, these results showed that Mo and Mφ respond similarly to LPS, but they display two distinct transcriptional programs in response to T. whipplei.

FIGURE 2.

T. whipplei triggers unusual host transcriptional program. A, Mo and Mφ were stimulated with T. whipplei or LPS for 6 h, and host responses were analyzed by microarrays. Each column represents a single array element and each row a separate experimental mRNA sample. Each expression measurement represents the ratio of fluorescence from the hybridized experimental sample (LPS or T. whipplei stimulated) to the reference sample (unstimulated). Data are represented by a color gradient from green (down-regulation) to red (up-regulation). We used MeV software for hierarchical clustering of mRNA samples. B, For T. whipplei-stimulated samples, data with respect to host response were selected and processed for hierarchical clustering algorithm from the MeV software. C, Mo (□) and Mφ (▪) were stimulated with T. whipplei for 6 h, and transcripts encoding IL-12p35 (IL12A), thioredoxin (TXN), glutaredoxin (GLRX), IL-16 (IL16), and IL-1β (IL1B) were quantified by qRT-PCR. Results are expressed as the ratio of expression levels in infected cells vs uninfected cells (n = 6). D, Mo (□) and Mφ (▪) were stimulated with T. whipplei for 6 h and cultured for 48 h. Apoptosis was measured by fluorescent annexin V assay (n = 4).

FIGURE 2.

T. whipplei triggers unusual host transcriptional program. A, Mo and Mφ were stimulated with T. whipplei or LPS for 6 h, and host responses were analyzed by microarrays. Each column represents a single array element and each row a separate experimental mRNA sample. Each expression measurement represents the ratio of fluorescence from the hybridized experimental sample (LPS or T. whipplei stimulated) to the reference sample (unstimulated). Data are represented by a color gradient from green (down-regulation) to red (up-regulation). We used MeV software for hierarchical clustering of mRNA samples. B, For T. whipplei-stimulated samples, data with respect to host response were selected and processed for hierarchical clustering algorithm from the MeV software. C, Mo (□) and Mφ (▪) were stimulated with T. whipplei for 6 h, and transcripts encoding IL-12p35 (IL12A), thioredoxin (TXN), glutaredoxin (GLRX), IL-16 (IL16), and IL-1β (IL1B) were quantified by qRT-PCR. Results are expressed as the ratio of expression levels in infected cells vs uninfected cells (n = 6). D, Mo (□) and Mφ (▪) were stimulated with T. whipplei for 6 h and cultured for 48 h. Apoptosis was measured by fluorescent annexin V assay (n = 4).

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In an attempt to understand the molecular basis of T. whipplei intracellular fate in Mo and Mφ, we investigated the genes that were modulated in response to the bacteria (Fig. 2,B). Few genes were up-regulated in Mo and Mφ stimulated by T. whipplei: FARP2, which encodes a member of Cdc42-GEF, a gene product with putative function (KIAA0590), the guanylate cyclase activator 1A, and GOS2, a putative cell cycle regulator. The expression of the gene encoding IL-12p35 (IL12A) was repressed in Mo and Mφ, as confirmed by qRT-PCR (Fig. 2,C). Twenty genes of host defense were up-regulated upon Mo stimulation (Table I) and transcriptionally silent or repressed in Mφ (Fig. 2,B). They consisted of genes involved in redox balance maintain such as TXNL, TXNL2, TXN, GLRX, and PRDX3, genes encoding the chemokines CXCL2, CCL4, CXCL1, and CXCL3, and genes playing a role in signal transduction such as TLR7, M6PR, JAK1, and IFI30. The up-regulated expression of TXN and GLRX genes was confirmed by qRT-PCR (Fig. 2,C). Conversely, up-regulated host defense genes in T. whipplei-stimulated Mφ were transcriptionally silent in Mo. They included genes encoding ILs such as IL-16 and IL-1β (Fig. 2,C), chemokines such as CCL15, CCL26, and XCL1, and genes known to be associated with Mφ deactivation such as Mφ scavenger receptor-1, BCL6, and INPPL1 (SHIP2) (Table II). Apoptosis-related genes were also overrepresented: they consisted of FAT tumor suppressor homologue 1 (Drosophila), death receptor 6, gene encoding caspase 6, apoptotic protease activating factor 1, Bcl2-antagonist/killer 1 (BAK1), p19, and death-associated protein 3. This proapoptotic transcriptional pattern resulted in the induction of apoptosis, as measured by fluorescent annexin V assay. Mφ apoptosis was detected 24 h after T. whipplei stimulation and 20% of Mφ became apoptotic after 48 h (Fig. 2,D). Mo, in which a proapoptotic transcriptional program was not induced, did not undergo apoptosis when they were stimulated with T. whipplei (Fig. 2,D). Finally, we measured the release of IL-16, IL-1β, and thioredoxin by Mo and Mφ. IL-16 was significantly secreted by T. whipplei-stimulated Mφ. This release was specific to Mφ because Mo did not secrete IL-16 (Fig. 3,A). Similarly, Il-1β levels were significantly higher in infected Mφ than in unstimulated ones (Fig. 3,B). In contrast, release of thioredoxin was up-regulated only in T. whipplei-stimulated Mo (Fig. 3 C). Taken together, these results showed that T. whipplei triggers an unusual transcriptional program in Mφ associating proapoptotic pattern and up-regulation of IL-16 and IL-1β, whereas the transcriptional program of Mo includes up-regulation of redox-associated molecules.

Table I.

Genes specifically up-regulated in T. whipplei-infected Moa

Gene SymbolGenBank Accession No.T. whipplei-Infected:Uninfected Mo RatioT. whipplei-Infected:Uninfected Mφ Ratio
TXNL AF003938 7.81 0.73 
CXCL2 M36820 6.09 1.45 
CCL4 J04130 5.79 1.31 
TXN X77584 5.67 0.86 
CCL4 AW964223 5.52 1.24 
CXCL1 NM_001511 4.89 2.19 
CXCL3 X53800 4.81 1.66 
TXNL2 AI674397 4.77 0.94 
TXN2 AI741767 4.40 0.70 
FTL BE301211 3.68 0.98 
LTB Y14768 3.33 1.00 
GLRX X76648 2.96 0.70 
IFI30 BE268378 2.89 1.16 
HLA-F AF055066 2.88 1.08 
JAK1 C06051 2.87 0.35 
TTRAP NM_016614 2.79 0.87 
TLR7 AF240467 2.77 1.29 
GLRX X76648 2.66 0.49 
M6PR AA812910 2.52 0.45 
PRDX3 D49396 2.50 0.38 
Gene SymbolGenBank Accession No.T. whipplei-Infected:Uninfected Mo RatioT. whipplei-Infected:Uninfected Mφ Ratio
TXNL AF003938 7.81 0.73 
CXCL2 M36820 6.09 1.45 
CCL4 J04130 5.79 1.31 
TXN X77584 5.67 0.86 
CCL4 AW964223 5.52 1.24 
CXCL1 NM_001511 4.89 2.19 
CXCL3 X53800 4.81 1.66 
TXNL2 AI674397 4.77 0.94 
TXN2 AI741767 4.40 0.70 
FTL BE301211 3.68 0.98 
LTB Y14768 3.33 1.00 
GLRX X76648 2.96 0.70 
IFI30 BE268378 2.89 1.16 
HLA-F AF055066 2.88 1.08 
JAK1 C06051 2.87 0.35 
TTRAP NM_016614 2.79 0.87 
TLR7 AF240467 2.77 1.29 
GLRX X76648 2.66 0.49 
M6PR AA812910 2.52 0.45 
PRDX3 D49396 2.50 0.38 
a

The level of expression is expressed in log ratio between T. whipplei-infected vs uninfected cells.

Table II.

Genes specifically up-regulated in T. whipplei-infected Mφa

Gene SymbolGenBank Accession No.T. whipplei-Infected:Uninfected Mφ RatioT. whipplei-Infected:Uninfected Mo Ratio
HFE U91328 8.11 0.34 
BCL6 U00115 5.87 1.23 
INPPL1 AW250964 5.75 1.10 
CD1C M28827 5.29 1.17 
IL16 AI652705 5.25 0.84 
IL1B MI5840 4.50 1.07 
IL1B X04500 4.44 1.03 
FAT NM_005245 4.00 0.72 
TNFRSF21 AF068868 3.86 0.79 
CASP6 U20536 3.64 0.96 
CCL15 AF088219 3.50 1.09 
APAF1 NM_001160 3.36 1.01 
BAK1 AI741331 3.00 0.60 
CCL26 AF096296 2.87 0.72 
HLA-DQB1 U83582 2.69 1.24 
CDKN2D U40343 2.67 0.95 
XCL1 AL031736 2.63 1.31 
MSR1 BAA02649 2.60 1.13 
MSR1 BAA02649 2.57 1.21 
DAP3 AA207194 2.50 1.26 
Gene SymbolGenBank Accession No.T. whipplei-Infected:Uninfected Mφ RatioT. whipplei-Infected:Uninfected Mo Ratio
HFE U91328 8.11 0.34 
BCL6 U00115 5.87 1.23 
INPPL1 AW250964 5.75 1.10 
CD1C M28827 5.29 1.17 
IL16 AI652705 5.25 0.84 
IL1B MI5840 4.50 1.07 
IL1B X04500 4.44 1.03 
FAT NM_005245 4.00 0.72 
TNFRSF21 AF068868 3.86 0.79 
CASP6 U20536 3.64 0.96 
CCL15 AF088219 3.50 1.09 
APAF1 NM_001160 3.36 1.01 
BAK1 AI741331 3.00 0.60 
CCL26 AF096296 2.87 0.72 
HLA-DQB1 U83582 2.69 1.24 
CDKN2D U40343 2.67 0.95 
XCL1 AL031736 2.63 1.31 
MSR1 BAA02649 2.60 1.13 
MSR1 BAA02649 2.57 1.21 
DAP3 AA207194 2.50 1.26 
a

The level of expression is expressed in log ratio between T. whipplei-infected vs uninfected cells. Several genes are repeated because they appear more than once on the array.

FIGURE 3.

T. whipplei affects cytokine production and redox status, and apoptosis. Mo (□) and Mφ (▪) were stimulated with T. whipplei for 16 h, and we assessed the presence of IL-16 (A), IL-1β (B), and thioredoxin (C) in culture supernatants by immunoassays (n = 5). ∗, p = 0.008.

FIGURE 3.

T. whipplei affects cytokine production and redox status, and apoptosis. Mo (□) and Mφ (▪) were stimulated with T. whipplei for 16 h, and we assessed the presence of IL-16 (A), IL-1β (B), and thioredoxin (C) in culture supernatants by immunoassays (n = 5). ∗, p = 0.008.

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The production of thioredoxin by Mo upon T. whipplei stimulation suggested that thioredoxin is involved in bacterial killing. We added thioredoxin to Mφ before T. whipplei infection and daily thereafter for 12 days. The effect of thioredoxin was found to be dose dependent (Fig. 4,A). A concentration as low as 0.1 ng/ml had no effect on T. whipplei replication. In the presence of 1 ng/ml thioredoxin, T. whipplei replication was significantly decreased by ∼30% after 12 days (p = 0.03). T. whipplei replication was inhibited by 50% in the presence of 10 ng/ml thioredoxin (p = 0.03 at day 12) and reached a plateau at higher concentrations (100 ng/ml thioredoxin). Adding inactive thioredoxin (10 ng/ml) had no significant effect on T. whipplei replication in Mφ (Fig. 4,B). To rule out the possibility that thioredoxin may have act directly on T. whipplei, human recombinant thioredoxin was added daily for 12 days in axenic T. whipplei cultures, and the number of bacterial DNA copies was determined daily. Adding thioredoxin to the culture medium (1 to 100 ng/ml) had no effect on T. whipplei replication (Fig. 4 C). The exogenous molecule remained active through the whole experimental procedure, as assessed by the reduction of insulin in presence of DTT (data not shown). The fact that biologically active thioredoxin had no effect on axenic T. whipplei culture, but inhibited its replication when added to Mφ, suggests that T. whipplei killing by Mo is partly related to thioredoxin.

FIGURE 4.

Thioredoxin partially inhibits T. whipplei replication in Mφ. A, Different concentrations of thioredoxin were added to infected Mφ daily (▪, 0 ng/ml; □, 0.1 ng/ml; ▨, 1 ng/ml; ▩, 10 ng/ml; ▦, 100 ng/ml). B, Thioredoxin (Trx) or maleimide-modified thioredoxin (mTrx) at 10 ng/ml was added every day to Mφ. C, Different concentrations of thioredoxin were added to T. whipplei cultured in axenic medium (○, 0 ng/ml; •, 1 ng/ml; ▵, 10 ng/ml; ▴, 100 ng/ml). Bacterial DNA copy number was determined by qPCR (n = 4).

FIGURE 4.

Thioredoxin partially inhibits T. whipplei replication in Mφ. A, Different concentrations of thioredoxin were added to infected Mφ daily (▪, 0 ng/ml; □, 0.1 ng/ml; ▨, 1 ng/ml; ▩, 10 ng/ml; ▦, 100 ng/ml). B, Thioredoxin (Trx) or maleimide-modified thioredoxin (mTrx) at 10 ng/ml was added every day to Mφ. C, Different concentrations of thioredoxin were added to T. whipplei cultured in axenic medium (○, 0 ng/ml; •, 1 ng/ml; ▵, 10 ng/ml; ▴, 100 ng/ml). Bacterial DNA copy number was determined by qPCR (n = 4).

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As IL-16 and IL-1β were specifically expressed and released by Mφ in which T. whipplei replicates, we wondered if they would have enabled bacterial replication in Mo that normally eliminated T. whipplei. IL-16 or IL-1β was added to Mo 18 h prior infection, and T. whipplei uptake and replication were assessed by qPCR. IL-16 increased T. whipplei uptake in a dose-dependent manner (Fig. 5,A). In the presence of 10 and 20 ng/ml IL-16, the bacterial uptake was significantly (p = 0.03) enhanced as compared with control values. IL-16 also increased T. whipplei replication in a dose-dependent manner (Fig. 5,B). Increased bacterial replication occurred without changes in bacterial uptake (compare Fig. 5, B and A). In the presence of 10 ng/ml IL-16, T. whipplei replication was maximal and decreased for higher concentrations of IL-16. When IL-16 was added after Mo infection, Mo eliminated T. whipplei as did untreated Mo (data not shown). In contrast to IL-16, IL-1β had no effect on T. whipplei replication. Indeed, adding exogenous IL-1β (concentrations ranging from 1 pg/ml to 1 ng/ml) to Mo culture before infection did not allow bacterial replication nor survival.

FIGURE 5.

IL-16 promotes T. whipplei replication. (A–C) Different concentrations of IL-16 (□, none; ▪, 2 ng/ml; ▦, 10 ng/ml; ▨, 20 ng/ml; ▦, 40 ng/ml) were added to Mo and Mφ 18 h prior infection. A, Mo uptake of T. whipplei was quantified after 4 h. Mo (B) and Mφ (C) were cultured for 9 and 12 days. The bacterial DNA copy number was determined by qPCR (n = 4). D, Infected Mφ were cultured in the presence of anti-IL-16 Abs (1 μg/ml added every 2 days during 12 days), and the copy number of bacterial DNA was determined by qPCR (n = 4) (E) Mo (□) and Mφ (▪) were incubated with IL-16 for 18 h and stimulated or not with T. whipplei for 6 h. Transcripts encoding IL-16 (IL16), IL-1β (IL1B), Bak1 (BAK1), caspase-8 (CASP8), thioredoxin (TXN), glutaredoxin (GLRX), IL-10 (IL10), and TGF-β1 (TGFB1) were quantified by qRT-PCR. Results are expressed as the ratio of expression levels in T. whipplei-infected vs uninfected cells following IL-16 treatment (n = 4).

FIGURE 5.

IL-16 promotes T. whipplei replication. (A–C) Different concentrations of IL-16 (□, none; ▪, 2 ng/ml; ▦, 10 ng/ml; ▨, 20 ng/ml; ▦, 40 ng/ml) were added to Mo and Mφ 18 h prior infection. A, Mo uptake of T. whipplei was quantified after 4 h. Mo (B) and Mφ (C) were cultured for 9 and 12 days. The bacterial DNA copy number was determined by qPCR (n = 4). D, Infected Mφ were cultured in the presence of anti-IL-16 Abs (1 μg/ml added every 2 days during 12 days), and the copy number of bacterial DNA was determined by qPCR (n = 4) (E) Mo (□) and Mφ (▪) were incubated with IL-16 for 18 h and stimulated or not with T. whipplei for 6 h. Transcripts encoding IL-16 (IL16), IL-1β (IL1B), Bak1 (BAK1), caspase-8 (CASP8), thioredoxin (TXN), glutaredoxin (GLRX), IL-10 (IL10), and TGF-β1 (TGFB1) were quantified by qRT-PCR. Results are expressed as the ratio of expression levels in T. whipplei-infected vs uninfected cells following IL-16 treatment (n = 4).

Close modal

We also added IL-16 to Mφ 18 h prior infection, and we studied T. whipplei replication (Fig. 5,C). Although T. whipplei uptake was slightly increased in response to IL-16 (data not shown), the replication of T. whipplei was significantly higher (p = 0.03) than in untreated Mφ, particularly when IL-16 was used at 10 ng/ml, with a number of copies of bacterial DNA doubling at day 12. When IL-16 was added after Mφ infection, the replication of T. whipplei did not change (data not shown). Finally, infected Mφ were treated with anti-IL-16 Abs (1 μg/ml), and T. whipplei replication was assessed at day 9 and 12 postinfection. Results clearly showed that adding anti-IL-16 Abs to infected Mφ completely abrogated bacterial replication (Fig. 5 D), demonstrating that IL-16 is a crucial mediator in T. whipplei replication.

We investigated further some putative mechanisms used by IL-16 favoring T. whipplei replication. Mo and Mφ were incubated with IL-16 (10 ng/ml) for 18 h and then stimulated or not by T. whipplei for 6 h. Their responses were determined by qRT-PCR targeting some genes already identified in T. whipplei-infected Mφ and not previously identified genes, such as IL-10 and TGF-β1, two cytokines known to inhibit the microbicidal machinery of Mo and Mφ. IL-16 abolished the up-regulation of thioredoxin- and glutaredoxin-encoding genes by Mo stimulated by T. whipplei and strongly repressed them in T. whipplei-stimulated Mφ (Fig. 5,E). In contrast, IL-16 induced a clear up-regulation of genes encoding IL-16, IL-1β, caspase-8, and Bak 1 in T. whipplei-stimulated Mφ but only up-regulated IL-1β gene in infected Mo. It must be noted that IL-16 also increased the expression of IL-10 and TGF-β1 genes in Mφ, but not in Mo, stimulated by T. whipplei (Fig. 5E). Finally, we wondered if IL-16 promoted Mo differentiation into Mφ, rendering them able to support T. whipplei replication. For that purpose, Mo were incubated with and without IL-16 (10 ng/ml) for 18 h, washed, cultured for 2 and 6 days, and assayed for CD16 and CD68. As revealed by flow cytometry, IL-16-pretreated Mo expressed higher levels of CD16 and CD68 than untreated ones after 2 days of culture (Table III). In contrast, CD16 and CD68 expression was similar in IL-16-treated and untreated cells after 6 days of culture (Table III), suggesting that IL-16 accelerated Mo differentiation into Mφ. Taken together, these results suggest that IL-16 exerts pleiotropic effects on Mo and Mφ.

Table III.

IL-16 effects on monocyte maturationa

Day 2Day 6
− IL-16+ IL-16− IL-16+ IL-16
CD16 31.6 ± 4.3 53.0 ± 5.1 79.3 ± 1.6 85.4 ± 3.6 
CD68 40.5 ± 4.7 61.8 ± 7.6 95.7 ± 0.9 91.5 ± 1.2 
Day 2Day 6
− IL-16+ IL-16− IL-16+ IL-16
CD16 31.6 ± 4.3 53.0 ± 5.1 79.3 ± 1.6 85.4 ± 3.6 
CD68 40.5 ± 4.7 61.8 ± 7.6 95.7 ± 0.9 91.5 ± 1.2 
a

Data are expressed as mean ± SD of the percentage of positive cells (n = 3).

IL-16 was associated with T. whipplei replication in Mφ from healthy individuals; therefore, we investigated its role in Mo and Mφ from WD patients. T. whipplei organisms were killed by Mo from WD patients (data not shown) as efficiently as those from healthy subjects. In patient’s Mφ, T. whipplei replication was associated with the clinical status of patients. Indeed, the number of bacterial DNA copies was significantly higher in Mφ from untreated patients than in control ones at day 12 (Fig. 6,A). The number of copies of bacterial DNA was lower in Mφ of successfully treated patients than in those of untreated patients and was similar to controls (Fig. 6,A). The production of IL-16 was assessed in culture supernatants from Mφ stimulated with T. whipplei. IL-16 amounts were similar in WD patients and controls (data not shown). Hence, IL-16 production by Mφ was not sufficient to account for T. whipplei replication. In contrast, circulating amounts of IL-16 were significantly higher in untreated WD patients than in control subjects and treated WD patients (Fig. 6 B). Finally, circulating IL-16 amounts were studied in patients before and after the establishment of successful treatment. IL-16 levels decreased within 3 mo to reach values measured in control subjects (data not shown). Taken together, these results showed that T. whipplei replication in Mφ and high levels of circulating IL-16 are associated in WD.

FIGURE 6.

IL-16 is critical in WD patients. A, Mφ from controls, untreated WD patients, and successfully treated WD patients were infected with T. whipplei for 4 h and cultured for 12 days. T. whipplei DNA copy number was determined by qPCR at day 12. B, Circulating IL-16 were measured on plasma from controls, untreated, and successfully treated patients. ∗ and ∗∗, p = 0.002 and p = 0.001, respectively.

FIGURE 6.

IL-16 is critical in WD patients. A, Mφ from controls, untreated WD patients, and successfully treated WD patients were infected with T. whipplei for 4 h and cultured for 12 days. T. whipplei DNA copy number was determined by qPCR at day 12. B, Circulating IL-16 were measured on plasma from controls, untreated, and successfully treated patients. ∗ and ∗∗, p = 0.002 and p = 0.001, respectively.

Close modal

Since the initial description of Whipple’s disease, it was suggested that deficient Mφ function was critical for the development of the disease, although the nature of this deficiency remained unknown. We showed in the present study that T. whipplei organisms were killed by Mo, whereas they replicated in Mφ. These results emphasized previous works in which some Mφ functions from WD patients were defective (13, 22).

The replication of T. whipplei in Mφ was associated with a specific gene expression pattern. This pattern was distinct from classical patterns observed in Mφ (23) stimulated by LPS (24, 25), Salmonella enterica serovar Typhimurium (26), Listeria monocytogenes (27), and Brucella abortus (28). It consisted of modulation of cytokine/chemokine genes and induction of an apoptotic program. Among cytokine/chemokine genes, the IL-16 encoding gene occupied an unexpected position. T. whipplei stimulated its expression and release by Mφ. IL-16 has been well known to be constitutively expressed by T cells, mast cells, dendritic cells, and circulating Mo (29). T. whipplei also induced the expression of the IL-1β encoding gene and the release of IL-1β by Mφ. As IL-1β induced the release of IL-16 in human fibroblasts (30), it was tempting to relate IL-1β and IL-16 overexpression and release by T. whipplei-stimulated Mφ. Nevertheless, IL-1β had no direct effect on T. whipplei replication in contrast to IL-16. The second major feature of transcriptional pattern of T. whipplei-stimulated Mφ was the up-regulation of genes involved in apoptosis. T. whipplei induced apoptosis of Mφ but not of Mo. The induction of apoptosis may enable the release of IL-16, as recently reported in another experimental model (29). Thus, one could relate the lack of T. whipplei-stimulated Mo apoptosis to that of IL-16 release (29). T. whipplei-stimulated Mφ had acquired some features of the alternatively activated Mφ through the up-regulation of Mφ scavenger receptors and the down-modulation of IL-12. The expression of the gene encoding IL-12p35 was down-modulated in Mφ and in Mo. This finding was consistent with previous reports in which the low production of IL-12 by Mo was associated with WD (14). Decreased IL-12 production may prevent the development of Th1 immune response and favor the shift toward Th2 response (31).

The killing of T. whipplei by Mo was associated with a transcriptional program in which the genes associated with bacterial replication were silent or down-modulated. Thioredoxin and glutaredoxin expression were specifically up-regulated in T. whipplei-infected Mo. This up-regulation has not been reported so far in response to other pathogens, except in Brucella-infected cells (24, 28). Thioredoxin pathway is potentially important for host response to T. whipplei, as it represents the only bacterium with a genome lacking homologues for thioredoxin and thioredoxin reductase genes, as well as glutaredoxin and glutathione reductase genes (7). Thioredoxin and glutaredoxin are antioxidant defense lines: they regulate cellular functions by controlling the intracellular redox status through oxidation of NADPH from oxidized thioredoxin and glutaredoxin. Adding thioredoxin to Mφ partly inhibited T. whipplei replication. Because thioredoxin was not active on axenic T. whipplei, it is likely that it stimulates the microbicidal program of Mφ. In accordance with this hypothesis, it has been demonstrated that thioredoxin activates NF-κB (32, 33) and AP-1 transcription (34, 35). Intracellular redox status also affects the cytokine release by Mφ and therefore influences the Th1/Th2 balance (36). Indeed, mice overexpressing human thioredoxin exhibit a long-term T cell polarization toward Th1 profile (37), depending on the enzymatic activity of thioredoxin (38). Finally, thioredoxin and/or glutaredoxin systems were shown antiapoptotic in mammalian cells (39).

The most important feature of our work was that exogenous IL-16 enabled T. whipplei replication in Mo as endogenous IL-16 did in Mφ. Adding IL-16 to Mo induced T. whipplei replication and even increased this bacterial replication in Mφ. Blocking IL-16 activity with anti-IL-16 Abs completely prevented T. whipplei replication in Mφ. IL-16 may have directly inhibited Mφ competence. IL-16 has been shown to interfere with cell-mediated immune response (40) and/or with the development of tolerogenic dendritic cells (41), probably through the binding of CD4 (42). First, we found that IL-16 down-modulated the expression of thioredoxin and glutaredoxin that was associated with T. whipplei killing by Mo. IL-16 also up-regulated its own expression and that of proapoptotic genes and immunoregulatory genes such as IL-10 and TGF-β1. These two latter genes have been shown to interfere with the acquisition of microbicidal competence. Second, IL-16 affected the maturation program of Mo. Indeed, it accelerated the Mo maturation into Mφ. Finally, the endogenous production of IL-16 was not sufficient to account for the replication of T. whipplei in Mφ from untreated patients even if bacterial replication was related to high IL-16 circulating levels found in these patients. This finding extends the role of IL-16 to the assessment of WD activity. IL-16 has been involved in diseases characterized by CD4+ T lymphocyte infiltrates, including atopic diseases (40) and sarcoidosis granulomas (43). It was increased in colonic tissues from patients with Crohn’s disease, another chronic intestinal disease (44). Hence, the response of Mo and Mφ to IL-16 is likely critical for T. whipplei replication in WD. This response was directly related to the activity of the disease because the successful treatment of WD patients was associated with decreased circulating levels of IL-16 and T. whipplei replication in Mφ, suggesting that elevated circulating IL-16 levels are a consequence of chronic infection.

The present study describes an unreported Mφ activation program in infectious diseases. T. whipplei replication was associated with IL-16 secretion and apoptosis of Mφ. Mo microbicidal activity was dependent on thioredoxin and regulated by IL-16. Increased amounts of circulating IL-16 in WD patients may support T. whipplei replication.

We are grateful to Prof. Philippe Sansonetti, Dr. Pablo Gluschankof, and Prof. Georges Grau for helpful suggestions and critical comments on an early draft.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This study was supported by the grant “Développement d’outils pour le diagnostic et le suivi des patients atteints de la maladie de Whipple” (PHRC 2001 UF 1658) funded through “Crédits ministériels Programme Hospitalier de Recherche Clinique” and through the Fifth Programme Cadre de Recherche Technologique of the European Community (Grant QRLT-2001-01049).

3

Abbreviations used in this paper: WD, Whipple’s disease; EST, expressed sequence tag; Mo, monocyte; Mφ, macrophage; qPCR, quantitative real-time PCR; qRT-PCR, quantitative real-time RT-PCR.

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