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Proteophosophoglycans regurgitated by Leishmania-infected sand flies target the L-arginine metabolism of host macrophages to promote parasite survival.

Rogers M, Kropf P, Choi BS, Dillon R, Podinovskaia M, Bates P, Müller I - PLoS Pathog. (2009)

Bottom Line: This early exacerbation was attributed to two fundamental properties of PSG: Firstly, PSG powerfully recruited macrophages to the dermal site of infection within 24 hours.Secondly, PSG enhanced alternative activation and arginase activity of host macrophages, thereby increasing L-arginine catabolism and the synthesis of polyamines essential for intracellular parasite growth.The increase in arginase activity promoted the intracellular growth of L. mexicana within classically activated macrophages, and inhibition of macrophage arginase completely ablated the early exacerbatory properties of PSG in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Imperial College of Science, Technology and Medicine, London, UK. matthew.rogers@imperial.ac.uk

ABSTRACT
All natural Leishmania infections start in the skin; however, little is known of the contribution made by the sand fly vector to the earliest events in mammalian infection, especially in inflamed skin that can rapidly kill invading parasites. During transmission sand flies regurgitate a proteophosphoglycan gel synthesized by the parasites inside the fly midgut, termed promastigote secretory gel (PSG). Regurgitated PSG can exacerbate cutaneous leishmaniasis. Here, we show that the amount of Leishmania mexicana PSG regurgitated by Lutzomyia longipalpis sand flies is proportional to the size of its original midgut infection and the number of parasites transmitted. Furthermore, PSG could exacerbate cutaneous L. mexicana infection for a wide range of doses (10-10,000 parasites) and enhance infection by as early as 48 hours in inflamed dermal air pouches. This early exacerbation was attributed to two fundamental properties of PSG: Firstly, PSG powerfully recruited macrophages to the dermal site of infection within 24 hours. Secondly, PSG enhanced alternative activation and arginase activity of host macrophages, thereby increasing L-arginine catabolism and the synthesis of polyamines essential for intracellular parasite growth. The increase in arginase activity promoted the intracellular growth of L. mexicana within classically activated macrophages, and inhibition of macrophage arginase completely ablated the early exacerbatory properties of PSG in vitro and in vivo. Thus, PSG is an essential component of the infectious sand fly bite for the early establishment of Leishmania in skin, which should be considered when designing and screening therapies against leishmaniasis.

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Exposure to PSG increases the alternative activation and arginase activity of macrophages.(A) 5×105 unstimulated, alternatively activated and classically activated BALB/c BMMΦ were incubated in the presence or absence of 1 µg L. mexicana PSG for 48 hours. Western blot of macrophage lysates (3 µg protein/lane) were probed for murine Arginase-1, Ym-1, iNOS and β-actin was used as loading control. (B&C) Arginase assay of L. mexicana-infected (B) or uninfected (C) macrophage lysates (unstimulated, AA or CA). (D) NO assay of infected macrophage culture supernatants. Macrophages were co-incubated with 1 µg L. mexicana PSG, 1 µg Lu. longipalpis sand fly saliva or 1 µg PSG and saliva for 48 hours. Data is representative of duplicate (A) or triplicate (B–D) experiments and normalised to the average unistimulated+saline control macrophage arginase/iNOs activity. Unless they are linked with a bar all test groups are compared to their relevant saline control; ns, not significant P>0.05; *, P<0.05; **, P<0.005; ***, P<0.0005.
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ppat-1000555-g005: Exposure to PSG increases the alternative activation and arginase activity of macrophages.(A) 5×105 unstimulated, alternatively activated and classically activated BALB/c BMMΦ were incubated in the presence or absence of 1 µg L. mexicana PSG for 48 hours. Western blot of macrophage lysates (3 µg protein/lane) were probed for murine Arginase-1, Ym-1, iNOS and β-actin was used as loading control. (B&C) Arginase assay of L. mexicana-infected (B) or uninfected (C) macrophage lysates (unstimulated, AA or CA). (D) NO assay of infected macrophage culture supernatants. Macrophages were co-incubated with 1 µg L. mexicana PSG, 1 µg Lu. longipalpis sand fly saliva or 1 µg PSG and saliva for 48 hours. Data is representative of duplicate (A) or triplicate (B–D) experiments and normalised to the average unistimulated+saline control macrophage arginase/iNOs activity. Unless they are linked with a bar all test groups are compared to their relevant saline control; ns, not significant P>0.05; *, P<0.05; **, P<0.005; ***, P<0.0005.

Mentions: Neither PSG nor sand fly saliva, alone or in combination, could affect the phagocytosis of L. mexicana metacyclic promastigotes into macrophages (Fig. S4A&B), or the biogenesis of the macrophage phagolysosome in vitro (Fig. S5A–L). Instead our results from air pouch macrophages (Fig. 3C&D) suggest that PSG may condition macrophages for enhanced parasite survival soon after sand fly delivery. Western blot revealed that PSG did not dampen classical activation in macrophages, as shown by unaltered levels of iNOS expression, instead, it enhanced the alternative activation of macrophages. This was shown by an increased arginase-1 and Ym-1 expression (Fig. 5A), and increased surface expression of CD206, the murine mannose receptor (Fig. S6) in IL-4 treated cells (AAMΦ). To examine this effect further, the activity of two key macrophage enzymes involved in Leishmania killing or Leishmania proliferation: iNOS and arginase were measured in response to PSG, saliva, PSG and saliva or deglycosylated PSG. We found that the arginase levels of L. mexicana-infected unstimulated, AAMΦ or CAMΦ in vitro significantly increased in the presence of PSG (Fig. 5B, P = 0.007–0.0003). The same effect was demonstrated for uninfected macrophages indicating that the PSG can enhance the alternatively activated state before infection (Fig. 5C, P = 0.045–0.003). By contrast, saliva from the vector Lu. longipalpis did not influence the arginase activity of macrophages under a range of activation states tested (unstimulated, CA, AA), or L. mexicana infection (Fig. 5B&C). Furthermore, no synergy between PSG and saliva was observed for arginase activity except for a mild additive effect in the CAMΦ (infected and uninfected) and in unstimulated, uninfected macrophages (Fig. 5B&C). Compared to the native PSG, deglycosylated PSG could not significantly increase arginase activities of macrophages under any of the activation stated tested in the presence or absence of infection (Fig. 5B&C). In CAMΦ, neither L. mexicana PSG nor Lu. longipalpis saliva, alone or in combination, in the presence or absence of infection could affect the activity of iNOS and the generation of the leishmanicidal metabolite NO (Fig. 5D). These results indicate that PSG can enhance the capacity of their host cells to support Leishmania infection by modulating their arginase activity.


Proteophosophoglycans regurgitated by Leishmania-infected sand flies target the L-arginine metabolism of host macrophages to promote parasite survival.

Rogers M, Kropf P, Choi BS, Dillon R, Podinovskaia M, Bates P, Müller I - PLoS Pathog. (2009)

Exposure to PSG increases the alternative activation and arginase activity of macrophages.(A) 5×105 unstimulated, alternatively activated and classically activated BALB/c BMMΦ were incubated in the presence or absence of 1 µg L. mexicana PSG for 48 hours. Western blot of macrophage lysates (3 µg protein/lane) were probed for murine Arginase-1, Ym-1, iNOS and β-actin was used as loading control. (B&C) Arginase assay of L. mexicana-infected (B) or uninfected (C) macrophage lysates (unstimulated, AA or CA). (D) NO assay of infected macrophage culture supernatants. Macrophages were co-incubated with 1 µg L. mexicana PSG, 1 µg Lu. longipalpis sand fly saliva or 1 µg PSG and saliva for 48 hours. Data is representative of duplicate (A) or triplicate (B–D) experiments and normalised to the average unistimulated+saline control macrophage arginase/iNOs activity. Unless they are linked with a bar all test groups are compared to their relevant saline control; ns, not significant P>0.05; *, P<0.05; **, P<0.005; ***, P<0.0005.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2722086&req=5

ppat-1000555-g005: Exposure to PSG increases the alternative activation and arginase activity of macrophages.(A) 5×105 unstimulated, alternatively activated and classically activated BALB/c BMMΦ were incubated in the presence or absence of 1 µg L. mexicana PSG for 48 hours. Western blot of macrophage lysates (3 µg protein/lane) were probed for murine Arginase-1, Ym-1, iNOS and β-actin was used as loading control. (B&C) Arginase assay of L. mexicana-infected (B) or uninfected (C) macrophage lysates (unstimulated, AA or CA). (D) NO assay of infected macrophage culture supernatants. Macrophages were co-incubated with 1 µg L. mexicana PSG, 1 µg Lu. longipalpis sand fly saliva or 1 µg PSG and saliva for 48 hours. Data is representative of duplicate (A) or triplicate (B–D) experiments and normalised to the average unistimulated+saline control macrophage arginase/iNOs activity. Unless they are linked with a bar all test groups are compared to their relevant saline control; ns, not significant P>0.05; *, P<0.05; **, P<0.005; ***, P<0.0005.
Mentions: Neither PSG nor sand fly saliva, alone or in combination, could affect the phagocytosis of L. mexicana metacyclic promastigotes into macrophages (Fig. S4A&B), or the biogenesis of the macrophage phagolysosome in vitro (Fig. S5A–L). Instead our results from air pouch macrophages (Fig. 3C&D) suggest that PSG may condition macrophages for enhanced parasite survival soon after sand fly delivery. Western blot revealed that PSG did not dampen classical activation in macrophages, as shown by unaltered levels of iNOS expression, instead, it enhanced the alternative activation of macrophages. This was shown by an increased arginase-1 and Ym-1 expression (Fig. 5A), and increased surface expression of CD206, the murine mannose receptor (Fig. S6) in IL-4 treated cells (AAMΦ). To examine this effect further, the activity of two key macrophage enzymes involved in Leishmania killing or Leishmania proliferation: iNOS and arginase were measured in response to PSG, saliva, PSG and saliva or deglycosylated PSG. We found that the arginase levels of L. mexicana-infected unstimulated, AAMΦ or CAMΦ in vitro significantly increased in the presence of PSG (Fig. 5B, P = 0.007–0.0003). The same effect was demonstrated for uninfected macrophages indicating that the PSG can enhance the alternatively activated state before infection (Fig. 5C, P = 0.045–0.003). By contrast, saliva from the vector Lu. longipalpis did not influence the arginase activity of macrophages under a range of activation states tested (unstimulated, CA, AA), or L. mexicana infection (Fig. 5B&C). Furthermore, no synergy between PSG and saliva was observed for arginase activity except for a mild additive effect in the CAMΦ (infected and uninfected) and in unstimulated, uninfected macrophages (Fig. 5B&C). Compared to the native PSG, deglycosylated PSG could not significantly increase arginase activities of macrophages under any of the activation stated tested in the presence or absence of infection (Fig. 5B&C). In CAMΦ, neither L. mexicana PSG nor Lu. longipalpis saliva, alone or in combination, in the presence or absence of infection could affect the activity of iNOS and the generation of the leishmanicidal metabolite NO (Fig. 5D). These results indicate that PSG can enhance the capacity of their host cells to support Leishmania infection by modulating their arginase activity.

Bottom Line: This early exacerbation was attributed to two fundamental properties of PSG: Firstly, PSG powerfully recruited macrophages to the dermal site of infection within 24 hours.Secondly, PSG enhanced alternative activation and arginase activity of host macrophages, thereby increasing L-arginine catabolism and the synthesis of polyamines essential for intracellular parasite growth.The increase in arginase activity promoted the intracellular growth of L. mexicana within classically activated macrophages, and inhibition of macrophage arginase completely ablated the early exacerbatory properties of PSG in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Imperial College of Science, Technology and Medicine, London, UK. matthew.rogers@imperial.ac.uk

ABSTRACT
All natural Leishmania infections start in the skin; however, little is known of the contribution made by the sand fly vector to the earliest events in mammalian infection, especially in inflamed skin that can rapidly kill invading parasites. During transmission sand flies regurgitate a proteophosphoglycan gel synthesized by the parasites inside the fly midgut, termed promastigote secretory gel (PSG). Regurgitated PSG can exacerbate cutaneous leishmaniasis. Here, we show that the amount of Leishmania mexicana PSG regurgitated by Lutzomyia longipalpis sand flies is proportional to the size of its original midgut infection and the number of parasites transmitted. Furthermore, PSG could exacerbate cutaneous L. mexicana infection for a wide range of doses (10-10,000 parasites) and enhance infection by as early as 48 hours in inflamed dermal air pouches. This early exacerbation was attributed to two fundamental properties of PSG: Firstly, PSG powerfully recruited macrophages to the dermal site of infection within 24 hours. Secondly, PSG enhanced alternative activation and arginase activity of host macrophages, thereby increasing L-arginine catabolism and the synthesis of polyamines essential for intracellular parasite growth. The increase in arginase activity promoted the intracellular growth of L. mexicana within classically activated macrophages, and inhibition of macrophage arginase completely ablated the early exacerbatory properties of PSG in vitro and in vivo. Thus, PSG is an essential component of the infectious sand fly bite for the early establishment of Leishmania in skin, which should be considered when designing and screening therapies against leishmaniasis.

Show MeSH
Related in: MedlinePlus