| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Unité des Rickettsies, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 6020, Institut Fédératif de Recherche 48, Université de la Méditerranée, Faculté de Médecine, Marseille, France
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
) 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 M inhibited bacterial replication. IL-16, which was up-regulated in M, enabled T. whipplei to replicate in monocytes and increased bacterial replication in M. In addition, anti-IL-16 Abs abolished T. whipplei replication in M. 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. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
) 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 M suggested that T. whipplei had a tropism for myeloid cells. It is well known that the microbicidal activity of monocytes (Mo) and M can be disarmed by immunoregulatory cytokines such as IL-4 or IL-10 and is stimulated by T cell-mediated signals such as IFN-
(11). T. whipplei multiplies after cytokine-mediated deactivation of Mo (12). The chronic evolution of WD evokes long-term persistence of T. whipplei in M, which requires the inhibition of M microbicidal competence. Although impaired microbicidal activity in M (13) and cell-mediated immunity, including defective production of IL-12 and IFN- (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 M 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 M, where its expression partly reduced T. whipplei replication. Conversely, IL-16 was found to be produced only by T. whipplei-infected M 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 M and the understanding of the WD pathophysiology.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
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.
Bacteria
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).
Infection procedure
Mo from circulating PBMC were isolated by Ficoll gradient (MSL; Eurobio) and adherence in flat-bottom 24-well plates (Nunc; PolyLabo). M were derived from Mo by a 7-day culture, as described previously (18). Mo and M (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 M 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-1
(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.
Quantitative real-time PCR (qPCR)
We lysed Mo and M, 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).
Synthesis of cDNA probes and hybridization
Mo and M 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.
Quantitative real-time RT-PCR (qRT-PCR)
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-1 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 -actin endogenous control (ACTBf, 5'-GGAAATCGTGCGTGACATTA-3', and ACTBr, 5'-AGGAAGGAAGGCTGGAAGAG-3') was determined as follows: fold change = 2–
Ct, where Ct = (CtTarget – CtActin)stimulated – (CtTarget – CtActin)unstimulated (21). Ct values were defined as the number of cycles for which the fluorescence signals were detected.
Apoptosis measurement
Mo and M 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).
Immunoassays
Mo and M (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-1. IL-16 and IL-1 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.
Electron microscopy
Infected Mo and M 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.
Flow cytometry
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.
Statistical analysis
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).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
and bacterial killing in Mo
First, we assessed the uptake of T. whipplei by human circulating Mo and Mo-derived M 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. 1A), although the number of T. whipplei DNA copies was significantly higher in M than in Mo at 2, 4, and 8 h. T. whipplei was found inside vacuoles in Mo (data not shown) and M (Fig. 1B) 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 M (Fig. 1C). As T. whipplei uptake was distinct in Mo and M (Fig. 1A), we wondered whether uptake differences would account for changes in survival pattern. Bacterial uptake by Mo was enhanced to levels observed in M by increasing bacterium-to-cell ratio to 200:1. This did not enable T. whipplei survival in Mo. In opposition, decreasing bacterial uptake in M (bacterium-to-cell ratio of 25:1) to the levels found in Mo did not impair T. whipplei replication (Fig. 1D). Hence, our results clearly showed that T. whipplei organisms replicate in M and are killed by Mo.
|
To determine whether T. whipplei survival or killing are associated with different gene expression programs, we monitored transcriptional responses of Mo and M 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 M (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 M. Hence, T. whipplei induced a higher transcriptional activity in M than in Mo. In addition, the transcriptional programs stimulated by T. whipplei were clearly distinct in Mo and M, whereas LPS induced a similar transcriptional pattern. Indeed, hierarchical clustering analysis revealed that Mo and M coclustered in response to LPS stimulation. Clustering algorithm placed the program of T. whipplei-stimulated M on a separate branch of the dendrogram (Fig. 2A). The transcriptional pattern of T. whipplei-stimulated Mo also differed from that elicited by LPS. Taken together, these results showed that Mo and M respond similarly to LPS, but they display two distinct transcriptional programs in response to T. whipplei.
|
In an attempt to understand the molecular basis of T. whipplei intracellular fate in Mo and M, we investigated the genes that were modulated in response to the bacteria (Fig. 2B). Few genes were up-regulated in Mo and M 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 M, as confirmed by qRT-PCR (Fig. 2C). Twenty genes of host defense were up-regulated upon Mo stimulation (Table I) and transcriptionally silent or repressed in M (Fig. 2B). 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. 2C). Conversely, up-regulated host defense genes in T. whipplei-stimulated M were transcriptionally silent in Mo. They included genes encoding ILs such as IL-16 and IL-1 (Fig. 2C), chemokines such as CCL15, CCL26, and XCL1, and genes known to be associated with M deactivation such as M 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. M apoptosis was detected 24 h after T. whipplei stimulation and 20% of M became apoptotic after 48 h (Fig. 2D). Mo, in which a proapoptotic transcriptional program was not induced, did not undergo apoptosis when they were stimulated with T. whipplei (Fig. 2D). Finally, we measured the release of IL-16, IL-1, and thioredoxin by Mo and M. IL-16 was significantly secreted by T. whipplei-stimulated M. This release was specific to M because Mo did not secrete IL-16 (Fig. 3A). Similarly, Il-1 levels were significantly higher in infected M than in unstimulated ones (Fig. 3B). In contrast, release of thioredoxin was up-regulated only in T. whipplei-stimulated Mo (Fig. 3C). Taken together, these results showed that T. whipplei triggers an unusual transcriptional program in M associating proapoptotic pattern and up-regulation of IL-16 and IL-1, whereas the transcriptional program of Mo includes up-regulation of redox-associated molecules.
|
|
|
The production of thioredoxin by Mo upon T. whipplei stimulation suggested that thioredoxin is involved in bacterial killing. We added thioredoxin to M before T. whipplei infection and daily thereafter for 12 days. The effect of thioredoxin was found to be dose dependent (Fig. 4A). 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 M (Fig. 4B). 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. 4C). 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 M, suggests that T. whipplei killing by Mo is partly related to thioredoxin.
|
As IL-16 and IL-1 were specifically expressed and released by M 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-1 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. 5A). 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. 5B). 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-1 had no effect on T. whipplei replication. Indeed, adding exogenous IL-1 (concentrations ranging from 1 pg/ml to 1 ng/ml) to Mo culture before infection did not allow bacterial replication nor survival.
|
18 h prior infection, and we studied T. whipplei replication (Fig. 5C). 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 M, 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 M infection, the replication of T. whipplei did not change (data not shown). Finally, infected M 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 M completely abrogated bacterial replication (Fig. 5D), 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 M 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 M and not previously identified genes, such as IL-10 and TGF-1, two cytokines known to inhibit the microbicidal machinery of Mo and M. 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 M (Fig. 5E). In contrast, IL-16 induced a clear up-regulation of genes encoding IL-16, IL-1, caspase-8, and Bak 1 in T. whipplei-stimulated M but only up-regulated IL-1 gene in infected Mo. It must be noted that IL-16 also increased the expression of IL-10 and TGF-1 genes in M, but not in Mo, stimulated by T. whipplei (Fig. 5 E). Finally, we wondered if IL-16 promoted Mo differentiation into M, 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 M. Taken together, these results suggest that IL-16 exerts pleiotropic effects on Mo and M.
|
IL-16 was associated with T. whipplei replication in M from healthy individuals; therefore, we investigated its role in Mo and M 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 M, T. whipplei replication was associated with the clinical status of patients. Indeed, the number of bacterial DNA copies was significantly higher in M from untreated patients than in control ones at day 12 (Fig. 6A). The number of copies of bacterial DNA was lower in M of successfully treated patients than in those of untreated patients and was similar to controls (Fig. 6A). The production of IL-16 was assessed in culture supernatants from M stimulated with T. whipplei. IL-16 amounts were similar in WD patients and controls (data not shown). Hence, IL-16 production by M 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. 6B). 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 M and high levels of circulating IL-16 are associated in WD.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
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 M. These results emphasized previous works in which some M functions from WD patients were defective (13, 22).
The replication of T. whipplei in M was associated with a specific gene expression pattern. This pattern was distinct from classical patterns observed in M (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 M. 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-1 encoding gene and the release of IL-1 by M. As IL-1 induced the release of IL-16 in human fibroblasts (30), it was tempting to relate IL-1 and IL-16 overexpression and release by T. whipplei-stimulated M. Nevertheless, IL-1 had no direct effect on T. whipplei replication in contrast to IL-16. The second major feature of transcriptional pattern of T. whipplei-stimulated M was the up-regulation of genes involved in apoptosis. T. whipplei induced apoptosis of M 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 M had acquired some features of the alternatively activated M through the up-regulation of M scavenger receptors and the down-modulation of IL-12. The expression of the gene encoding IL-12p35 was down-modulated in M 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 M partly inhibited T. whipplei replication. Because thioredoxin was not active on axenic T. whipplei, it is likely that it stimulates the microbicidal program of M. In accordance with this hypothesis, it has been demonstrated that thioredoxin activates NF-
B (32, 33) and AP-1 transcription (34, 35). Intracellular redox status also affects the cytokine release by M 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 M. Adding IL-16 to Mo induced T. whipplei replication and even increased this bacterial replication in M. Blocking IL-16 activity with anti-IL-16 Abs completely prevented T. whipplei replication in M. IL-16 may have directly inhibited M 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-1. 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 M. Finally, the endogenous production of IL-16 was not sufficient to account for the replication of T. whipplei in M 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 M 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 M, suggesting that elevated circulating IL-16 levels are a consequence of chronic infection.
The present study describes an unreported M activation program in infectious diseases. T. whipplei replication was associated with IL-16 secretion and apoptosis of M. 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.
|
Acknowledgments |
|---|
|
Disclosures |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
|
Footnotes |
|---|
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). 
2 Address correspondence and reprint requests to Dr. Jean-Louis Mege, Unité des Rickettsies, Faculté de Médecine, 27 Boulevard J. Moulin, 13385 Marseille Cedex 5, France. E-mail address: Jean-Louis.Mege{at}medecine.univ-mrs.fr
3 Abbreviations used in this paper: WD, Whipple’s disease; EST, expressed sequence tag; Mo, monocyte; M, macrophage; qPCR, quantitative real-time PCR; qRT-PCR, quantitative real-time RT-PCR.
Received for publication April 15, 2005. Accepted for publication July 19, 2005.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
-chain expressing mononuclear blood cells and transient inhibitory serum factors in Whipple’s disease. Clin. Immunol. Immunopathol. 72:217.-226. [Medline]
. Ann. Intern. Med. 129:875.-877. B in the immune system. Annu. Rev. Immunol. 12:141.-179. [Medline]
B activation. J. Biol. Chem. 272:30952.-30961. Related articles in The JI:
This article has been cited by other articles:
|
|
 |
|
  M. Benoit, B. Desnues, and J.-L. Mege Macrophage Polarization in Bacterial Infections J. Immunol., September 15, 2008; 181(6): 3733 - 3739. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Fenollar, X. Puechal, and D. Raoult Whipple's Disease N. Engl. J. Med., January 4, 2007; 356(1): 55 - 66. [Full Text] [PDF]
|
|
|
|
 |
|
 S. Genot, A. Stein, H. Lepidi, M. Ihrig, D. Raoult, and J.-L. Mege Murine Model of Infection by Tropheryma whipplei. Infect. Immun., August 1, 2006; 74(8): 4915 - 4917. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Moos, D. Kunkel, T. Marth, G. E. Feurle, B. LaScola, R. Ignatius, M. Zeitz, and T. Schneider Reduced Peripheral and Mucosal Tropheryma whipplei-Specific Th1 Response in Patients with Whipple's Disease J. Immunol., August 1, 2006; 177(3): 2015 - 2022. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Desnues, M. Ihrig, D. Raoult, and J.-L. Mege Whipple's Disease: a Macrophage Disease Clin. Vaccine Immunol., February 1, 2006; 13(2): 170 - 178. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||
) and M
) 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, M
: 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.
, 1 ng/ml; 
, 10 ng/ml; 
, 0 ng/ml; •, 1 ng/ml; 
, 10 ng/ml; 
, 100 ng/ml). Bacterial DNA copy number was determined by qPCR (n = 4).
