Macrophage activation to kill Leishmania tropica: kinetics of macrophage response to lymphokines that induce antimicrobial activities against amastigotes

Lymphokine (LK) treatment of resident peritoneal macrophages from C3H/HeN mice induced two antimicrobial activities against Leishmania tropica: increased resistance of activated macrophages to infection with amastigotes and intracellular destruction of those parasites that entered activated cells. The onset and duration of these two antimicrobial activities were quite different. Resistance to infection was observed as early as 4 hr after the addition of LK, became maximal at 8 hr, and persisted in a subpopulation of treated cells for as long as 72 hr. In contrast, intracellular killing occurred with as little as 4 hr of LK treatment after infection, and maximal killing was observed in cultures exposed to LK 24 hr. Intracellular killing capacity of lymphokine-treated cells was progressively lost in macrophages treated longer than 12 hr before exposure to parasites. This decay in ability to destroy intracellular L. tropica was also seen in macrophages cultured longer than 12 hr before LK treatment, and may reflect loss of macrophage responsiveness to LK with increasing time in vitro. Thus, treatment of macrophages with lymphokines induced both a stable change in cell-parasite interactions, resistance to infection, and a short-lived capacity to destroy intracellular amastigotes.     

Differential susceptibility of activated macrophage cytotoxic effector reactions to the suppressive effects of transforming growth factor-beta 1

We examined the effects of TGF-beta 1 on induction of several activated macrophage antimicrobial activities against the protozoan parasite Leishmania, and on induction of tumoricidal activity against the fibrosarcoma tumor target 1023. TGF-beta by itself did not affect the viability of either the intracellular or extracellular target in concentrations up to 200 ng/ml. As little as 1 ng/ml TGF-beta, however, suppressed more than 70% of the intracellular killing activity of macrophages treated with lymphokines. In contrast, more than 100 ng/ml TGF-beta was required to suppress intracellular killing by cells activated with an equivalent amount of recombinant IFN-gamma. Addition of TGF-beta for up to 30 min after exposure to activation factors significantly reduced macrophage killing of intracellular parasites. Pretreatment of macrophages with TGF-beta was even more effective: treatment of cells with TGF-beta for 4 h before addition of activation factors abolished all macrophage intracellular killing activity. Regardless of treatment sequence, however, TGF-beta had absolutely no effect, at any concentration tested, on activated macrophage resistance to infection induced by lymphokines or by the cooperative interaction of IFN-gamma and IL-4. Effects of TGF-beta on tumoricidal activity of activated macrophages was intermediate to that of its effects on intracellular killing or resistance to infection. Lymphokine-induced tumor cytotoxicity was marginally (25%) affected by TGF-beta; 200 ng/ml was able to suppress IFN-gamma-induced tumoricidal activity by 40%. Thus, TGF-beta dramatically suppressed certain activated macrophage cytotoxic effector reactions, but was only partially or not at all effective against others, even when the same activation agent (IFN-gamma) was used. The biochemical target for TGF-beta suppressive activity in these reactions may be the pathway for nitric oxide production from L-arginine, because TGF-beta also inhibited the generation of nitric oxide by cytokine-activated macrophages.     

Macrophage activation to kill Leishmania tropica: characterization of P/J mouse macrophage defects for lymphokine-induced antimicrobial activities against Leishmania tropica amastigotes

Macrophages from P/J mice demonstrated both quantitative and qualitative defects in lymphokine (LK)-induced activated macrophage antileishmanial effector reactions: a) these cells recognized the same LK signals that generated resistance to infection in responsive C3H/HeN macrophages, but more signal was required to observe maximal activity; b) LK-induced intracellular destruction of Leishmania tropica by P/J macrophages was minimal (less than 20%), and was induced by only one of three LK signals that regulate antimicrobial activities in C3H/HeN macrophages. The defective microbicidal activity of P/J macrophages observed with LK activation in vitro could also be demonstrated in vivo. Macrophages from P/J mice exposed to the macrophage-activating agent Mycobacterium bovis strain BCG in vivo were capable of restricting the intracellular replication of L. tropica but could not eliminate intracellular parasites, even with further incubation with LK during the 72-hr culture period. The defect of P/J macrophages for intracellular destruction of L. tropica, then, occurred in the activation sequence before the triggering stage that characterizes the macrophage defect of C3H/HeJ mice. Genetic regulation of the P/J macrophage defect appears to be by a single autosomal gene, with defective microbicidal activity as a recessive trait in these animals.     

Effect of BMAP-28 antimicrobial peptides on Leishmania major promastigote and amastigote growth: role of leishmanolysin in parasite survival

Background

Protozoan parasites, such as Leishmania, still pose an enormous public health problem in many countries throughout the world. Current measures are outdated and have some associated drug resistance, prompting the search into novel therapies. Several innovative approaches are under investigation, including the utilization of host defence peptides (HDPs) as emerging anti-parasitic therapies. HDPs are characterised by their small size, amphipathic nature and cationicity, which induce permeabilization of cell membranes, whilst modulating the immune response of the host. Recently, members of the cathelicidin family of HDPs have demonstrated significant antimicrobial activities against various parasites including Leishmania. The cathelicidin bovine myeloid antimicrobial peptide 28 (BMAP-28) has broad antimicrobial activities and confers protection in animal models of bacterial infection or sepsis. We tested the effectiveness of the use of BMAP-28 and two of its isomers the D-amino acid form (D-BMAP-28) and the retro-inverso form (RI-BMAP-28), as anti-leishmanial agents against the promastigote and amastigote intracellular Leishmania major lifecycle stages.

Methodology/Principal Findings

An MTS viability assay was utilized to show the potent antiparasitic activity of BMAP-28 and its protease resistant isomers against L. major promastigotes in vitro. Cell membrane permeability assays, caspase 3/7, Tunel assays and morphologic studies suggested that this was a late stage apoptotic cell death with early osmotic cell lysis caused by the antimicrobial peptides. Furthermore, BMAP-28 and its isomers demonstrated anti-leishmanial activities against intracellular amastigotes within a macrophage infection model.

Conclusions/Significance

Interestingly, D-BMAP-28 appears to be the most potent antiparasitic of the three isomers against wild type L. major promastigotes and amastigotes. These exciting results suggest that BMAP-28 and its protease resistant isomers have significant therapeutic potential as novel anti-leishmanials.     

LXR-dependent gene expression is important for macrophage survival and the innate immune response

The liver X receptors (LXRs) are nuclear receptors with established roles in the regulation of lipid metabolism. We now show that LXR signaling not only regulates macrophage cholesterol metabolism but also impacts antimicrobial responses. Mice lacking LXRs are highly susceptible to infection with the intracellular bacteria Listeria monocytogenes (LM). Bone marrow transplant studies point to altered macrophage function as the major determinant of susceptibility. LXR-null macrophages undergo accelerated apoptosis when challenged with LM and exhibit defective bacterial clearance in vivo. These defects result, at least in part, from loss of regulation of the antiapoptotic factor SPalpha, a direct target for regulation by LXRalpha. Expression of LXRalpha or SPalpha in macrophages inhibits apoptosis in the setting of LM infection. Our results demonstrate that LXR-dependent gene expression plays an unexpected role in innate immunity and suggest that common nuclear receptor pathways mediate macrophage responses to modified lipoproteins and intracellular pathogens.     

The Trypanosoma cruzi protease cruzain mediates immune evasion

Trypanosoma cruzi is the causative agent of Chagas' disease. Novel chemotherapy with the drug K11777 targets the major cysteine protease cruzain and disrupts amastigote intracellular development. Nevertheless, the biological role of the protease in infection and pathogenesis remains unclear as cruzain gene knockout failed due to genetic redundancy. A role for the T. cruzi cysteine protease cruzain in immune evasion was elucidated in a comparative study of parental wild type- and cruzain-deficient parasites. Wild type T. cruzi did not activate host macrophages during early infection (<60 min) and no increase in ～P iκB was detected. The signaling factor NF-κB P65 colocalized with cruzain on the cell surface of intracellular wild type parasites, and was proteolytically cleaved. No significant IL-12 expression occurred in macrophages infected with wild type T. cruzi and treated with LPS and BFA, confirming impairment of macrophage activation pathways. In contrast, cruzain-deficient parasites induced macrophage activation, detectable iκB phosphorylation, and nuclear NF-κB P65 localization. These parasites were unable to develop intracellularly and survive within macrophages. IL 12 expression levels in macrophages infected with cruzain-deficient T. cruzi were comparable to LPS activated controls. Thus cruzain hinders macrophage activation during the early (<60 min) stages of infection, by interruption of the NF-κB P65 mediated signaling pathway. These early events allow T. cruzi survival and replication, and may lead to the spread of infection in acute Chagas' disease.     

Discovery of a novel Toxoplasma gondii conoid-associated protein important for parasite resistance to reactive nitrogen intermediates

Toxoplasma gondii modifies its host cell to suppress its ability to become activated in response to IFN-γ and TNF-α and to develop intracellular antimicrobial effectors, including NO. Mechanisms used by T. gondii to modulate activation of its infected host cell likely underlie its ability to hijack monocytes and dendritic cells during infection to disseminate to the brain and CNS where it converts to bradyzoites contained in tissue cysts to establish persistent infection. To identify T. gondii genes important for resistance to the effects of host cell activation, we developed an in vitro murine macrophage infection and activation model to identify parasite insertional mutants that have a fitness defect in infected macrophages following activation but normal invasion and replication in naive macrophages. We identified 14 independent T. gondii insertional mutants out of >8000 screened that share a defect in their ability to survive macrophage activation due to macrophage production of reactive nitrogen intermediates (RNIs). These mutants have been designated counter-immune mutants. We successfully used one of these mutants to identify a T. gondii cytoplasmic and conoid-associated protein important for parasite resistance to macrophage RNIs. Deletion of the entire gene or just the region encoding the protein in wild-type parasites recapitulated the RNI-resistance defect in the counter-immune mutant, confirming the role of the protein in resistance to macrophage RNIs.     

Hypoxia modulates expression of the 70-kD heat shock protein and reduces<Emphasis Type="Italic">Leishmania</Emphasis> infection in macrophages

Hypoxia, a microenvironmental factor present in diseased tissues, has been recognized as a specific metabolic stimulus or a signal of cellular response. Experimental hypoxia has been reported to induce adaptation in macrophages such as differential migration, elevation of proinflammatory cytokines and glycolytic enzyme activities, and decreased phagocytosis of inert particles. In this study we demonstrate that although exposure to hypoxia (5% O₂, 5% CO₂, and balanced N₂) did not change macrophage viability, or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cleavage and proliferation, it significantly reduced expression of the 70-kD heat shock protein (HSP70), which was restored to prehypoxia levels after reoxygenation. The influence of low oxygen tension on macrophage functional activity was also studied, i.e. the ability of these cells to maintain or resist infection by a microorganism. We demonstrate that macrophages from two different sources (a murine cell line and primary cells) exposed to hypoxia were efficiently infected withLeishmania amazonensis, but after 24 h showed a reduction in the percentage of infected cells and of the number of intracellular parasites per macrophage, indicating that hypoxia induced macrophages to kill the intracellular parasites. These results support the notion that hypoxia, a microenvironmental factor, can modulate macrophage protein expression and functional activity.     

Colony-stimulating factors and regulation of macrophage tumoricidal and microbicidal activities

Conditioned medium from antigen- or mitogen-stimulated spleen cells, lymphokines, contained factors that induced formation of granulocyte and macrophage colonies in cultures of bone marrow cells (CSF). Lymphokines also contained factors that induced macrophage non-specific tumoricidal activity against fibrosarcoma 1023, antibody-dependent tumoricidal activity against lymphoma 18-8, and antimicrobial activities against amastigotes of the protozoan parasite, Leishmania tropica. The factors that regulated macrophage effector functions, however, were different from those that induced colony formation, and could be distinguished from CSF by Sephadex gel chromatography or heat sensitivity. To further analyze a role for CSF in induction of macrophage effector activities, conditioned medium from several nonlymphoid cell sources (L-929, WEHI-3, and endotoxin-treated lung cells) were assayed for CSF activities and capacity to induce tumoricidal and microbicidal activities. Conditioned medium that contained either macrophages CSF (CSF-1) or the factor that induced formation of both macrophage and granulocyte colonies failed to activate macrophages for effector activities against fibrosarcoma 1023, lymphoma 18-8, and L. tropica amastigotes (either resistance to infection or intracellular destruction). These data suggest that CSF has no direct role in activation of macrophages for tumoricidal and microbicidal activities against these targets.     

Suppression of macrophage lysosomal enzymes after Leishmania donovani infection

In order to have an insight into the role of host lysosomal enzymes in the intracellular survival of Leishmania parasites, the activities of beta-galactosidase, alpha-mannosidase, and N-acetyl-beta-D-glucosaminidase were studied in peritoneal macrophages of hamsters infected with L. donovani. There was a significant decrease of all three lysosomal enzymes after infection. Heat-killed or formalin-treated parasites failed to inhibit the enzymes, instead a slight stimulation was observed. Purified excreted factor from promastigotes had no effect on the enzymes except beta-galactosidase which was inhibited up to 20%. Inhibition of enzymes was not due to increased secretion after infection. The absence of induction of any endogenous macrophage inhibitor was confirmed by mixed experiments. The levels of 5'-nucleotidase and lactate dehydrogenase remained unchanged after infection. Thus, the inhibition of lysosomal enzymes appears to be the effect of infection process and reflects to actua decrease rather than increased secretion or the action of any inhibitors present in Leishmania promastigotes.     

Leishmania amazonensis Amastigotes Highly Express a Tryparedoxin Peroxidase Isoform That Increases Parasite Resistance to Macrophage Antimicrobial Defenses and Fosters Parasite Virulence

Professional phagocytes generate a myriad of antimicrobial molecules to kill invading microorganisms, of which nitrogen oxides are integral in controlling the obligate intracellular pathogen Leishmania. Although reactive nitrogen species produced by the inducible nitric oxide synthase (iNOS) can promote the clearance of intracellular parasites, some Leishmania species/stages are relatively resistant to iNOS-mediated antimicrobial activity. The underlying mechanism for this resistance remains largely uncharacterized. Here, we show that the amastigote form of L. amazonensis is hyper-resistant to the antimicrobial actions of cytokine-activated murine and human macrophages as compared to its promastigote counterpart. Amastigotes exhibit a marked ability to directly counter the cytotoxicity of peroxynitrite (ONOO⁻), a leishmanicidal oxidant that is generated during infection through the combined enzymatic activities of NADPH oxidase and iNOS. The enhanced antinitrosative defense of amastigotes correlates with the increased expression of a tryparedoxin peroxidase (TXNPx) isoform that is also upregulated in response to iNOS enzymatic activity within infected macrophages. Accordingly, ectopic over-expression of the TXNPx isoform by L. amazonensis promastigotes significantly enhances parasite resistance against ONOO⁻ cytotoxicity. Moreover, TXNPx-overexpressing parasites exhibit greater intra-macrophage survival, and increased parasite growth and lesion development in a murine model of leishmaniasis. Our investigations indicate that TXNPx isoforms contribute to Leishmania''s ability to adapt to and antagonize the hostile microenvironment of cytokine-activated macrophages, and provide a mechanistic explanation for persistent infection in experimental and human leishmaniasis.     

Host-cell cyclophilin is important for the intracellular replication of Leishmania major

The antiparasitic effects of cyclosporin A were examined in leishmanial infection by analysing the role of CsA-binding proteins (cyclophilins) in the host–parasite interaction. We hypothesized that the leishmanicidal effects of CsA on Leishmania major infected macrophages might be mediated through a cyclophilin of either the parasite or the host cell. Two cyclophilins (20 and 22 kDa) were purified from L. major parasites and N-terminally sequenced. Although enzyme activity of these cyclophilins was inhibited by CsA, pretreatment of L. major parasites with CsA did not result in reduction of a subsequent macrophage infection, arguing against a role of L. major cyclophilins as infectivity potentiators. However, host-cell cyclophilin A (CypA) was found to be critically involved in the intracellular replication of L. major parasites in murine macrophages. An antisense oligonucleotide to murine CypA was constructed and added to cultures of peritoneal macrophages prior to infection with L. major parasites. This treatment strongly reduced the expression of CypA in macrophages and resulted in the inhibition of the intracellular replication of L. major amastigotes. These data indicate that interaction of amastigotes with host-cell cyclophilin is an important part of the intracellular replication machinery of L. major and define, for the first time, a direct involvement of a cyclophilin in the survival strategies of an intracellular parasite.     

Effect of hypoxia on macrophage infection by Leishmania amazonensis

In the present study, we compared the effect of 5% oxygen tension (hypoxia) with a normal tension of 21% oxygen (normoxia) on macrophage infection by the protozoan parasite Leishmania amazonensis. Macrophages from different sources (human cell line U937, murine cell line J774, and murine peritoneal macrophages) exposed to hypoxia showed a reduction of the percentage of infected cells and the number of intracellular parasites per cell. Observations on the kinetics of infection indicated that hypoxia did not depress L. amazonensis phagocytosis but induced macrophages to reduce intracellular parasitism. Furthermore, hypoxia did not act synergistically with gamma-interferon and bacterial lipopolysaccharides in macrophages to induce killing of parasites. Experiments also indicated no correlation between nitric oxide production and control of infection in macrophages under hypoxic condition. Thus, we have provided the first evidence that hypoxia, which occurs in various pathological conditions, can alter macrophage susceptibility to a parasitic infection.     

Role of protein kinase C-alpha in the control of infection by intracellular pathogens in macrophages.

The protein kinase C (PKC) family regulates macrophage function involved in host defense against infection. In this study, we investigated the role of macrophage PKC-alpha in the uptake and subsequent fate of Leishmania donovani promastigotes and Legionella pneumophila infections. To this end, we used clones of the murine macrophage cell line RAW 264.7 overexpressing a dominant-negative (DN) mutant of PKC-alpha. While phagocytosis of L. donovani promastigotes was not affected by DN PKC-alpha overexpression, their intracellular survival was enhanced by 10- to 20-fold at 48 h postinfection. Intracellular survival of a L. donovani mutant defective in lipophosphoglycan repeating units synthesis, which normally is rapidly degraded in phagolysosomes, was enhanced by 100-fold at 48 h postinfection. However, IFN-gamma-induced leishmanicidal activity was not affected by DN PKC-alpha overexpression. Similar to macrophages from genetically resistant C57BL/6 mice, control RAW 264.7 cells were not permissive for the intracellular replication of Legionella pneumophila. In contrast, DN PKC-alpha-overexpressing RAW 264.7 clones were phenotypically similar to macrophages from genetically susceptible A/J mice, as they allowed intracellular replication of L. pneumophila. Permissiveness to L. pneumophila was not the consequence of a general defect in the microbicidal capacities because killing of a temperature-sensitive mutant of Pseudomonas aeruginosa was normal in DN PKC-alpha-overexpressing RAW 264.7 clones. Collectively, these results support a role for PKC-alpha in the regulation of innate macrophage functions involved in the control of infection by intracellular parasites.     

Flavone-resistant Leishmania donovani Overexpresses LdMRP2 Transporter in the Parasite and Activates Host MRP2 on Macrophages to Circumvent the Flavone-mediated Cell Death

In parasites, ATP-binding cassette (ABC) transporters represent an important family of proteins related to drug resistance and other biological activities. Resistance of leishmanial parasites to therapeutic drugs continues to escalate in developing countries, and in many instances, it is due to overexpressed ABC efflux pumps. Progressively adapted baicalein (BLN)-resistant parasites (pB²⁵R) show overexpression of a novel ABC transporter, which was classified as ABCC2 or Leishmania donovani multidrug resistance protein 2 (LdMRP2). The protein is primarily localized in the flagellar pocket region and in internal vesicles. Overexpressed LdABCC2 confers substantial BLN resistance to the parasites by rapid drug efflux. The BLN-resistant promastigotes when transformed into amastigotes in macrophage cells cannot be cured by treatment of macrophages with BLN. Amastigote resistance is concomitant with the overexpression of macrophage MRP2 transporter. Reporter analysis and site-directed mutagenesis assays demonstrated that antioxidant response element 1 is activated upon infection. The expression of this phase II detoxifying gene is regulated by NFE2-related factor 2 (Nrf2)-mediated antioxidant response element activation. In view of the fact that the signaling pathway of phosphoinositol 3-kinase controls microfilament rearrangement and translocation of actin-associated proteins, the current study correlates with the intricate pathway of phosphoinositol 3-kinase-mediated nuclear translocation of Nrf2, which activates MRP2 expression in macrophages upon infection by the parasites. In contrast, phalloidin, an agent that prevents depolymerization of actin filaments, inhibits Nrf2 translocation and Mrp2 gene activation by pB²⁵R infection. Taken together, these results provide insight into the mechanisms by which resistant clinical isolates of L. donovani induce intracellular events relevant to drug resistance.     

Defensins enable macrophages to inhibit the intracellular proliferation of Listeria monocytogenes

Listeria monocytogenes is a facultative intracellular pathogen that infects a large diversity of host cells, including macrophages. To avoid the phagosome microbicidal environment, L. monocytogenes secretes a pore-forming toxin (listeriolysin O, LLO) that releases the bacterium into the cytoplasm. We hypothesized that the α-defensins (HNPs) and/or humanized θ-defensin (RC-1) peptides produced by human and non-human primate neutrophils, respectively, cooperate with macrophages to control L. monocytogenes infection. Our results establish that HNP-1 and RC-1 enable macrophages to control L. monocytogenes intracellular growth by inhibiting phagosomal escape, as a consequence, bacteria remain trapped in a LAMP-1-positive phagosome. Importantly, HNP-1 interaction with macrophages and RC-1 interaction with bacteria are required to prevent macrophage infection. In accordance with these results, RC-1 is a more potent anti-listerial peptide than HNP-1 and HNP-1 is acquired by macrophages and trafficked to the phagocytosed bacteria. Finally, HNP-1 and RC-1 antimicrobial activity is complemented by their ability to prevent LLO function through two mechanisms, blocking LLO-dependent perforation of macrophage membranes and the release of LLO from the bacteria. In conclusion, at the site of infection the cooperation between antimicrobial peptides, such as HNP-1, and macrophages likely plays a critical role in the innate immune defence against L. monocytogenes.     

Role of macrophage phospholipase D in natural and CpG-induced antimycobacterial activity

The present study addresses the differential ability of macrophages to control intracellular growth of non-pathogenic Mycobacterium smegmatis (Msm) and pathogenic M. tuberculosis (MTB). Results reported herein show that 3 h post infection, intracellular Msm, but not MTB, was significantly killed by macrophages. As the role of human macrophage phospholipase D (PLD) in the activation of antimicrobial mechanisms has been documented, we hypothesised the role of such enzyme in antimycobacterial mechanisms. To this aim, macrophage PLD activity was analysed at different times after exposure with either pathogenic MTB or non-pathogenic Msm. Results showed that, starting from 15 min after mycobacterial exposure, MTB did not induce macrophage PLD activity, whereas the environmental non-pathogenic Msm stably increased it. The direct contribution of PLD in intracellular mycobacterial killing was also analysed by inhibiting enzymatic activity with ethanol or calphostin C. Results show that PLD inhibition significantly increases intracellular Msm replication. In order to see whether the innate PLD-mediated antimicrobial mechanisms against MTB are also induced after CpG ODN stimulation, the role of PLD has been analysed in the course of CpG-mediated intracellular MTB killing. CpG DNA increased PLD activity in both uninfected and MTB-infected macrophages, and the inhibition of PLD activity resulted in a significant reduction of CpG-induced MTB killing. Taken together, our data suggest a relationship between host PLD activation and the macrophage ability to control intracellular mycobacterial growth.     

Stimulation by granulocyte-macrophage colony-stimulating factor of Leishmania tropica killing by macrophages.

The intracellular protozoan parasite Leishmania tropica was found to survive unharmed and to multiply for several days in normal mouse peritoneal macrophages. In contrast, when infected monolayers were treated with GM-CSF, there was a continuous decrease in the percentage of infected cells, reaching less than 10% on day 4 in culture, compared to about 30% in normal controls. Microscopic observations showed an increased number of dead parasites in GM-CSF treated infected cells. Within 5 hr of incubation with GM-CSF, almost 40% of intracellular parasites showed morphologic damage, compared to less than 10% in untreated cells. Pretreatment of macrophage monolayers with pure GM-CSF before infection led to an increased level of phagocytosis of L. tropica parasites as reflected by the percentage of infected cells and the increased number of parasites in each infected cell. GM-CSF treated cultures showed 73% infected cells containing a mean of five parasites per cell, as compared to controls in which only about 50% of macrophages were infected with only two parasites per cell. The number of dead parasites per cell was 5-fold higher in the GM-CSF treated cultures at 2 hr. After 24 hr the percentage of infected GM-CSF treated cells was less than one-third that in the control cultures.     

Leishmania infection inhibits macrophage motility by altering F‐actin dynamics and the expression of adhesion complex proteins

Leishmania is an intracellular protozoan parasite that causes a broad spectrum of clinical manifestations, ranging from self‐healing skin lesions to fatal visceralizing disease. As the host cells of choice for all species of Leishmania, macrophages are critical for the establishment of infections. How macrophages contribute to parasite homing to specific tissues and how parasites modulate macrophage function are still poorly understood. In this study, we show that Leishmania amazonensis infection inhibits macrophage roaming motility. The reduction in macrophage speed is not dependent on particle load or on factors released by infected macrophages. L. amazonensis‐infected macrophages also show reduced directional migration in response to the chemokine MCP‐1. We found that infected macrophages have lower levels of total paxillin, phosphorylated paxillin, and phosphorylated focal adhesion kinase when compared to noninfected macrophages, indicating abnormalities in the formation of signaling adhesion complexes that regulate motility. Analysis of the dynamics of actin polymerization at peripheral sites also revealed a markedly enhanced F‐actin turnover frequency in L. amazonensis‐infected macrophages. Thus, Leishmania infection inhibits macrophage motility by altering actin dynamics and impairing the expression of proteins that function in plasma membrane–extracellular matrix interactions.