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IL-16 promotes T. whipplei replication by inhibiting phagosome conversion and modulating macrophage activation.

Ghigo E, Barry AO, Pretat L, Al Moussawi K, Desnues B, Capo C, Kornfeld H, Mege JL - PLoS ONE (2010)

Bottom Line: Second, the full transcriptional response of murine macrophages to T. whipplei showed that T. whipplei specifically modulated the expression of 231 probes in IL-16(-/-) macrophages.Gene Ontology analysis revealed that 10 of 13 over-represented terms were linked to immune responses, including proinflammatory transcriptional factors of the NF-κB family.Our results demonstrated a previously unreported function for IL-16 in promoting bacterial replication through inhibited phagolysosome biogenesis and modulated macrophage activation program.

View Article: PubMed Central - PubMed

Affiliation: URMITE, CNRS UMR 6236-IRD 3R198, Université de la Méditerranée, Marseille, France.

ABSTRACT
The replication of Tropheryma whipplei (the agent of Whipple's disease) within human macrophages is associated with the expression of IL-16, a cytokine known for its chemotactic and inflammatory properties. In this study, we asked whether IL-16 acts on T. whipplei replication by interfering with the endocytic pathway. We observed that in macrophages, T. whipplei was located within late phagosomes that were unable to fuse with lysosomes; in monocytes, T. whipplei was eliminated in phagolysosomes. Moreover, adding IL-16 to monocytes induced bacterial replication and inhibited phagolysosome formation. On the other hand, blocking IL-16 activity, either with anti-IL-16 antibodies in human macrophages or by using murine IL-16(-/-) bone marrow-derived macrophages, inhibited T. whipplei replication and rescued phagolysosome biogenesis. Furthermore, we propose that IL-16-mediated interference with the endocytic pathway is likely related to macrophage activation. First, IFNγ induced T. whipplei elimination and phagolysosome formation and inhibited IL-16 production by macrophages. Second, the full transcriptional response of murine macrophages to T. whipplei showed that T. whipplei specifically modulated the expression of 231 probes in IL-16(-/-) macrophages. Gene Ontology analysis revealed that 10 of 13 over-represented terms were linked to immune responses, including proinflammatory transcriptional factors of the NF-κB family. Our results demonstrated a previously unreported function for IL-16 in promoting bacterial replication through inhibited phagolysosome biogenesis and modulated macrophage activation program.

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Effect of exogenous IL-16 on T. whipplei fate in macrophages.Macrophages were treated with rhIL-16 as described in Figure 3. (A) T. whipplei uptake (inset) and replication were determined by qPCR. The results are expressed as the mean ± SEM of four independent experiments performed in triplicate. (B) The intracellular localization of T. whipplei was analyzed by indirect immunofluorescence and laser scanning microscopy at day 12. Scale bars represent 5 µm. (C) The percentage of organisms that colocalized with Lamp-1 or cathepsin D, respectively, was determined. More than 300 phagosomes were examined per experimental condition, and the results are expressed as the mean ± SEM of four independent experiments. p<0.05. (D) The localization of organisms with p62, a specific marker for autophagosomes, was assessed by indirect immunofluorescence and laser scanning microscopy. More than 200 phagosomes were examined in two independent experiments.
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pone-0013561-g004: Effect of exogenous IL-16 on T. whipplei fate in macrophages.Macrophages were treated with rhIL-16 as described in Figure 3. (A) T. whipplei uptake (inset) and replication were determined by qPCR. The results are expressed as the mean ± SEM of four independent experiments performed in triplicate. (B) The intracellular localization of T. whipplei was analyzed by indirect immunofluorescence and laser scanning microscopy at day 12. Scale bars represent 5 µm. (C) The percentage of organisms that colocalized with Lamp-1 or cathepsin D, respectively, was determined. More than 300 phagosomes were examined per experimental condition, and the results are expressed as the mean ± SEM of four independent experiments. p<0.05. (D) The localization of organisms with p62, a specific marker for autophagosomes, was assessed by indirect immunofluorescence and laser scanning microscopy. More than 200 phagosomes were examined in two independent experiments.

Mentions: Given that IL-16 release is related to T. whipplei replication [6], we asked if exogenous IL-16 inhibits the conversion of T. whipplei phagosomes to phagolysosomes. To address this question, monocytes were treated for 16 hours with 10 ng/ml of recombinant human (rh) IL-16 prior to T. whipplei infection [6]. The pretreatment of monocytes with IL-16 did not significantly alter bacterial uptake (Figure 3A, inset). T. whipplei replication occurred to an extent similar to that observed in macrophages (comparison between Figure 3A and Figure 1) as published before [6]. However, T. whipplei colocalized with Lamp-1 but not with cathepsin D in IL-16-treated monocytes (Figure 3B). The percentage of phagosomes containing T. whipplei colocalizing with cathepsin D fell significantly (p<0.05) (two-fold) between days 0 and 1. At day 12, T. whipplei was only present in IL-16-treated cells, in which it resided in phagosomes associated with Lamp-1, but not with cathepsin D (Figure 3C). We also studied the effect of exogenous IL-16 on the intracellular fate of T. whipplei in macrophages. IL-16 did not affect T. whipplei uptake but it significantly (p<0.05) increased bacterial replication (Figure 4A) as published before [6]. It also inhibited the acquisition of cathepsin D by T. whipplei phagosomes (Figure 4, B and C). Next, we wondered whether IL-16 re-routed T. whipplei phagosomes towards the autophagosome pathway. We found that Ab directed against p62, a marker for autophagosomes [35], did not colocalize with T. whipplei (Figure 4D). Finally, we investigated if the effect of IL-16 on T. whipplei trafficking was specific using latex beads. Latex beads were located within phagolysosomes at days 1 and 12 post-ingestion. IL-16 did not modify the latex beads localization with either of the two markers (Figure S1), demonstrating that IL-16 specifically acted on T. whipplei trafficking. Taken together, these data show that incubating monocytes or macrophages with exogenous IL-16 can increase T. whipplei replication within phagosomes that are unable to be converted to phagolysosomes.


IL-16 promotes T. whipplei replication by inhibiting phagosome conversion and modulating macrophage activation.

Ghigo E, Barry AO, Pretat L, Al Moussawi K, Desnues B, Capo C, Kornfeld H, Mege JL - PLoS ONE (2010)

Effect of exogenous IL-16 on T. whipplei fate in macrophages.Macrophages were treated with rhIL-16 as described in Figure 3. (A) T. whipplei uptake (inset) and replication were determined by qPCR. The results are expressed as the mean ± SEM of four independent experiments performed in triplicate. (B) The intracellular localization of T. whipplei was analyzed by indirect immunofluorescence and laser scanning microscopy at day 12. Scale bars represent 5 µm. (C) The percentage of organisms that colocalized with Lamp-1 or cathepsin D, respectively, was determined. More than 300 phagosomes were examined per experimental condition, and the results are expressed as the mean ± SEM of four independent experiments. p<0.05. (D) The localization of organisms with p62, a specific marker for autophagosomes, was assessed by indirect immunofluorescence and laser scanning microscopy. More than 200 phagosomes were examined in two independent experiments.
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pone-0013561-g004: Effect of exogenous IL-16 on T. whipplei fate in macrophages.Macrophages were treated with rhIL-16 as described in Figure 3. (A) T. whipplei uptake (inset) and replication were determined by qPCR. The results are expressed as the mean ± SEM of four independent experiments performed in triplicate. (B) The intracellular localization of T. whipplei was analyzed by indirect immunofluorescence and laser scanning microscopy at day 12. Scale bars represent 5 µm. (C) The percentage of organisms that colocalized with Lamp-1 or cathepsin D, respectively, was determined. More than 300 phagosomes were examined per experimental condition, and the results are expressed as the mean ± SEM of four independent experiments. p<0.05. (D) The localization of organisms with p62, a specific marker for autophagosomes, was assessed by indirect immunofluorescence and laser scanning microscopy. More than 200 phagosomes were examined in two independent experiments.
Mentions: Given that IL-16 release is related to T. whipplei replication [6], we asked if exogenous IL-16 inhibits the conversion of T. whipplei phagosomes to phagolysosomes. To address this question, monocytes were treated for 16 hours with 10 ng/ml of recombinant human (rh) IL-16 prior to T. whipplei infection [6]. The pretreatment of monocytes with IL-16 did not significantly alter bacterial uptake (Figure 3A, inset). T. whipplei replication occurred to an extent similar to that observed in macrophages (comparison between Figure 3A and Figure 1) as published before [6]. However, T. whipplei colocalized with Lamp-1 but not with cathepsin D in IL-16-treated monocytes (Figure 3B). The percentage of phagosomes containing T. whipplei colocalizing with cathepsin D fell significantly (p<0.05) (two-fold) between days 0 and 1. At day 12, T. whipplei was only present in IL-16-treated cells, in which it resided in phagosomes associated with Lamp-1, but not with cathepsin D (Figure 3C). We also studied the effect of exogenous IL-16 on the intracellular fate of T. whipplei in macrophages. IL-16 did not affect T. whipplei uptake but it significantly (p<0.05) increased bacterial replication (Figure 4A) as published before [6]. It also inhibited the acquisition of cathepsin D by T. whipplei phagosomes (Figure 4, B and C). Next, we wondered whether IL-16 re-routed T. whipplei phagosomes towards the autophagosome pathway. We found that Ab directed against p62, a marker for autophagosomes [35], did not colocalize with T. whipplei (Figure 4D). Finally, we investigated if the effect of IL-16 on T. whipplei trafficking was specific using latex beads. Latex beads were located within phagolysosomes at days 1 and 12 post-ingestion. IL-16 did not modify the latex beads localization with either of the two markers (Figure S1), demonstrating that IL-16 specifically acted on T. whipplei trafficking. Taken together, these data show that incubating monocytes or macrophages with exogenous IL-16 can increase T. whipplei replication within phagosomes that are unable to be converted to phagolysosomes.

Bottom Line: Second, the full transcriptional response of murine macrophages to T. whipplei showed that T. whipplei specifically modulated the expression of 231 probes in IL-16(-/-) macrophages.Gene Ontology analysis revealed that 10 of 13 over-represented terms were linked to immune responses, including proinflammatory transcriptional factors of the NF-κB family.Our results demonstrated a previously unreported function for IL-16 in promoting bacterial replication through inhibited phagolysosome biogenesis and modulated macrophage activation program.

View Article: PubMed Central - PubMed

Affiliation: URMITE, CNRS UMR 6236-IRD 3R198, Université de la Méditerranée, Marseille, France.

ABSTRACT
The replication of Tropheryma whipplei (the agent of Whipple's disease) within human macrophages is associated with the expression of IL-16, a cytokine known for its chemotactic and inflammatory properties. In this study, we asked whether IL-16 acts on T. whipplei replication by interfering with the endocytic pathway. We observed that in macrophages, T. whipplei was located within late phagosomes that were unable to fuse with lysosomes; in monocytes, T. whipplei was eliminated in phagolysosomes. Moreover, adding IL-16 to monocytes induced bacterial replication and inhibited phagolysosome formation. On the other hand, blocking IL-16 activity, either with anti-IL-16 antibodies in human macrophages or by using murine IL-16(-/-) bone marrow-derived macrophages, inhibited T. whipplei replication and rescued phagolysosome biogenesis. Furthermore, we propose that IL-16-mediated interference with the endocytic pathway is likely related to macrophage activation. First, IFNγ induced T. whipplei elimination and phagolysosome formation and inhibited IL-16 production by macrophages. Second, the full transcriptional response of murine macrophages to T. whipplei showed that T. whipplei specifically modulated the expression of 231 probes in IL-16(-/-) macrophages. Gene Ontology analysis revealed that 10 of 13 over-represented terms were linked to immune responses, including proinflammatory transcriptional factors of the NF-κB family. Our results demonstrated a previously unreported function for IL-16 in promoting bacterial replication through inhibited phagolysosome biogenesis and modulated macrophage activation program.

Show MeSH
Related in: MedlinePlus