Primary Heligmosomoides polygyrus bakeri infection induces myeloid-derived suppressor cells that suppress CD4+ Th2 responses and promote chronic infection.

Primary infection with the gastrointestinal nematode Heligmosomoides polygyrus bakeri is chronic in C57BL/6 (B6) mice whereas challenge infection is rapidly eliminated. F4/80-CD11b+Gr+ cells, presumed to be neutrophils, were reported to accumulate around encysting larvae in intestinal tissue during primary infection, but their exact identity and role remain unclear. We observed significant increases in F4/80-CD11bhiGr1hi cells in mesenteric lymph nodes (MLNs) and spleen after primary but not challenge infection; a high proportion of these cells expressed Ly6G and Ly6C. These cells, which phenotypically resemble myeloid-derived suppressor cells (MDSC), increased in lamina propria (LP) early during primary infection. Increased MDSC were associated with low numbers of alternatively activated macrophages (AAMØ) in LP and CD4+GATA3+ T cells and AAMØ in MLN and spleen. Purified CD11c-CD11b+Gr1+ cells from H. polygyrus bakeri-infected mice suppressed OVA-specific CD4+ T-cell proliferation via a nitric oxide-dependent mechanism and parasite-specific IL-4 secretion in vitro. Adoptive transfer of CD11c-CD11b+Gr1+ cells from mice with primary infection resulted in significantly higher adult worm burdens and increased egg production in naïve B6 recipients infected with H. polygyrus bakeri. Altogether, these findings indicate that primary H. polygyrus bakeri infection induces a novel subset of MDSC that suppress CD4+ Th2 responses and promote chronic infection.

Genetic control of susceptibility to infection with Plasmodium chabaudi chabaudi AS in inbred mouse strains.

To identify genetic effects modulating the blood stage replication of the malarial parasite, we phenotyped a group of 25 inbred mouse strains for susceptibility to Plasmodium chabaudi chabaudi AS infection (peak parasitemia, survival). A broad spectrum of responses was observed, with strains such as C57BL/6J being the most resistant (low parasitemia, 100% survival) and strains such as NZW/LacJ and C3HeB/FeJ being extremely susceptible (very high parasitemia and uniform lethality). A number of strains showed intermediate phenotypes and gender-specific effects, suggestive of rich genetic diversity in response to malaria in inbred strains. An F2 progeny was generated from SM/J (susceptible) and C57BL/6J (resistant) parental strains, and was phenotyped for susceptibility to P. chabaudi chabaudi AS. A whole-genome scan in these animals identified the Char1 locus (LOD=7.40) on chromosome 9 as a key regulator of parasite density and pointed to a conserved 0.4-Mb haplotype at Char1 that segregates with susceptibility/resistance to infection. In addition, a second locus was detected in [SM/J × C57BL/6J] F2 mice on the X chromosome (LOD=4.26), which was given the temporary designation Char11. These studies identify a conserved role of Char1 in regulating response to malaria in inbred mouse strains, and provide a prioritized 0.4-Mb interval for the search of positional candidates.

Caspase-12 deficiency enhances cytokine responses but does not protect against lethal Plasmodium yoelii 17XL infection.

To investigate the effect of caspase-12 deficiency on IFN-γ- independent control of blood-stage malaria, we compared lethal Plasmodium yoelii 17XL infection in wild-type C57BL / 6J and caspase-12-/-mice. Infected caspase-12-/- mice exhibited higher parasitaemia than WT mice on days 8 and 9 post-inoculation, but all WT and caspase-12-/- mice succumbed by day 10. In addition, infected caspase-12-/-mice had significantly elevated levels of IFN-γ, TNF, IL-18,and IL-10 in sera compared to infected WT mice. At the terminal stage of disease, there were no differences in cytokine levels in the tissues of infected WT and caspase-12-/- mice. However, liver pathology was more severe in infected caspase-12-/- mice compared to infected WT mice. Together, these findings indicate that although caspase-12 deficiency results in enhanced pro-inflammatory and immunoregulatory cytokine levels in sera during P. yoelii 17XL infection, these responses are not essential for protection against lethal malaria infection.

Identification of a novel cerebral malaria susceptibility locus (Berr5) on mouse chromosome 19.

Cerebral malaria (CM) is an acute, generally lethal condition characterized by high fever, seizures and coma. The genetic component to CM can be investigated in mouse models that vary in degree of susceptibility to infection with Plasmodium berghei ANKA. Using survival time to measure susceptibility in an informative F2 cross (n=257), we identified linkage to chromosome 19 (Berr5 (Berghei resistance locus 5), LOD=4.69) controlling, in part, the differential response between resistant BALB/c and susceptible C57BL/6 progenitors. BALB/c alleles convey increased survival through the cerebral phase of infection but have no quantitative effect on parasitemia during the later, anemic phase. The Berr5 locus colocalizes with three other immune loci, including Trl-4 (tuberculosis resistance), Tsiq2 (T-cell secretion of IL-4) and Eae19 (experimental allergic encephalitis 19), suggesting the possibility of a common genetic effect underlying these phenotypes. Potential positional candidates include the family of Ifit1-3 (interferon-inducible protein with tetratricopeptide repeats 1-3) and Fas.

Mapping of Char10, a novel malaria susceptibility locus on mouse chromosome 9.

Resistance to blood-stage malaria in AcB55 and AcB61 is caused by a loss of function mutation in pyruvate kinase (Pklr(I90N)). Likewise, pyruvate kinase (PK) deficiency in humans is protective against Plasmodium replication in vitro. We identified a third AcB strain, AcB62 that also carries the Pklr(I90N) mutation. However, AcB62 mice were susceptible to P.chabaudi infection and showed high levels of parasite replication (54-62% peak parasitemia). AcB62 mice showed the hallmarks of PK deficiency-associated anemia similar to AcB55/61 with reticulocytosis, splenic red pulp expansion, tissue iron overload, and increased expression of iron metabolism proteins. This suggests that malaria susceptibility in AcB62 is not because of absence of PK deficiency-associated pathophysiology. To map novel genetic factors affecting malaria susceptibility in AcB62, we generated an informative F2 population using AcB62 (Pklr(I90N)) and CBA-Pk(slc) (Pklr(G338D)) as progenitors and identified a novel locus on chromosome 9 (Char10; LOD=7.24) that controls peak parasitemia. A weaker linkage to the Pklr region of chromosome 3 (LOD=3.7) was also detected, a finding that may reflect the segregation of the two defective Pklr alleles. AcB62 alleles at both loci are associated with higher peak parasitemia. These results identify Char10 as a novel locus modulating severity of malaria in the context of PK deficiency.

Modulation of malaria-induced immunopathology by concurrent gastrointestinal nematode infection in mice.

We investigated malaria-associated pathology in mice co-infected with Heligmosomoides polygyrus (Hp) and Plasmodium chabaudi AS (Pc). Despite higher peak parasitemia, co-infected wild-type (WT) C57BL/6 mice displayed similar body weight losses, malarial anaemia, and tissue damage but less severe hypothermia and hypoglycaemia, and earlier reticulocytosis than Pc-infected WT mice. Co-infected STAT6(-/-) mice, deficient in nematode-induced Th2 responses, experienced similar peak parasitemias and generally suffered malaria-associated pathology to a similar degree as co-infected WT mice. These data indicate a complex relationship amongst helminths, malaria and host immune responses resulting in modulation of some but not all aspects of malaria-associated pathology.

Incremental expression of Tlr4 correlates with mouse resistance to Salmonella infection and fine regulation of relevant immune genes.

The mouse response to Salmonella Typhimurium infection is partly controlled through detection of the bacterium lipopolysaccharide by the host pattern recognition receptor, Toll-like receptor 4 (Tlr4). Mice deficient in Tlr4 signaling are extremely susceptible to Salmonella infection with a 1,000-fold reduction in LD(50). In a previous study, we showed, using transgenic mice carrying one, three, six and >30 copies of Tlr4, that the level of expression of this gene influences the outcome of Salmonella infection, with a plateau effect starting at three copies. In the present study, we further investigate the impact of Tlr4 during Salmonella infection in mice expressing Tlr4 at slightly sub-normal, normal and slightly supra-normal levels by comparing host responses in mice carrying one, two and three copies of Tlr4 on the same genetic background. We describe in detail the in vivo host response to pathogenic Salmonella and show for the first time, in this narrow range of Tlr4 expression, an incremental protective effect against Salmonella due to improved control of bacterial growth in target organs and increased expression of important immune response genes in the spleen.

Antigen presentation and dendritic cell biology in malaria.

Dendritic cells (DCs) are important both in amplifying the innate immune response and in initiating adaptive immunity and shaping the type of T helper (Th) response. Although the role of DCs in immune responses to many intracellular pathogens has been delineated and research is underway to identify the mechanisms involved, relatively little is known concerning the role of DCs in immunity to malaria. In this review, we provide an overview and summary of previous and current studies aimed to investigate the role of DCs as antigen presenting cells (APCs). In addition, the role of DCs in inducing innate and adaptive immunity to blood-stage malaria is discussed and, where information is available, the mechanisms involved are presented. Data from studies in humans infected with Plasmodium falciparum, the major human parasite responsible for the high morbidity and mortality associated with malaria throughout many regions of the developing world, as well as data from experimental mouse models are presented. Overall, the data from these studies are conflicting. The possible reasons for these differences, including the use of different parasite species and parasite strains in the mouse studies, are discussed. Nevertheless, together the data have important implications for development of an effective malaria vaccine since the selection of appropriate Plasmodium antigens and/or adjuvants, targeting innate immune responses involving DCs, may provide optimal protection against malaria. It is hoped that this review promotes more investigation among malariologists and immunologists alike on DCs and malaria.

Early interactions between blood-stage plasmodium parasites and the immune system.

Accumulating evidence provides strong support for the importance of innate immunity in shaping the subsequent adaptive immune response to blood-stage Plasmodium parasites, the causative agents of malaria. Early interactions between blood-stage parasites and cells of the innate immune system, including dendritic cells, monocytes/macrophages, natural killer (NK) cells, NKT cells, and gamma6 T cells, are important in the timely control of parasite replication and in the subsequent elimination and resolution of the infection. The major role of innate immunity appears to be the production of immunoregulatory cytokines, such as interleukin (IL)-12 and interferon (IFN)-gamma, which are critical for the development of type 1 immune responses involving CD4+ Thl cells, B cells, and effector cells which mediate cell-mediated and antibody-dependent adaptive immune responses. In addition, it is likely that cells of the innate immune system, especially dendritic cells, serve as antigen-presenting cells. Here, we review recent data from rodent models of blood-stage malaria and from human studies, and outline the early interactions of infected red blood cells with the innate immune system. We compare and contrast the results derived from studies in infected laboratory mice and humans. These host species are sufficiently different with respect to the identity of the infecting Plasmodium species, the resulting pathologies, and immune responses, particularly where the innate immune response is concerned. The implications of these findings for the development of an effective and safe malaria vaccine are also discussed.

Phenotypic expression of pyruvate kinase deficiency and protection against malaria in a mouse model.

The recombinant congenic mouse strains AcB55 and AcB61 are extremely resistant to malaria (Plasmodium chabaudi AS) despite the presence of susceptibility alleles at the known Char1/Char2 resistance loci. Resistance in AcB55 and AcB61 is controlled by a locus on chromosome 3 (Char4) shown to be allelic with or tightly linked to a loss-of-function mutation in pyruvate kinase (Pklr). AcB55 and AcB61 show important splenomegaly prior to infection caused by the expansion of the red pulp, and display histological signs of extramedullary erythropoiesis in the liver. Examination of splenic cell populations by flow cytometry demonstrates elevated numbers of TER119-positive erythroid precursor cells (>30% of total spleen cells), while RNA expression studies show elevated expression of erythrocyte-specific transcripts such as globin, transferrin receptor, and Nramp2/Slc11a2 in the spleen of both strains. Hematological profiling in both strains is consistent with the presence of anemia as evidenced by low total erythrocyte counts, decreased hemoglobin, as well as abnormally high numbers of circulating reticulocytes (15-20%). These results strongly suggest that the mutant Pklr allele (Pklr(269A)) of AcB55/61 strains causes hemolytic anemia compensated by constitutive erythropoiesis, which in turn protects the mice against P. chabaudi infection. The possible molecular basis of the Pklr protective effect is discussed and is under current investigation in these two strains.

Susceptibility to malaria as a complex trait: big pressure from a tiny creature.

Malaria, which is a major infectious disease worldwide, is caused by the Plasmodium parasite, one of the longest-known parasites infecting humans. The malaria situation is complicated by the emergence of drug resistance and the lack of an effective vaccine. Genetic factors play a key role in disease susceptibility, progression and outcome. Interestingly, an increasing large number of polymorphisms associated with resistance and susceptibility in humans have been found in proteins from erythrocytes, the site of Plasmodium replication. Some of these deleterious alleles have been selected by direct genetic pressure from the parasite in endemic areas of malaria. A number of additional gene effects have been mapped both in humans and in mice using population studies and experimental models of malaria, respectively. These recent studies have started to reveal additional aspects of the complex host-parasite interactions.

Complex genetic control of susceptibility to malaria in mice.

Malaria is a major infectious disease worldwide, with over 1 million deaths in African children every year. The molecular pathways of pathogenesis of the Plasmodium parasite and the host mechanisms of defense against this infection remain poorly understood. Epidemiological studies, together with linkage analyses in endemic areas have clearly pointed at a genetic component of innate susceptibility and severity of disease. In humans, this genetic trait is complex, and has been studied in a mouse experimental model over the past few years. Inbred strains of mice show different degrees of susceptibility to infection with Plasmodium chabaudi, and the genetic component of these inter-strain differences has been studied in standard informative backcross and F2 populations, as well as in recombinant inbred strains and more recently, in recombinant congenic strains. These studies have shown that genetic susceptibility to malaria is also complex in mice, and have led to the mapping of major susceptibility Char (Chabaudi resistance) loci, located on chromosomes 9 (Char1), 8 (Char2), 17 (Char3) and 3 (Char4).

Identification of a new malaria susceptibility locus (Char4) in recombinant congenic strains of mice.

The genetic component of susceptibility to malaria is complex, both in humans and in the mouse model of infection. Two murine loci on chromosomes 8 (Pchr/Char2) and 9 (Char1) have previously been mapped in F(2) crosses, and play an important role in regulating blood parasitemia and survival to infection with Plasmodium chabaudi. These loci explain only part of the interstrain phenotypic variance, and their penetrance and expressivity vary in different inbred strains. Novel loci regulating response to P. chabaudi infection were investigated by using an alternative strategy based on a newly derived set of AcB/BcA recombinant congenic strains bred from malaria-susceptible A/J (A) and resistant C57BL/6J (B6). One of the AcB strains, AcB55, is shown to be highly resistant to infection despite 83% susceptible A genomic composition, including susceptibility alleles at Char1 and Pchr/Char2. Early onset of parasite clearance in AcB55 is associated with lower peak parasitemia and absence of mortality. Linkage analysis in an informative (AcB55 x A)F(2) population, using peak parasitemia as a quantitative trait, located a new B6-derived resistance locus on chromosome 3 (lod score = 6.57) that we designate Char4. A second, suggestive linkage on chromosome 10 (lod score = 2.53) shows additive effect with Char4 on peak parasitemia. Char4 maps to a small congenic B6 fragment in AcB55 that should facilitate the search for candidate genes. Our findings provide an entry point for parallel association studies in humans between the syntenic 4q21-4q25 region and susceptibility to disease in endemic areas of malaria.

Modulation of host responses to blood-stage malaria by interleukin-12: from therapy to adjuvant activity.

This review focuses on the role of interleukin (IL)-12, a proinflammatory cytokine with pleiotropic effects as a potent immunoregulatory molecule and hematopoietic growth factor, in infection with Plasmodium parasites, the causative agents of malaria. IL-12 has been demonstrated to have profound effects on the immune response to blood-stage malaria, to induce protection, and to alleviate malarial anemia. In combination with an anti-malarial drug, IL-12 is effective in an established malaria infection. This cytokine also has potent immune effects as a malaria vaccine adjuvant. However, IL-12 can also mediate pathology during blood-stage malaria.

Granulocyte-macrophage colony-stimulating factor-deficient mice have impaired resistance to blood-stage malaria.

The contribution of granulocyte-macrophage colony-stimulating factor (GM-CSF), a hematopoietic and immunoregulatory cytokine, to resistance to blood-stage malaria was investigated by infecting GM-CSF-deficient (knockout [KO]) mice with Plasmodium chabaudi AS. KO mice were more susceptible to infection than wild-type (WT) mice, as evidenced by higher peak parasitemia, recurrent recrudescent parasitemia, and high mortality. P. chabaudi AS-infected KO mice had impaired splenomegaly and lower leukocytosis but equivalent levels of anemia compared to infected WT mice. Both bone marrow and splenic erythropoiesis were normal in infected KO mice. However, granulocyte-macrophage colony formation was significantly decreased in these tissues of uninfected and infected KO mice, and the numbers of macrophages in the spleen and peritoneal cavity were significantly lower than in infected WT mice. Serum levels of gamma interferon (IFN-gamma) were found to be significantly higher in uninfected KO mice, and the level of this cytokine was not increased during infection. In contrast, IFN-gamma levels were significantly above normal levels in infected WT mice. During infection, tumor necrosis factor alpha (TNF-alpha) levels were significantly increased in KO mice and were significantly higher than TNF-alpha levels in infected WT mice. Our results indicate that GM-CSF contributes to resistance to P. chabaudi AS infection and that it is involved in the development of splenomegaly, leukocytosis, and granulocyte-macrophage hematopoiesis. GM-CSF may also regulate IFN-gamma and TNF-alpha production and activity in response to infection. The abnormal responses seen in infected KO mice may be due to the lack of GM-CSF during development, to the lack of GM-CSF in the infected mature mice, or to both.

Mouse models of chronic lung infection with Pseudomonas aeruginosa: models for the study of cystic fibrosis.

The discovery of the CFTR gene in 1989 has lead to rapid progress in understanding the molecular basis of cystic fibrosis (CF) and the biological properties of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. However, more than 10 years later, recurrent lung infections with Pseudomonas aeruginosa, which lead to chronic lung disease and eventual respiratory failure, remain the major cause of morbidity and mortality among CF patients. A distinguishing feature of lung disease in CF is an exaggerated and persistent inflammatory response, characterized by the accumulation of excessive numbers of neutrophils and dysregulated cytokine production. The events leading to the establishment of lung infection with P. aeruginosa, especially the inflammatory and immunological events, and the relation between the CF defect and infection, remain largely undefined. Progress in this area has been hampered by the lack of a suitable animal model. An exciting achievement in the past few years has been the development of a number of variants of CFTR-deficient mice which exhibit defective cAMP-mediated Cl(-) conductance and have a range of clinical phenotypes from mild to severe. In parallel, a model of chronic P. aeruginosa lung infection has been established in genetically and immunologically well-defined inbred mouse strains which differ in susceptibility to this infection in the lung. BALB/c mice are resistant, while DBA/2 mice are extremely susceptible, with high mortality within 3 days of infection. C57BL/6 and A/J mice are relatively susceptible and experience low mortality. Furthermore, the bacterial load correlates with the magnitude and quality of the inflammatory response in the infected lungs of BALB/c and C57BL/6 mice. Although results of infection studies in CFTR-deficient mice have been variable, C57BL/6-Cftr(m1UNC)/Cftr(m1UNC) knockout mice compared to littermate control mice are highly susceptible to chronic P. aeruginosa infection in the lung. The availability of CFTR knockout mice and non-CF inbred mice differing in susceptibility to chronic P. aeruginosa infection offers useful tools for progress in understanding the genesis of chronic P. aeruginosa infection and the ensuing inflammation in the CF lung, as well as the relation between the CF defect and infection. Information generated from these studies will provide the rationale for the development of novel immunomodulatory measures capable of ameliorating or modulating the chronic inflammation associated with CF lung disease.

Identification and characterization of naturally occurring variants of the macrophage scavenger receptor (SR-A).

The scavenger receptor (SR) family comprises a group of cell surface proteins functionally defined by their ability to bind chemically modified lipoproteins. In macrophages, the class A Type I and Type II SRs (SR-AI/II) are thought to play a key role in adherence to and phagocytosis of infectious agents. Immunoprecipitation studies show that the rat anti-SR-AI/II monoclonal antibody 2F8 detects the mature, trimeric form of the receptor expressed in peritoneal macrophages from A/J, but not from C57Bl/6J (B6) mice. Subsequent sequencing of cDNA and genomic clones indicates that SR-AI and AII of A/J and B6 mice differ in sequence at nine positions, two in the cytoplasmic domain and seven in the extracellular spacer and alpha-helical coiled coil domains. These sequence polymorphisms are non-conservative and produce distinct receptor molecules that differ by four charged residues and alter recognition of the receptor by the monoclonal 2F8 antibody. The B6 SR-AI/II haplotype appears unique, since most inbred strains analyzed show the A/J-type haplotype. Interestingly, several of the B6 polymorphic variant residues are conserved in human and bovine receptors, suggesting a recent divergence of the A/J haplotype. Initial studies in CHO-derived cells expressing individual receptor isoforms indicate that the A/J and B6 receptors are stable and can mature into oligomers expressed in the membrane fractions of these cells. In these transfectants, no major functional differences were detected between receptors of the two haplotypes with respect to internalization and degradation of (125)I-labeled acetylated LDL. However, since SR-AI/II recognizes a large number of structurally unrelated anionic molecules, the possibility that different haplotypes may affect either binding and release of other ligands, or receptor recycling, cannot be excluded.