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Aluminium: a natural adjuvant in Leishmania transmission via sand flies?
Transactions of the Royal Society of Tropical Medicine and Hygiene (2008) 102, 1140—1142
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Aluminium: a natural adjuvant in Leishmania transmission via sand flies? Rhayza Maingon a, Amandeep Khela a, Christopher Sampson a, Richard Ward a, Karen Walker b, Christopher Exley c,∗ a
Centre for Applied Entomology and Parasitology — Research Institute for Science and Technology in Medicine, Keele University, Staffordshire, UK b School of Life Sciences, Keele University, Staffordshire, UK c Birchall Centre for Inorganic Chemistry and Materials Science, Lennard-Jones Laboratories, Keele University, Staffordshire, ST5 5BG, UK Received 6 February 2008; received in revised form 3 April 2008; accepted 3 April 2008 Available online 19 May 2008
KEYWORDS Leishmania; Tropism; Aluminium; Sand flies; Adjuvant; Salivary gland
Summary Genetically identical Leishmania chagasi/infantum parasites cause both atypical cutaneous leishmaniasis and visceral leishmaniasis. In this report we have tested the first part of a hypothesis that states that the form of this disease that is manifested depends upon the adjuvant-like activity of aluminium of dietary origin accumulated in the salivary gland of the sand fly vector. In sand flies fed aluminium-supplemented sucrose we have used histochemistry to qualitatively identify aluminium in their salivary glands and graphite furnace atomic absorption spectrometry to quantify the aluminium content of dissected salivary glands. Aluminium may be acting as a natural adjuvant in some forms of leishmaniasis. © 2008 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.
1. Introduction Leishmania—human interaction ranges from asymptomatic infection to a number of clinical presentations, including benign cutaneous self-healing lesions and fatal visceral pathologies. The form taken by the disease might depend upon factors contributed by the Leishmania species, by the host immune response, by sand fly genetic variability and
∗
Corresponding author. E-mail address: [email protected] (C. Exley)..
by, as yet, unconfirmed environmental agents. In respect of the latter it has been reported that in Central America, apparently genetically identical Le. chagasi/infantum parasites cause both atypical cutaneous leishmaniasis (ACL) and visceral leishmaniasis (VL) (Noyes et al., 1997). A possible explanation of why there should be two such extreme responses to the same parasite has centred upon the suggestion that the contamination of the skin of one geographically discrete population by inorganic particles of volcanic origin may be having an immunomodulatory effect with concomitant repercussions for disease progression (Convit et al., 2005, 2006). The authors attributed the immunomodulatory effect of the inorganic particles to the fact that they were
0035-9203/$ — see front matter © 2008 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.trstmh.2008.04.012
Aluminium in sand flies predominantly composed of aluminium and silicon, both of which are known to affect the immune response. This hypothesis led the research reported here on how synergisms among the parasite, the host response and the sand fly vector might be linked to the environment through the vector’s diet — and specifically, the accumulation of aluminium of dietary origin in the sand fly salivary gland. Aluminium salts are the most widely used adjuvants in vaccination (Brewer, 2006), including against Leishmania spp. (Rosado-Vallado et al., 2005), although their proantigenicity is not fully understood. As a major constituent of saliva is the glycopeptide mucin, which binds aluminium with some avidity (Exley, 1998), it is likely that aluminium accumulated in sand fly salivary glands might act as a ‘natural’ adjuvant and thereby alter the host immune response to the transmission of Leishmania spp. parasite via this vector. This report describes results obtained during the first test of our hypothesis, namely to determine if sand flies fed an aluminium-rich diet accumulate aluminium in their salivary glands.
2. Materials and methods
1141 groups were dissected under a stereoscope, placed into PBS solution at ∼1 salivary gland/l of buffer and stored at −20 ◦ C. Thawed salivary gland samples were digested in 14 mol/l HNO3 and their total aluminium content measured by graphite furnace atomic absorption spectrometry (GFAAS) using a previously validated programme (Schneider and Exley, 2001). Each digest was measured five times and the mean accepted if the %rsd was <10%. An unpaired Student’s t test was used to compare the mean concentration of aluminium in the salivary glands of sand flies fed normal and aluminium-supplemented sugar feeds.
3. Results and Discussion The longevity of sand flies fed only sucrose or aluminiumsupplemented sucrose was similar (10 ± 3 d post-eclosion), indicating that aluminium did not significantly influence their survival under the laboratory rearing conditions used in this study. Sand flies fed aluminium-supplemented sucrose stained positively for aluminium (Figure 1A), with particularly dense staining associated with salivary glands,
2.1. Aluminium-fed sand flies A laboratory-reared population of approximately 130 generations of Lutzomyia longipalpis s.l. (Diptera: Psychodidae; the main vector of Le. chagasi in Latin America) established from flies field-caught at Jacobina, Bahia State, in Brazil, was reared as described (Modi and Tesh, 1983). Emerged flies were separated into sugar-fed control and sugar + aluminium-fed experimental groups (∼60 flies per group) and were fed ad libitum until day 4 post-emergence, on 70% (w/v) sucrose or 70% (w/v) sucrose containing 1 mmol/l aluminium, respectively. Separate triplicate paired (sugar-fed and sugar + aluminium-fed) fly groups were used for histochemical detection of aluminium (20 females per group), and measurement of the aluminium content of salivary glands (30 females per group). All solutions were prepared in ultra-pure water (conductivity <0.067 S cm−1 ) (Elga UK Ltd, Marlow, Bucks, UK) to avoid contamination.
2.2. Aluminium histochemical detection Individual female flies were preserved in formaldehyde, ethanol dehydrated, embedded in LR white resin under vacuum and cut longitudinally into 2 m-thick sections as previously detailed (Brazil et al., 2003). Histological sections were stained specifically for aluminium with a modified Harris’ haematoxylin stain, from which the usual mordant, alum, was omitted. Aluminium present in tissue acts as an in situ mordant, and its presence is indicated under the light microscope (Olympus B × 50) as a vivid purple stain (Exley, 1998).
2.3. Aluminium quantification in sand fly salivary glands Salivary glands from individual flies belonging to the sugarfed control and the sugar + aluminium-fed experimental
Figure 1 Aluminium-specific histochemical staining of sand fly sections showing: (A) intense aluminium staining of salivary gland (kidney-shaped structure at the centre of the image) of sand fly fed an aluminium-supplemented diet; and (B) no aluminium staining of salivary gland of sand fly fed a nonaluminium-supplemented diet.
1142 whereas sand flies fed normal sucrose showed little positive staining for aluminium (Figure 1B). Clearly, although sand flies exposed to aluminium in their diet accumulated it in other regions of the body, the presence of aluminium in the salivary glands was particularly pronounced, and this observation was supported by its measurement in salivary gland preparations. The total aluminium in salivary gland preparations taken from flies fed the aluminiumsupplemented sucrose was statistically higher (P < 0.05), at 25.3 ± 9.20 mg/l compared with 9.4 ± 2.32 mg/l in the salivary gland preparations taken from control flies. These new preliminary findings present the opportunity to propose an intriguing and importantly testable hypothesis linking the environment and the host response to vector-transmitted parasitic disease. In this context (ser-thr) repeat-rich ‘mucins’ [including an Aedes aegypti metal-responsive gut mucin (Rayms-Keller et al., 2000)] have been identified in the salivary and/or gut transcriptomes and/or proteomes from a number of insect vectors of disease including the malaria vector Anopheles gambiae and the sand fly focus of this study, Lu. longipalpis (Shen et al., 1999; Valenzuela et al., 2004). Furthermore, a (ser-pro) repeat-rich ‘mucin’-like filamentous proteophosphoglycan (ffPG), secreted by the Leptomonas differentiation stage of Le. mexicana (and to a lesser extent by Le. chagasi), in experimentally infected Lu. longipalpis sand flies, was shown to enhance Leishmania virulence in vivo (Rogers et al., 2004). It is tempting to speculate that aluminium-mucin interactions might influence Leishmania virulence and infection outcome directly or indirectly by immunomodulation of the host’s response. If such speculation were found to be relevant to Leishmania spp. transmission via sand flies, then it might also be true for other parasites and other vectors. Indeed, might the concept of environmental aluminium acting in the capacity of a natural adjuvant in such forms of disease transmission even form the basis of a future ‘biological’ therapy? Authors’ contributions: CE contributed the original hypothesis, was a major contributor to the project’s experimental design, and carried out the measurements of total aluminium in salivary gland preparations; RM and RW provided sandfly populations and expertise in dissecting sandfly salivary glands; AK and CS ran the project on a day to day basis contributing to all aspects of the experimental work; KW carried out the detailed histochemistry; RM, CE and RW wrote the article. All authors read and approved the final manuscript. CE is guarantor of the paper. Acknowledgments: We are grateful to Dr Pam Taylor for laboratory sand fly rearing, to Madhuka Ariyarathne for preliminary experiments on the effect of aluminium on sand fly survival rates, and to Ziad Taha for his help with dissection of salivary glands.
R. Maingon et al. Funding: None. Conflicts of interests: None declared. Ethical approval: Not required.
References Brazil, B.G., Linonet, J., Brazil, R.P., 2003. Preparation of sandfly (Diptera: Psychodidae: Phlebotominae) specimens for histological studies. Mem. Inst. Oswaldo Cruz 98, 287—290. Brewer, J.M., 2006. How do aluminium adjuvants work? Immunol. Lett. 102, 10—15. Convit, J., Ulrich, M., Perez, M., Hung, J., Castillo, J., Rojas, H., Viquez, A., Araya, L.N., De Lima, H., 2005. Atypical cutaneous leishmaniasis in Central America: possible interaction between infectious and environmental elements. Trans. R. Soc. Trop. Med. Hyg. 99, 13—17. Convit, J., Ulrich, M., Castillo, J., De Lima, H., Perez, M., Caballero, N., Hung, J., Arana, B., Perez, P., 2006. Inorganic particles in the skin of inhabitants of volcanic areas of Central America: their possible immunomodulatory influence in leishmaniasis and leprosy. Trans. R. Soc. Trop. Med. Hyg. 100, 734— 739. Exley, C., 1998. The precipitation of mucin by aluminium. J. Inorg. Biochem. 70, 195—206. Modi, G.B., Tesh, R.B., 1983. A simple technique for mass rearing Lutzomyia longipalpis and Phlebotomus papatasi (Diptera: Psychodidae) in the laboratory. J. Med. Entomol. 20, 568— 569. Noyes, H., Chance, M., Ponce, C., Ponce, E., Maingon, R., 1997. Leishmania chagasi: genotypically similar parasites from Honduras cause both visceral and cutaneous leishmaniasis in humans. Exp. Parasitol. 85, 264—273. Rayms-Keller, A., McGaw, M., Oray, C., Carlson, J.O., Beaty, B.J., 2000. Molecular cloning and characterization of a metal responsive Aedes aegypti intestinal mucin cDNA. Insect Mol. Biol. 9, 419—426. Rogers, M.E., Ilg, T., Nikolaev, A.V., Fergusson, M.A., Bates, P.A., 2004. Transmission of cutaneous leishmaniasis by sand flies is enhanced by regurgitation of fPPG. Nature 430, 463— 467. Rosado-Vallado, M., Mut-Martin, M., Garcia-Miss Mdel, R., Dumonteil, E., 2005. Aluminium phosphate potentiates the efficacy of DNA vaccines against Leishmania mexicana. Vaccine 23, 5372—5379. Schneider, C., Exley, C., 2001. Silicic acid (Si(OH)4 ) is a significant influence upon the atomic absorption signal of aluminium measured by graphite furnace atomic absorption spectrometry (GFAAS). J. Inorg. Biochem. 87, 45—50. Shen, Z., Dimopoulos, G., Kafatos, F.C., Jacobs-Lorena, M., 1999. A cell surface mucin specifically expressed in the midgut of the malaria mosquito Anopheles gambiae. Proc. Natl. Acad. Sci. USA 96, 5610—5615. Valenzuela, J.G., Garfield, M., Rowton, E.D., Pham, V.M., 2004. Identification of the most abundant secreted proteins from the salivary glands of the sand fly Lutzomyia longipalpis, vector of Leishmania chagasi. J. Exp. Biol. 207, 3717—3729.
available at www.sciencedirect.com
journal homepage: www.elsevierhealth.com/journals/trst
Aluminium: a natural adjuvant in Leishmania transmission via sand flies? Rhayza Maingon a, Amandeep Khela a, Christopher Sampson a, Richard Ward a, Karen Walker b, Christopher Exley c,∗ a
Centre for Applied Entomology and Parasitology — Research Institute for Science and Technology in Medicine, Keele University, Staffordshire, UK b School of Life Sciences, Keele University, Staffordshire, UK c Birchall Centre for Inorganic Chemistry and Materials Science, Lennard-Jones Laboratories, Keele University, Staffordshire, ST5 5BG, UK Received 6 February 2008; received in revised form 3 April 2008; accepted 3 April 2008 Available online 19 May 2008
KEYWORDS Leishmania; Tropism; Aluminium; Sand flies; Adjuvant; Salivary gland
Summary Genetically identical Leishmania chagasi/infantum parasites cause both atypical cutaneous leishmaniasis and visceral leishmaniasis. In this report we have tested the first part of a hypothesis that states that the form of this disease that is manifested depends upon the adjuvant-like activity of aluminium of dietary origin accumulated in the salivary gland of the sand fly vector. In sand flies fed aluminium-supplemented sucrose we have used histochemistry to qualitatively identify aluminium in their salivary glands and graphite furnace atomic absorption spectrometry to quantify the aluminium content of dissected salivary glands. Aluminium may be acting as a natural adjuvant in some forms of leishmaniasis. © 2008 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.
1. Introduction Leishmania—human interaction ranges from asymptomatic infection to a number of clinical presentations, including benign cutaneous self-healing lesions and fatal visceral pathologies. The form taken by the disease might depend upon factors contributed by the Leishmania species, by the host immune response, by sand fly genetic variability and
∗
Corresponding author. E-mail address: [email protected] (C. Exley)..
by, as yet, unconfirmed environmental agents. In respect of the latter it has been reported that in Central America, apparently genetically identical Le. chagasi/infantum parasites cause both atypical cutaneous leishmaniasis (ACL) and visceral leishmaniasis (VL) (Noyes et al., 1997). A possible explanation of why there should be two such extreme responses to the same parasite has centred upon the suggestion that the contamination of the skin of one geographically discrete population by inorganic particles of volcanic origin may be having an immunomodulatory effect with concomitant repercussions for disease progression (Convit et al., 2005, 2006). The authors attributed the immunomodulatory effect of the inorganic particles to the fact that they were
0035-9203/$ — see front matter © 2008 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.trstmh.2008.04.012
Aluminium in sand flies predominantly composed of aluminium and silicon, both of which are known to affect the immune response. This hypothesis led the research reported here on how synergisms among the parasite, the host response and the sand fly vector might be linked to the environment through the vector’s diet — and specifically, the accumulation of aluminium of dietary origin in the sand fly salivary gland. Aluminium salts are the most widely used adjuvants in vaccination (Brewer, 2006), including against Leishmania spp. (Rosado-Vallado et al., 2005), although their proantigenicity is not fully understood. As a major constituent of saliva is the glycopeptide mucin, which binds aluminium with some avidity (Exley, 1998), it is likely that aluminium accumulated in sand fly salivary glands might act as a ‘natural’ adjuvant and thereby alter the host immune response to the transmission of Leishmania spp. parasite via this vector. This report describes results obtained during the first test of our hypothesis, namely to determine if sand flies fed an aluminium-rich diet accumulate aluminium in their salivary glands.
2. Materials and methods
1141 groups were dissected under a stereoscope, placed into PBS solution at ∼1 salivary gland/l of buffer and stored at −20 ◦ C. Thawed salivary gland samples were digested in 14 mol/l HNO3 and their total aluminium content measured by graphite furnace atomic absorption spectrometry (GFAAS) using a previously validated programme (Schneider and Exley, 2001). Each digest was measured five times and the mean accepted if the %rsd was <10%. An unpaired Student’s t test was used to compare the mean concentration of aluminium in the salivary glands of sand flies fed normal and aluminium-supplemented sugar feeds.
3. Results and Discussion The longevity of sand flies fed only sucrose or aluminiumsupplemented sucrose was similar (10 ± 3 d post-eclosion), indicating that aluminium did not significantly influence their survival under the laboratory rearing conditions used in this study. Sand flies fed aluminium-supplemented sucrose stained positively for aluminium (Figure 1A), with particularly dense staining associated with salivary glands,
2.1. Aluminium-fed sand flies A laboratory-reared population of approximately 130 generations of Lutzomyia longipalpis s.l. (Diptera: Psychodidae; the main vector of Le. chagasi in Latin America) established from flies field-caught at Jacobina, Bahia State, in Brazil, was reared as described (Modi and Tesh, 1983). Emerged flies were separated into sugar-fed control and sugar + aluminium-fed experimental groups (∼60 flies per group) and were fed ad libitum until day 4 post-emergence, on 70% (w/v) sucrose or 70% (w/v) sucrose containing 1 mmol/l aluminium, respectively. Separate triplicate paired (sugar-fed and sugar + aluminium-fed) fly groups were used for histochemical detection of aluminium (20 females per group), and measurement of the aluminium content of salivary glands (30 females per group). All solutions were prepared in ultra-pure water (conductivity <0.067 S cm−1 ) (Elga UK Ltd, Marlow, Bucks, UK) to avoid contamination.
2.2. Aluminium histochemical detection Individual female flies were preserved in formaldehyde, ethanol dehydrated, embedded in LR white resin under vacuum and cut longitudinally into 2 m-thick sections as previously detailed (Brazil et al., 2003). Histological sections were stained specifically for aluminium with a modified Harris’ haematoxylin stain, from which the usual mordant, alum, was omitted. Aluminium present in tissue acts as an in situ mordant, and its presence is indicated under the light microscope (Olympus B × 50) as a vivid purple stain (Exley, 1998).
2.3. Aluminium quantification in sand fly salivary glands Salivary glands from individual flies belonging to the sugarfed control and the sugar + aluminium-fed experimental
Figure 1 Aluminium-specific histochemical staining of sand fly sections showing: (A) intense aluminium staining of salivary gland (kidney-shaped structure at the centre of the image) of sand fly fed an aluminium-supplemented diet; and (B) no aluminium staining of salivary gland of sand fly fed a nonaluminium-supplemented diet.
1142 whereas sand flies fed normal sucrose showed little positive staining for aluminium (Figure 1B). Clearly, although sand flies exposed to aluminium in their diet accumulated it in other regions of the body, the presence of aluminium in the salivary glands was particularly pronounced, and this observation was supported by its measurement in salivary gland preparations. The total aluminium in salivary gland preparations taken from flies fed the aluminiumsupplemented sucrose was statistically higher (P < 0.05), at 25.3 ± 9.20 mg/l compared with 9.4 ± 2.32 mg/l in the salivary gland preparations taken from control flies. These new preliminary findings present the opportunity to propose an intriguing and importantly testable hypothesis linking the environment and the host response to vector-transmitted parasitic disease. In this context (ser-thr) repeat-rich ‘mucins’ [including an Aedes aegypti metal-responsive gut mucin (Rayms-Keller et al., 2000)] have been identified in the salivary and/or gut transcriptomes and/or proteomes from a number of insect vectors of disease including the malaria vector Anopheles gambiae and the sand fly focus of this study, Lu. longipalpis (Shen et al., 1999; Valenzuela et al., 2004). Furthermore, a (ser-pro) repeat-rich ‘mucin’-like filamentous proteophosphoglycan (ffPG), secreted by the Leptomonas differentiation stage of Le. mexicana (and to a lesser extent by Le. chagasi), in experimentally infected Lu. longipalpis sand flies, was shown to enhance Leishmania virulence in vivo (Rogers et al., 2004). It is tempting to speculate that aluminium-mucin interactions might influence Leishmania virulence and infection outcome directly or indirectly by immunomodulation of the host’s response. If such speculation were found to be relevant to Leishmania spp. transmission via sand flies, then it might also be true for other parasites and other vectors. Indeed, might the concept of environmental aluminium acting in the capacity of a natural adjuvant in such forms of disease transmission even form the basis of a future ‘biological’ therapy? Authors’ contributions: CE contributed the original hypothesis, was a major contributor to the project’s experimental design, and carried out the measurements of total aluminium in salivary gland preparations; RM and RW provided sandfly populations and expertise in dissecting sandfly salivary glands; AK and CS ran the project on a day to day basis contributing to all aspects of the experimental work; KW carried out the detailed histochemistry; RM, CE and RW wrote the article. All authors read and approved the final manuscript. CE is guarantor of the paper. Acknowledgments: We are grateful to Dr Pam Taylor for laboratory sand fly rearing, to Madhuka Ariyarathne for preliminary experiments on the effect of aluminium on sand fly survival rates, and to Ziad Taha for his help with dissection of salivary glands.
R. Maingon et al. Funding: None. Conflicts of interests: None declared. Ethical approval: Not required.
References Brazil, B.G., Linonet, J., Brazil, R.P., 2003. Preparation of sandfly (Diptera: Psychodidae: Phlebotominae) specimens for histological studies. Mem. Inst. Oswaldo Cruz 98, 287—290. Brewer, J.M., 2006. How do aluminium adjuvants work? Immunol. Lett. 102, 10—15. Convit, J., Ulrich, M., Perez, M., Hung, J., Castillo, J., Rojas, H., Viquez, A., Araya, L.N., De Lima, H., 2005. Atypical cutaneous leishmaniasis in Central America: possible interaction between infectious and environmental elements. Trans. R. Soc. Trop. Med. Hyg. 99, 13—17. Convit, J., Ulrich, M., Castillo, J., De Lima, H., Perez, M., Caballero, N., Hung, J., Arana, B., Perez, P., 2006. Inorganic particles in the skin of inhabitants of volcanic areas of Central America: their possible immunomodulatory influence in leishmaniasis and leprosy. Trans. R. Soc. Trop. Med. Hyg. 100, 734— 739. Exley, C., 1998. The precipitation of mucin by aluminium. J. Inorg. Biochem. 70, 195—206. Modi, G.B., Tesh, R.B., 1983. A simple technique for mass rearing Lutzomyia longipalpis and Phlebotomus papatasi (Diptera: Psychodidae) in the laboratory. J. Med. Entomol. 20, 568— 569. Noyes, H., Chance, M., Ponce, C., Ponce, E., Maingon, R., 1997. Leishmania chagasi: genotypically similar parasites from Honduras cause both visceral and cutaneous leishmaniasis in humans. Exp. Parasitol. 85, 264—273. Rayms-Keller, A., McGaw, M., Oray, C., Carlson, J.O., Beaty, B.J., 2000. Molecular cloning and characterization of a metal responsive Aedes aegypti intestinal mucin cDNA. Insect Mol. Biol. 9, 419—426. Rogers, M.E., Ilg, T., Nikolaev, A.V., Fergusson, M.A., Bates, P.A., 2004. Transmission of cutaneous leishmaniasis by sand flies is enhanced by regurgitation of fPPG. Nature 430, 463— 467. Rosado-Vallado, M., Mut-Martin, M., Garcia-Miss Mdel, R., Dumonteil, E., 2005. Aluminium phosphate potentiates the efficacy of DNA vaccines against Leishmania mexicana. Vaccine 23, 5372—5379. Schneider, C., Exley, C., 2001. Silicic acid (Si(OH)4 ) is a significant influence upon the atomic absorption signal of aluminium measured by graphite furnace atomic absorption spectrometry (GFAAS). J. Inorg. Biochem. 87, 45—50. Shen, Z., Dimopoulos, G., Kafatos, F.C., Jacobs-Lorena, M., 1999. A cell surface mucin specifically expressed in the midgut of the malaria mosquito Anopheles gambiae. Proc. Natl. Acad. Sci. USA 96, 5610—5615. Valenzuela, J.G., Garfield, M., Rowton, E.D., Pham, V.M., 2004. Identification of the most abundant secreted proteins from the salivary glands of the sand fly Lutzomyia longipalpis, vector of Leishmania chagasi. J. Exp. Biol. 207, 3717—3729.