Mary E. Wilson

Oxidative Responses of Human and Murine Macrophages During Phagocytosis of Leishmania chagasi

Leishmania chagasi, the cause of South American visceral leishmaniasis, must survive antimicrobial responses of host macrophages to establish infection. Macrophage oxidative responses have been shown to diminish in the presence of intracellular leishmania. However, using electron spin resonance we demonstrated that murine and human macrophages produce O2− during phagocytosis of opsonized promastigotes. Addition of the O2− scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl to cultures resulted in increased infection, suggesting that O2− enhances macrophage leishmanicidal activity. The importance of NO· produced by inducible NO synthase (iNOS) in controlling murine leishmaniasis is established, but its role in human macrophages has been debated. We detected NO· in supernatants from murine, but not human, macrophages infected with L. chagasi. Nonetheless, the iNOS inhibitor NG-monomethyl-l-arginine inhibited IFN-γ-mediated intracellular killing by both murine and human macrophages. According to RNase protection assay and immunohistochemistry, iNOS mRNA and protein were expressed at higher levels in bone marrow of patients with visceral leishmaniasis than in controls. The iNOS protein also increased upon infection of human macrophages with L. chagasi promastigotes in vitro in the presence of IFN-γ. These data suggest that O2− and NO· each contribute to intracellular killing of L. chagasi in human and murine macrophages.

The major surface protease (MSP or GP63) of Leishmania sp. Biosynthesis, regulation of expression, and function

Leishmania sp. are digenetic protozoa that cause an estimated 1.5-2 million new cases of leishmaniasis per year worldwide. Among the molecular factors that contribute to Leishmania sp. virulence and pathogenesis is the major surface protease, alternately called MSP, GP63, leishmanolysin, EC3.4.24.36, and PSP, which is the most abundant surface protein of leishmania promastigotes. Recent studies using gene knockout, antisense RNA and overexpression mutants have demonstrated a role for MSP in resistance of promastigotes to complement-mediated lysis and either a direct or indirect role in receptor-mediated uptake of leishmania. The MSP gene clusters in different Leishmania sp. include multiple distinct MSPs that tend to fall into three classes, which can be distinguished by their sequences and by their differential expression in parasite life stages. Regulated expression of MSP class gene products during the parasite life cycle occurs at several levels involving both mRNA and protein metabolism. In this review we summarize advances in MSP research over the past decade, including organization of the gene families, crystal structure of the protein, regulation of mRNA and protein expression, biosynthesis and possible functions. The MSPs exquisitely demonstrate the multiple levels of post-transcriptional gene regulation that occur in Leishmania sp. and other trypanosomatid protozoa.

Identifying Functional MicroRNAs in Macrophages with Polarized Phenotypes

Background: Macrophages that respond to external stimuli assume a spectrum of activation states. Results: Expression of several miRNAs was altered significantly in different macrophage activation states. Functional activities on cytokine/chemokine expression were shown. Conclusion: miRNAs influenced macrophage differentiation toward distinct activation patterns. Significance: Profiling miRNA guide and passenger strands can reveal miRNA changes in differentiated cells responding to acute stimuli. Macrophages respond to external stimuli with rapid changes in expression of many genes. Different combinations of external stimuli lead to distinct polarized activation patterns, resulting in a spectrum of possible macrophage activation phenotypes. MicroRNAs (miRNAs) are small, noncoding RNAs that can repress the expression of many target genes. We hypothesized that miRNAs play a role in macrophage polarization. miRNA expression profiles were determined in monocyte-derived macrophages (MDMs) incubated in conditions causing activation toward M1, M2a, M2b, or M2c phenotypes. One miRNA guide strand and seven miRNA passenger strands were significantly altered. Changes were confirmed in MDMs from six separate donors. The amplitude of miRNA expression changes in MDMs was smaller than described studies of monocytes responding to inflammatory stimuli. Further investigation revealed this correlated with higher basal miRNA expression in MDMs compared with monocytes. The regulation of M1- and M2b-responsive miRNAs (miR-27a, miR-29b, miR-125a, miR-146a, miR-155, and miR-222) was similar in differentiated THP-1 cells and primary MDMs. Studies in this model revealed cross-talk between IFNγ- and LPS-associated pathways regulating miRNA expression. Furthermore, expression of M1-associated transcripts was increased in THP-1 cells transfected with mimics of miR-29b, miR-125a-5p, or miR-155. The apparent inflammatory property of miR-29b and miR-125a-5p can be at least partially explained by repression of TNFAIP3, a negative regulator of NF-κB signaling. Overall, these data suggest miRNAs can contribute to changes in macrophage gene expression that occur in different exogenous activating conditions.

Activation of TGF-β by Leishmania chagasi: Importance for Parasite Survival in Macrophages

TGF-β is a potent regulatory cytokine that suppresses expression of inducible NO synthase and IFN-γ, and suppresses Th1 and Th2 cell development. We examined whether functionally active TGF-β is present in the local environment surrounding the invading protozoan Leishmania chagasi. Our prior data showed that TGF-β levels are significantly increased in L. chagasi-infected mice. In the current study, we found TGF-β was also abundant in bone marrows of humans with acute visceral leishmaniasis but not in those of uninfected controls. Furthermore, L. chagasi infection caused an increase in biologically active TGF-β in human macrophage cultures without changing the total TGF-β. Therefore, we investigated the means through which leishmania could augment activated but not total TGF-β. Incubation of latent TGF-β with Leishmania sp. promastigotes caused active TGF-β to be released from the latent complex. In contrast, the nonpathogenic protozoan Crithidia fasciculata could not activate TGF-β. TGF-β activation by leishmania was prevented by inhibitors of cysteine proteases and by the specific cathepsin B inhibitor CA074. Physiologic concentrations of TGF-β inhibited killing of intracellular L. chagasi in macrophages, although the phagocytosis-induced respiratory burst remained intact. In contrast, supraphysiologic concentrations of TGF-β had no effect on parasite survival. We hypothesize that the combined effect of abundant TGF-β stores at extracellular sites during infection, and the ability of the parasite to activate TGF-β in its local environment, leads to high levels of active TGF-β in the vicinity of the infected macrophage. Locally activated TGF-β could, in turn, enhance parasite survival through its effects on innate and adaptive immune responses.

Hydrogen peroxide-mediated toxicity for Leishmania donovani chagasi promastigotes. Role of hydroxyl radical and protection by heat shock.

Leishmania must survive despite exposure to the toxic oxidant hydrogen peroxide (H2O2) during phagocytosis by macrophages. We investigated the mechanism of H2O2 toxicity for L. donovani chagasi promastigotes, and factors responsible for their relative H2O2 resistance. There was a dose-dependent toxic effect of H2O2 for promastigotes isolated during logarithmic phase of growth. In contrast, stationary phase promastigotes were less susceptible to H2O2 toxicity, and more infectious for BALB/c mice. By spin trapping we found that hydroxyl radical (.OH) was generated after exposure of promastigotes to H2O2, and the amount of .OH was greater with log-phase than with stationary-phase promastigotes. .OH was generated after the addition of H2O2 to the cytosol but not the membranes of fractionated promastigotes, and the magnitude of .OH was greater in log than in stationary promastigote cytosol. Deferoxamine inhibition suggested that intracellular promastigote iron catalyzes .OH formation via the Fenton reaction. Furthermore, exposure of log-phase promastigotes to heat shock induced a relative H2O2-resistant state, which was not associated with a decrease in .OH formation but which required ongoing transcription. Thus, growth to stationary phase and heat shock both induce a state of relative H2O2 resistance, but these are probably due to different resistance mechanisms.

Association between the Tumor Necrosis Factor Locus and the Clinical Outcome of Leishmania chagasi Infection

ABSTRACT A periurban outbreak of visceral leishmaniasis (VL) caused by the protozoan Leishmania chagasi is ongoing outside Natal, northeast Brazil. Manifestations range from asymptomatic infection to disseminated visceral disease. Literature reports suggest that both genetic and environmental factors influence the outcome of infection. Due to the association of the tumor necrosis factor (TNF) locus with other infectious diseases, we examined whether polymorphic alleles at this locus are associated with the outcome of L. chagasi infection. Neighborhoods with ongoing transmission were identified through patients admitted to local hospitals. Altogether, 1,024 individuals from 183 families were classified with the following disease phenotypes: (i) symptomatic VL, (ii) asymptomatic infection (positive delayed-type hypersensitivity [DTH+]), or (iii) no evidence of infection (DTH−). Genotypes were determined at a microsatellite marker (MSM) upstream of the TNFB gene encoding TNF-β and at a restriction fragment length polymorphism (RFLP) at position −307 in the promoter of the TNFA gene encoding TNF-α. Analyses showed that the distribution of TNFA RFLP alleles (TNF1 and TNF2) and the TNF MSM alleles (TNFa1 to TNFa15) differed between individuals with VL and those with DTH+ phenotypes. TNF1 was transmitted more frequently than expected from heterozygous parents to DTH+ offspring (P = 0.0006), and haplotypes containing TNF2 were associated with symptomatic VL (P = 0.0265, transmission disequilibrium test). Resting serum TNF-α levels were higher in TNF1/2 heterozygotes than in TNF1/1 homozygotes (P < 0.05). These data led us to hypothesize that an individuals genotype at the TNF locus may be associated with whether he or she develops asymptomatic or symptomatic disease after L. chagasi infection. The results preliminarily suggest that this may be the case, and follow-up with larger populations is needed for verification.

TGF-β Mediates CTLA-4 Suppression of Cellular Immunity in Murine Kalaazar

Recent studies indicate important roles for CTLA-4 engagement in T cells, and for TGF-β production in the immunopathogenesis of murine kalaazar or visceral leishmaniasis, but a functional link between these two pathways in helping intracellular parasite growth is unknown. Here we report that Ag or anti-CD3 activation of splenic CD4+ T cells from visceral leishmaniasis leads to intense CTLA-4-mediated TGF-β1 production, as assessed either by CTLA-4 blockade or by direct CTLA-4 cross-linkage. Production of TGF-β1 accounted for the reciprocal regulation of IFN-γ production by CTLA-4 engagement. Following CD4+ T cell activation, intracellular growth of Leishmania chagasi in cocultured splenic macrophages required both CTLA-4 function and TGF-β1 secretion. Cross-linkage of CTLA-4 markedly increased L. chagasi replication in cocultures of infected macrophages and activated CD4+ T cells, and parasite growth could be completely blocked with neutralizing anti-TGF-β1 Ab. Exogenous addition of rTGF-β1 restored parasite growth in cultures protected from parasitism by CTLA-4 blockade. These results indicate that the negative costimulatory receptor CTLA-4 is critically involved in TGF-β production and in intracellular parasite replication seen in murine kalaazar.

Inducible Resistance to Oxidant Stress in the Protozoan Leishmania chagasi

Leishmania sp. protozoa are introduced into a mammalian skin by a sandfly vector, whereupon they encounter increased temperature and toxic oxidants generated during phagocytosis. We studied the effects of 37 °C “heat shock” or sublethal menadione, which generates superoxide and hydrogen peroxide, on Leishmania chagasi virulence. Both heat and menadione caused parasites to become more resistant to H2O2-mediated toxicity. Peroxide resistance was also induced as promastigotes developed in culture from logarithmic to their virulent stationary phase form. Peroxide resistance was not associated with an increase in reduced thiols (trypanothione and glutathione) or increased activity of ornithine decarboxylase, which is rate-limiting in trypanothione synthesis. Membrane lipophosphoglycan increased in size as parasites developed to stationary phase but not after environmental exposures. Instead, parasites underwent a heat shock response upon exposure to heat or sublethal menadione, detected by increased levels of HSP70. Transfection of promastigotes withL. chagasi HSP70 caused a heat-inducible increase in resistance to peroxide, implying it is involved in antioxidant defense. We conclude that leishmania have redundant mechanisms for resisting toxic oxidants. Some are induced during developmental change and others are induced in response to environmental stress.

Serial Quantitative PCR Assay for Detection, Species Discrimination, and Quantification of Leishmania spp. in Human Samples

ABSTRACT The Leishmania species cause a variety of human disease syndromes. Methods for diagnosis and species differentiation are insensitive and many require invasive sampling. Although quantitative PCR (qPCR) methods are reported for leishmania detection, no systematic method to quantify parasites and determine the species in clinical specimens is established. We developed a serial qPCR strategy to identify and rapidly differentiate Leishmania species and quantify parasites in clinical or environmental specimens. SYBR green qPCR is mainly employed, with corresponding TaqMan assays for validation. The screening primers recognize kinetoplast minicircle DNA of all Leishmania species. Species identification employs further qPCR set(s) individualized for geographic regions, combining species-discriminating probes with melt curve analysis. The assay was sufficient to detect Leishmania parasites, make species determinations, and quantify Leishmania spp. in sera, cutaneous biopsy specimens, or cultured isolates from subjects from Bangladesh or Brazil with different forms of leishmaniasis. The multicopy kinetoplast DNA (kDNA) probes were the most sensitive and useful for quantification based on promastigote standard curves. To test their validity for quantification, kDNA copy numbers were compared between Leishmania species, isolates, and life stages using qPCR. Maxicircle and minicircle copy numbers differed up to 6-fold between Leishmania species, but the differences were smaller between strains of the same species. Amastigote and promastigote leishmania life stages retained similar numbers of kDNA maxi- or minicircles. Thus, serial qPCR is useful for leishmania detection and species determination and for absolute quantification when compared to a standard curve from the same Leishmania species.

An Effect of Parasite-Encoded Arginase on the Outcome of Murine Cutaneous Leishmaniasis

Classical activation of macrophages infected with Leishmania species results in expression and activation of inducible NO synthase (iNOS) leading to intracellular parasite killing. Macrophages can contrastingly undergo alternative activation with increased arginase activity, metabolism of arginine along the polyamine pathway, and consequent parasite survival. An active role for parasite-encoded arginase in host microbicidal responses has not previously been documented. To test the hypothesis that parasite-encoded arginase can influence macrophage responses to intracellular Leishmania, a comparative genetic approach featuring arginase-deficient mutants of L. mexicana lacking both alleles of the gene encoding arginase (Δarg), as well as wild-type and complemented Δarg controls (Δarg[pArg]), was implemented. The studies showed: 1) the absence of parasite arginase resulted in a significantly attenuated infection of mice (p < 0.05); 2) poorer survival of Δarg in mouse macrophages than controls correlated with greater NO generation; 3) the difference between Δarg or control intracellular survival was abrogated in iNOS-deficient macrophages, suggesting iNOS activity was responsible for increased Δarg killing; 4) consistently, immunohistochemistry showed enhanced nitrotyrosine modifications in tissues of mice infected with Δarg compared with control parasites. Furthermore, 5) in the face of decreased parasite survival, lymph node cells draining cutaneous lesions of Δarg parasites produced more IFN-γ and less IL-4 and IL-10 than controls. These data intimate that parasite-encoded arginase of Leishmania mexicana subverts macrophage microbicidal activity by diverting arginine away from iNOS.