Infection by and protective immune responses against Plasmodium berghei ANKA are not affected in macrophage scavenger receptors A deficient mice
© Cunha-Rodrigues et al; licensee BioMed Central Ltd. 2006
Received: 29 June 2006
Accepted: 16 August 2006
Published: 16 August 2006
Scavenger receptors (SRs) recognize endogenous molecules modified by pathological processes as well as components of diverse microorganisms. Mice deficient for both SR-AI and II are more susceptible to infections by a variety of bacterial and viral pathogens.
Here we show that SR-A deficient mice and wild type mice are equally susceptible to malaria infection both during liver and blood stages. Moreover, like wild type mice, SR-A deficient mice are able to mount a protective immune response against radiation attenuated sporozoites.
Our results do not reveal a function of SR-A I and II receptors in the Plasmodium berghei ANKA infection, both in the development of CM and parasitemia control. Moreover, these receptors appear not to be required for the establishment of a protective immune response against the malaria liver stages.
Malaria infection starts in the mammalian host with the injection of Plasmodium sporozoites by a mosquito bite. Sporozoites travel to the liver where they cross the sinusoidal wall through Kupffer cells and then migrate through several hepatocytes before they establish an infection with the formation of a parasitophorous vacuole [1, 2]. Within the vacuole the sporozoites develop and generate millions of merozoites that are released into the bloodstream. With the infection of erythrocytes the clinical phase of malaria begins. In any given year, more than a million children die as a result of malaria infection. The death from infection is largely due to an acute syndrome known as cerebral malaria (CM). The neurological manifestations of CM include headache, agitation, psychosis, seizures and impaired consciousness that lead to coma and death .
The class A macrophage scavenger receptor (SR-A) is the prototypic member of a large family of membrane receptors that bind oxidized low density lipoprotein and a wide variety of other ligands many of which are derived from apoptotic cells and pathogens . The SR-A receptors occur in two different forms that are generated by alternative splicing of the primary transcript: SR-AI and SR-AII. Both receptors have nearly identical ligand binding properties . They are expressed primarily by mature cells of the myelomonocytic lineage such as Kupffer cells in the liver, glial cells in the brain and macrophages that are resident in or recruited to various tissues. They are also expressed by sinusoidal endothelial cells in the liver . SR-A receptors appear to have beneficial and pathological functions. They mediate phagocytosis of apoptotic cells , and have been implicated in atherogenesis, the clearance of debris during acute neuronal degeneration  as well as in innate immunity and antigen presentation . SR-A receptors bind lipopolysaccharide from Gram-negative and lipoteichoic acid from Gram-positive bacteria . SR-AI and II receptor deficient mice (SR-A-/-) are more susceptible to infections by a variety of pathogenic microorganisms such as Listeria monocytogenes , Staphylococcus aureus , Bacillus Calmette-Guérin  and herpes simplex virus .
In the present work we examined the role of SR-A receptors in three different aspects of a malaria infection, namely first, in the establishment of a primary liver infection; second, in the protective immune response against the liver stages; and third, in the development of the cerebral pathology associated with blood stages of infection.
Results and discussion
SR-A receptor functions have been implicated in both the innate and the adaptive branches of the immune response. To determine whether SR-A receptors play a role in immune responses against malaria, we used a well-established model of immunization against the liver stages of malaria infection. Inoculation with radiation attenuated sporozoites (RAS) results in protection against a subsequent challenge with infective sporozoites, both in mice and humans [12, 13]. Irradiated sporozoites invade hepatocytes but do not develop normally and thus fail to establish an erythrocytic infection . SR-A-/- mice as well as their SR-A+/+ littermates  were immunized with 5 × 104 Plasmodium berghei RAS and challenged 10 days later with 104 infective sporozoites. Forty two hours after the challenge the level of protection was measured by quantifying the parasite load in the liver using qRT-PCR, as described above. RAS immunization of SR-A+/+ mice, as expected, clearly reduced the level of infection measured 40 hours after a challenge with infective sporozoites. The same level of protection was achieved in SR-A-/- mice (Fig. 1B). This shows that the lack of SR-A receptors does not affect the establishment of a protective immune response against RAS.
Taken together our data suggests that the lack of SR-A receptors has no influence in the malaria outcome during the blood stages of infection.
The results shown here do not reveal a function of SR-A I and II receptors in the Plasmodium berghei ANKA infection, in the development of CM or parasitemia control. Moreover, these receptors appear not to be required for the establishment of a protective immune response against the malaria liver stages in the well-established model of immunity against RAS. It has been shown that mice deficient in another member of the scavenger receptor family, CD36, have no differences at the liver infection . Moreover, recent experiments with mice deficient in CD36, indicate that this receptor is involved in the sequestration of Plasmodium berghei ANKA to the lung and adipose tissues but not in the development of cerebral malaria . In view of the complex and overlapping functions of different scavenger receptors it is conceivable that the true role of individual receptors is not revealed in experiments with receptor deficient mice unless a specific function of the receptor is analyzed . Whether SR-A receptors do have such specific functions in malaria infections of mice or humans remains to be elucidated.
Plasmodium berghei ANKA sporozoites were obtained from dissection of infected Anopheles stephensi mosquito salivary glands from the IMM insectary production. Male, 7–9 weeks old, SR-A-/- and their control littermate mice generously provided by Dr. T. Kodama (University of Tokyo)  were infected by intravenous injection of Plasmodium berghei (2 × 104) sporozoites or by intraperitoneal injection of Plasmodium berghei ANKA or P. yoelii 17 × (106) infected erythrocytes (originally, provided by Dr. A. Waters and C. Janse, Leiden University, or Dr. David Walliker, Edimburgh University, respectively).
Parasite quantification in the liver
40 hours post infection with sporozoites livers were removed, homogenized and total RNA was extracted (RNeasy Mini kit Quiagen). cDNA was obtained by reverse transcription (First-strand cDNA synthesis kit, Roche). Real-time PCR, using primers specific for Plasmodium berghei 18S rRNA (5'-AAGCATTAAATAAAGCGAATACATCCTTAC-3' and 5'-GGAGATTGGTTTTGACGTTTATGTG-3'), was used for quantification of parasite load in the livers of mice 40 hours after sporozoite inoculation.
Immunizations with radition attenuated sorozoites was perfomed by injecting 5 × 104 γ-irradiated (16,000 rads) Plasmodium berghei ANKA sporozoites followed by challenge (10 days later) with 104 infective sporozoites, both by intravenous injection. Parasite burden in the liver was measured as described in the previous section.
Following blood stage infection and pathology
Cerebral complications were monitored by ataxia, paralysis, deviation of the head and convulsions, symptoms leading to coma and death. Blood peripheral parasitemia was determined by counting parasites in Giemsa stained thin blood films.
We thank Werner Haas for critically reading this manuscript. MCR supported by Fundação para a Ciência e Tecnologia (BD/8435/2002) performed liver and blood stage infections, immunization assays, qPCR assays, data analysis and participated both in the study design and manuscript drafting. SP performed mice genotyping, RNA extractions, blood parasitemia quantification. MF participated in the study design. MMM conceived the study, participated in its design and coordination as well as manuscript drafting. MMM supervises MCR and SP. MMM is a fellow of the EMBO YIP and is a Howard Hughes Medical Institute International Research Scholar. The work was supported by Fundação para a Ciência e Tecnologia (Grants POCTI/SAU-MMO/60930/2004 to MMM) and European Science Foundation (Grant EURYI 2004 to MMM). All authors read and approved the final manuscript.
- Mota MM, Pradel G, Vanderberg JP, Hafalla JC, Frevert U, Nussenzweig RS, Nussenzweig V, Rodriguez A: Migration of Plasmodium sporozoites through cells before infection. Science. 2001, 291 (5501): 141-144. 10.1126/science.291.5501.141.View ArticlePubMedGoogle Scholar
- Frevert U, Engelmann S, Zougbede S, Stange J, Ng B, Matuschewski K, Liebes L, Yee H: Intravital observation of Plasmodium berghei sporozoite infection of the liver. PLoS Biol. 2005, 3 (6): e192-10.1371/journal.pbio.0030192.PubMed CentralView ArticlePubMedGoogle Scholar
- Newton CR, Warrell DA: Neurological manifestations of falciparum malaria. Ann Neurol. 1998, 43 (6): 695-702. 10.1002/ana.410430603.View ArticlePubMedGoogle Scholar
- Gough PJ, Gordon S: The role of scavenger receptors in the innate immune system. Microbes Infect. 2000, 2 (3): 305-311. 10.1016/S1286-4579(00)00297-5.View ArticlePubMedGoogle Scholar
- Hughes DA, Fraser IP, Gordon S: Murine macrophage scavenger receptor: in vivo expression and function as receptor for macrophage adhesion in lymphoid and non-lymphoid organs. Eur J Immunol. 1995, 25 (2): 466-473.View ArticlePubMedGoogle Scholar
- Platt N, Gordon S: Is the class A macrophage scavenger receptor (SR-A) multifunctional? - The mouse's tale. J Clin Invest. 2001, 108 (5): 649-654. 10.1172/JCI200113903.PubMed CentralView ArticlePubMedGoogle Scholar
- Bell MD, Lopez-Gonzalez R, Lawson L, Hughes D, Fraser I, Gordon S, Perry VH: Upregulation of the macrophage scavenger receptor in response to different forms of injury in the CNS. J Neurocytol. 1994, 23 (10): 605-613. 10.1007/BF01191555.View ArticlePubMedGoogle Scholar
- Suzuki H, Kurihara Y, Takeya M, Kamada N, Kataoka M, Jishage K, Ueda O, Sakaguchi H, Higashi T, Suzuki T, Takashima Y, Kawabe Y, Cynshi O, Wada Y, Honda M, Kurihara H, Aburatani H, Doi T, Matsumoto A, Azuma S, Noda T, Toyoda Y, Itakura H, Yazaki Y, Kodama T: A role for macrophage scavenger receptors in atherosclerosis and susceptibility to infection. Nature. 1997, 386 (6622): 292-296. 10.1038/386292a0.View ArticlePubMedGoogle Scholar
- Thomas CA, Li Y, Kodama T, Suzuki H, Silverstein SC, El Khoury J: Protection from lethal gram-positive infection by macrophage scavenger receptor-dependent phagocytosis. J Exp Med. 2000, 191 (1): 147-156. 10.1084/jem.191.1.147.PubMed CentralView ArticlePubMedGoogle Scholar
- Haworth R, Platt N, Keshav S, Hughes D, Darley E, Suzuki H, Kurihara Y, Kodama T, Gordon S: The macrophage scavenger receptor type A is expressed by activated macrophages and protects the host against lethal endotoxic shock. J Exp Med. 1997, 186 (9): 1431-1439. 10.1084/jem.186.9.1431.PubMed CentralView ArticlePubMedGoogle Scholar
- Kumar KA, Oliveira GA, Edelman R, Nardin E, Nussenzweig V: Quantitative Plasmodium sporozoite neutralization assay (TSNA). J Immunol Methods. 2004, 292 (1-2): 157-164. 10.1016/j.jim.2004.06.017.View ArticlePubMedGoogle Scholar
- Rieckmann KH, Beaudoin RL, Cassells JS, Sell KW: Use of attenuated sporozoites in the immunization of human volunteers against falciparum malaria. Bull World Health Organ. 1979, 57 Suppl 1: 261-265.PubMedGoogle Scholar
- Nussenzweig RS, Vanderberg J, Most H, Orton C: Protective immunity produced by the injection of x-irradiated sporozoites of plasmodium berghei. Nature. 1967, 216 (111): 160-162. 10.1038/216160a0.View ArticlePubMedGoogle Scholar
- Nardin EH, Nussenzweig RS: T cell responses to pre-erythrocytic stages of malaria: role in protection and vaccine development against pre-erythrocytic stages. Annu Rev Immunol. 1993, 11: 687-727. 10.1146/annurev.iy.11.040193.003351.View ArticlePubMedGoogle Scholar
- Schofield L, Grau GE: Immunological processes in malaria pathogenesis. Nat Rev Immunol. 2005, 5 (9): 722-735. 10.1038/nri1686.View ArticlePubMedGoogle Scholar
- Nogami S, Watanabe J, Nakagaki K, Nakata K, Suzuki H, Suzuki H, Fujisawa M, Kodama T, Kojima S: Involvement of macrophage scavenger receptors in protection against murine malaria. Am J Trop Med Hyg. 1998, 59 (5): 843-845.PubMedGoogle Scholar
- Inoue M, Xuan X, Fujisaki K, Igarashi I, Suzuki H: Short report: role of type I/II scavenger receptors in malarial infection in C57BL/6J mice. Am J Trop Med Hyg. 2006, 75 (1): 178-181.PubMedGoogle Scholar
- Sinnis P, Febbraio M: Plasmodium yoelii sporozoites infect CD36-deficient mice. Exp Parasitol. 2002, 100 (1): 12-16. 10.1006/expr.2001.4676.View ArticlePubMedGoogle Scholar
- Franke-Fayard B, Janse CJ, Cunha-Rodrigues M, Ramesar J, Buscher P, Que I, Lowik C, Voshol PJ, den Boer MA, van Duinen SG, Febbraio M, Mota MM, Waters AP: Murine malaria parasite sequestration: CD36 is the major receptor, but cerebral pathology is unlinked to sequestration. Proc Natl Acad Sci U S A. 2005, 102 (32): 11468-11473. 10.1073/pnas.0503386102.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.