IL-13 pre-treatment of murine peritoneal macrophages increases their anti-Toxoplasma gondii activity induced by lipopolysaccharides

Th1 cytokines and microbial lipopolysaccharides (LPS) activate macrophages to produce inflammatory mediators and effector molecules. Althrough Th2 cytokines often have an opposite action to Th1 cytokines and down-modulate the inflammatory response of macrophages, they can induce a distinct alternative activation that is beneficial in host defence. In this study, we report that IL-13 enhances the anti-Toxoplasma activity of LPS-activated murine macrophages. The inhibition of parasite proliferation was not related to reduced Toxoplasma gondii penetration into the cells, nor to the conversion of tachyzoites into bradyzoites. Used alone, IL-13 triggers the polarisation of macrophages towards type 2. However, in LPS-activated macrophages, we show the priming capacity of this cytokine to enhance the expression of inducible nitric oxide synthase (iNOS), a major marker of type 1 macrophages. This effect of IL-13 was not dependent on the activation state of macrophages (resident versus thioglycolate-elicited) or the timing of pre-treatment. We demonstrate a correlation between the enhancement of NO production and upgrading of the microbicidal effectiveness of the macrophages. Thus, both Th2 and Th1 cytokines could activate macrophages to control infections.     

Leishmania inhibits STAT1-mediated IFN-gamma signaling in macrophages: increased tyrosine phosphorylation of dominant negative STAT1beta by Leishmania mexicana

Previous studies have demonstrated that Leishmania donovani attenuates STAT1-mediated signaling in macrophages; however it is not clear whether other species of Leishmania, which cause cutaneous disease, also interfere with macrophage IFN-gamma signaling. Therefore, we determined the effect of Leishmania major and Leishmania mexicana infection on STAT1-mediated IFN-gamma signaling pathway in J774A.1 and RAW264.7 macrophages. We found that both L. major and L. mexicana suppressed IFNgammaRalpha (alpha subunit of interferon gamma receptor) and IFN-gammaRbeta (beta subunit of interferon gamma receptor) expression, reduced levels of total Jak1 and Jak2, and down-regulated IFN-gamma-induced Jak1, Jak2 and STAT1 activation. The effect of L. mexicana infection on Jak1, Jak2 and STAT1 activation was more profound when compared with L. major. Although tyrosine phosphorylation of STAT1alpha was decreased in IFN-gamma stimulated macrophages infected with L. major or L. mexicana, those infected with L. mexicana showed a significant increase in phosphorylation of the dominant negative STAT1beta. These findings indicate that L. major and L. mexicana attenuate STAT1-mediated IFN-gamma signaling in macrophages. Furthermore, they also demonstrate that L. mexicana preferentially enhances tyrosine phosphorylation of dominant negative STAT1beta, which may be one of the several survival mechanisms used by this parasite to evade the host defense mechanisms.     

Modulation of inducible nitric oxide synthase induction by prostaglandin E2 in macrophages: distinct susceptibility in murine J774 and RAW 264.7 macrophages

Prostaglandin E2 (PGE2) is the major cyclooxygenase metabolite in macrophages with complex proinflammatory and immunoregulatory properties. In the present study, we have compared the modulatory role of PGE2/cAMP-dependent signaling on induced nitric oxide (NO) production in two murine macrophages, J774 and RAW 264.7. With no effect on NO release by itself, PGE2 co-addition with lipopolysaccharide (LPS) resulted in a concentration-dependent enhancement in NO release and inducible NO synthase induction in J774, but not in RAW 264.7, macrophages. The potentiation effect of PGE2 in J774 cells was still seen when applied within 9 h after LPS treatment. Whereas RAW 264.7 macrophages release PGE2 with greater extent than J774 macrophages in response to LPS, indomethacin and NS-398, upon abolishing LPS-induced PGE2 release, caused a more obvious inhibition of NO release from J774 than RAW 264.7 cells. Thus, we suggest a higher positive modulatory role of PGE2--either endogenous or exogenous--on NO formation in J774 cells. Supporting these findings, exogenous PGE2 triggers cAMP formation in J774 cells with higher potency and efficacy. Of interest, dBcAMP also elicits higher sensitivity in potentiating NO release in J774 cells. We conclude that the opposite effect of PGE2/cAMP signaling on macrophage NO induction depends on its signaling efficacy and might be associated with the difference in endogenous PGE2 levels.     

Saliva from Lutzomyia longipalpis induces CC chemokine ligand 2/monocyte chemoattractant protein-1 expression and macrophage recruitment

Saliva of bloodfeeding arthropods has been incriminated in facilitating the establishment of parasite in their host. We report on the leukocyte chemoattractive effect of salivary gland homogenate (SGH) from Lutzomyia longipalpis on saliva-induced inflammation in an air pouch model. SGH (0.5 pair/animal) was inoculated in the air pouch formed in the back of BALB/c or C57BL/6 mice. L. longipalpis SGH induced a significant influx of macrophages in BALB/c but not in C57BL/6 mice. SGH-induced cell recruitment reached a peak at 12 h after inoculation and was higher than that induced by the LPS control. This differential cell recruitment in BALB/c mice was directly correlated to an increase in CCL2/MCP-1 expression in the air pouch lining tissue. In fact, treatment with bindarit, an inhibitor of CCL2/MCP-1 synthesis, and also with a specific anti-MCP-1 mAb resulted in drastic reduction of macrophage recruitment and inhibition of CCL2/MCP-1 expression in the lining tissue. CCL2/MCP-1 production was also seen in vitro when J774 murine macrophages were exposed to L. longipalpis SGH. The SGH effect was abrogated by preincubation with serum containing anti-SGH IgG Abs as well as in mice previously sensitized with L. longipalpis bites. Interestingly, the combination of SGH with Leishmania chagasi induced an increased recruitment of neutrophils and macrophages when compared with L. chagasi alone. Taken together these results suggest that SGH not only induces the recruitment of a greater number of macrophages by enhancing CCL2/MCP-1 production but also synergizes with L. chagasi to recruit more inflammatory cells to the site of inoculation.     

Pharmacological evaluation of anti-leishmanial activity by in vivo nitric oxide modulation in Balb/c mice infected with Leishmania major MRHO/IR/75/ER: an Iranian strain of cutaneous leishmaniasis

Cutaneous leishmaniasis with a variation in its clinical signs is still one of the health problems in the world, region and Iran. Immune responses against leishmania consist of cytokines, immune cells and mediators. Macrophages participate actively in the inflammatory response by releasing chemokines and mediators including nitric oxide (NO) and reactive oxygen and nitrogen intermediates. This study investigates whether NO had anti-leishmanial effects and/or mediated pathology in mice infected with Leishmania major MRHO/IR/75/ER (IR/75). NO inducer lipopolysaccharide (LPS) and NO donor S-nitrosoglutathione (SNOG) were used for their ability to increase RNI and to modify leishmania infection in susceptible Balb/c mice, in order to evaluate the effects of NO production on size and lesion macroscopy, delay of lesion formation and proliferation of amastigotes inside macrophages. Liver, spleen and lymph nodes were also studied as target organs to detect amastigotes. In addition to plasma, liver and spleen suspensions were investigated for NO induction by using Griess microassay. Statistical analysis of data revealed an association between increases in NO level with the pathology of disease in Balb/c mice infected with L. major IR/75. The survival of leishmania parasite inside the macrophages and its proliferation was affected by LPS and SNOG-treatments. An inconsistent relationship was evident between the NO modulation and pathological changes in treated Balb/c mice infected with L. major IR/75.     

Toxoplasma gondii infection of activated J774-A1 macrophages causes inducible nitric oxide synthase degradation by the proteasome pathway

Classically activated macrophages produce nitric oxide (NO), which is a potent microbicidal agent. NO production is catalyzed by inducible nitric oxide synthase (iNOS), which uses arginine as substrate producing NO and citruline. However, it has been demonstrated that NO production is inhibited after macrophage infection of Toxoplasma gondii, the agent of toxoplasmosis, due to iNOS degradation. Three possible iNOS degradation pathways have been described in activated macrophages: proteasome, calpain and lysosomal. To identify the iNOS degradation pathway after T. gondii infection, J774-A1 macrophage cell line was activated with lipopolysaccharide and interferon-gamma for 24 h, treated with the following inhibitors, lactacystin (proteasome), calpeptin (calpain), or concanamycin A (lysosomal), and infected with the parasite. NO production and iNOS expression were evaluated after 2 and 6 h of infection. iNOS was degraded in J774-A1 macrophages infected with T. gondii. However, treatment with lactacystin maintained iNOS expression in J774-A1 macrophages infected for 2 h by T. gondii, and after 6 h iNOS was localized in aggresomes. iNOS was degraded after parasite infection of J774-A1 macrophages treated with calpeptin or concanamycin A. NO production confirmed iNOS expression profiles. These results indicate that T. gondii infection of J774-A1 macrophages caused iNOS degradation by the proteasome pathway.     

Red blood cell lysate modulates the expression of extracellular matrix proteins in dermal fibroblasts

Trichinella spiralis is a zoonotic nematode and food borne parasite and infection with T. spiralis leads to suppression of the host immune response and other immunopathologies. The excretory/secretory (ES) products of T. spiralis play important roles in the process of immunomodulation. However, the mechanisms and related molecules are unknown. Macrophages, a target for immunomodulation by the helminth parasite, play a critical role in initiating and modulating the host immune response to parasite infection. In this study, we examined the effect of ES products from different stages of T. spiralis on modulating J774A.1 macrophage activities. ES products from different stages of T. spiralis reduced the capacity of macrophages to express pro-inflammatory cytokines (tumor necrosis factor α, interleukin-1β , interleukin-6 , and interleukin-12) in response to lipopolysaccharide (LPS) challenge. However, only ES products from 3-day-old adult worms and 5-day-old adult worms/new-born larvae significantly inhibited inducible nitric oxide synthase gene expression in LPS-induced macrophages. In addition, ES products alone boosted the expression of anti-inflammatory cytokines interleukin-10 and transforming growth factor-β and effector molecule arginase 1 in J774A.1 macrophages. Signal transduction studies showed that ES products significantly inhibited nuclear factor-κB translocation into the nucleus and the phosphorylation of both extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase in LPS-stimulated J774A.1 macrophages. These results suggest that ES products regulate host immune response at the macrophage level through inhibition of pro-inflammatory cytokines production and induction of macrophage toward the alternative phenotype, which maybe important for worm survival and host health.     

Leishmania major amastigotes initiate the L-arginine-dependent killing mechanism in IFN-gamma-stimulated macrophages by induction of tumor necrosis factor-alpha

Macrophages exposed to IFN-gamma and infected with amastigotes of Leishmania major develop the capacity to eliminate the intracellular pathogen. This antimicrobial activity of activated macrophages correlates with the initiation of nitrogen oxidation of L-arginine, yet other reports suggest that two signals are required for induction of this biochemical pathway for effector activity. In the present studies, macrophages treated with up to 100 U/ml IFN-gamma, or 100 ng LPS, or 10(7) amastigotes produced minimal quantities (less than 9 microM) of NO2- and failed to develop cytotoxic effector activities. In contrast, the combination of IFN-gamma and either LPS (greater than 0.1 ng) or amastigotes (10(6) induced high concentrations (much greater than 30 microM) of NO2- and macrophage cytotoxicity against intra- and extracellular targets. The induction of nitrogen oxidation by amastigotes could be dissociated from LPS-induced events by 1) performing the assays in the presence of polymyxin B (which blocked LPS effects, but not amastigote effects), 2) determining the threshold of IFN-gamma required to prime cells for subsequent trigger (1 U/ml for LPS trigger effects; 10-fold higher for amastigotes), and 3) determining the heat sensitivity of the two trigger agents (amastigote effects abolished at 100 degrees C; LPS effects unaffected at this temperature). Further, culture fluids from amastigote-infected macrophages did not contain detectable LPS (less than 6 pg/ml). Possible parasite and cell-associated factors that could contribute to the induction of nitrogen oxidation and cytotoxic activity of IFN-gamma treated macrophages were examined: only certain intact microorganisms, LPS from a variety of bacteria, and the cytokine TNF alpha were effective. Both NO2- production and intracellular killing were abolished by the addition of anti-TNF-alpha mAb in the assay. TNF-alpha was produced by amastigote-infected macrophages and IFN-gamma dramatically enhanced secretion of this cytokine; IFN-gamma alone had no effect. Endogenous TNF-alpha produced during infection of macrophages with L. major acted in an autocrine fashion to trigger the production of L-arginine-derived toxic nitrogen intermediates that killed the intracellular parasites.     

Leishmania amazonensis infection does not inhibit systemic nitric oxide levels elicited by lipopolysaccharide in vivo

Leishmaniasis is a parasitic disease that leads to chronic inflammation. Macrophages, depending on their activation state, are either hosts or killers of the parasites. Downregulation of nitric oxide (NO) synthesis by the parasite infecting the macrophages has been proposed to be an important evading mechanism based on in vitro studies. We confirmed inhibition of NO release by macrophages infected with Leishmania amazonensis in vitro. To examine the role of the parasite in regulating NO production in vivo, we monitored systemic NO levels elicited by challenging naive and L. amazonensis-infected BALB/c mice with lipopolysaccharide (LPS). Animals were challenged after 1, 2, 6, and 9 wk of infection. NO production was monitored by electron paramagnetic resonance spectroscopy as the levels of hemoglobin nitrosyl complexes (HbNO) present in the animal's blood. No significant differences in HbNO levels were observed between LPS-treated naive and inoculated mice at any time during infection. To control for increased macrophage numbers in infected mice, naive mice were injected with a macrophage cell line before LPS challenge; this treatment did not increase produced NO levels. The results argue against a major role for the parasite in downregulating NO production in vivo.     

Macrophage interactions with neutrophils regulate Leishmania major infection

Macrophages are host cells for the pathogenic parasite Leishmania major. Neutrophils die and are ingested by macrophages in the tissues. We investigated the role of macrophage interactions with inflammatory neutrophils in control of L. major infection. Coculture of dead exudate neutrophils exacerbated parasite growth in infected macrophages from susceptible BALB, but killed intracellular L. major in resistant B6 mice. Coinjection of dead neutrophils amplified L. major replication in vivo in BALB, but prevented parasite growth in B6 mice. Neutrophil depletion reduced parasite load in infected BALB, but exacerbated infection in B6 mice. Exacerbated growth of L. major required PGE(2) and TGF-beta production by macrophages, while parasite killing depended on neutrophil elastase and TNF-alpha production. These results indicate that macrophage interactions with dead neutrophils play a previously unrecognized role in host responses to L. major infection.     

Studies on the inactivation of superoxide dismutase activity by nitric oxide from rat peritoneal macrophages

Rat peritoneal macrophages stimulated with lipopolysaccharide (LPS) and Phorbol myristate acetate (PMA) generated increased levels of superoxide anions (O2ú-) by 122% as compared to those stimulated with PMA alone. However, Nitric oxide (NO) synthase inhibitors-n-monomethyl arginine (nMMA) or spermine-HCI lowered the enhanced levels of O2ú- released by LPS treated macrophages. The Superoxide dismutase (SOD) activity in LPS treated macrophages was 51% lower than that observed in resident cells. NO synthase inhibitors prevented the loss of SOD activity in LPS treated cells. Exogenously added SOD during sensitization of cells with LPS also inactivated the enzyme. This inactivation of SOD is inhibited by Nitric oxide synthase inhibitors. PMA alone did not affect SOD activity. NO synthase inhibitors also did not affect PMA activated superoxide anion generation in macrophages. These studies indicate that nitric oxide generated by LPS treated macrophages can inactivate SOD activity.     

Leishmania Eukaryotic Initiation Factor (LeIF) Inhibits Parasite Growth in Murine Macrophages

The leishmaniases constitute neglected global public health problems that require adequate control measures, prophylactic clinical vaccines and effective and non-toxic drug treatments. In this study, we explored the potential of Leishmania infantum eukaryotic initiation factor (LieIF), an exosomal protein, as a novel anti-infective therapeutic molecule. More specifically, we assessed the efficacy of recombinant LieIF, in combination with recombinant IFN-γ, in eliminating intracellular L. donovani parasites in an in vitro macrophage model. J774A.1 macrophages were initially treated with LieIF/IFN-γ prior to in vitro infection with L. donovani stationary phase promastigotes (pre-infection treatment), and resistance to infection was observed 72 h after infection. J774A.1 macrophages were also treated with LieIF/IFN-γ after L. donovani infection (post-infection treatment), and resistance to infection was also observed at both time points tested (19 h and 72 h) after infection. To elucidate the LieIF/IFN-γ-induced mechanism(s) that mediate the reduction of intracellular parasite growth, we examined the generation of potent microbicidal molecules, such as nitric oxide (NO) and reactive oxygen species (ROS), within infected macrophages. Furthermore, macrophages pre-treated with LieIF/IFN-γ showed a clear up-regulation in macrophage inflammatory protein 1α (MIP-1α) as well as tumor necrosis factor alpha (TNF-α) expression. However, significant different protein levels were not detected. In addition, macrophages pre-treated with LieIF/IFN-γ combined with anti-TNF-α monoclonal antibody produced significantly lower amounts of ROS. These data suggest that during the pre-treatment state, LieIF induces intramacrophage parasite growth inhibition through the production of TNF-α, which induces microbicidal activity by stimulating NO and ROS production. The mechanisms of NO and ROS production when macrophages are treated with LieIF after infection are probably different. Overall, these results indicate that LieIF is a good candidate for use as an anti-leishmanial molecule.     

The response of testicular leukocytes to lipopolysaccharide-induced inflammation: further evidence for heterogeneity of the testicular macrophage population

The majority of macrophages in the rat testis can be identified by the tissue-resident macrophage marker ED2. A smaller population of intratesticular macrophages do not express the ED2 antigen but are positive for the monocyte/macrophage marker ED1. Treatment of adult rats with the inflammatory stimulus lipopolysaccharide (LPS) had no effect on the number of testicular resident (ED2(+)) macrophages but caused a transient increase in ED1(+)ED2(-) monocyte-like macrophages (an average three-fold increase 12 h later). In both control and LPS-treated rat testes, a majority of macrophages that expressed ED1 and all Leydig cells were immuno-positive for the inducible isoform of nitric oxide synthase (iNOS). However, less than 6% of ED2(+) macrophages showed any iNOS expression, even after LPS treatment. This deficiency was confirmed by the finding that isolated ED2(+) testicular macrophages (>98% pure) stimulated with LPS did not produce NO in vitro. In contrast, resident macrophages from the peritoneum showed the expected NO response, and purified Leydig cells produced significant NO regardless of the presence or absence of LPS. Collectively, these data indicate the presence of at least two macrophage subsets in the adult rat testis: (1) the ED2(+) resident macrophages, which do not alter following LPS-treatment and mostly do not express iNOS or produce NO in response to an inflammatory stimulus, and (2) the ED1(+)ED2(-) monocyte-like macrophages, which increase in number after LPS-treatment and express iNOS even in the absence of exogenous inflammatory stimulation. It is highly probable that these different subsets have different functional roles within the testis.     

Oxalomalate affects the inducible nitric oxide synthase expression and activity

Inducible nitric oxide synthase (iNOS) is an homodimeric enzyme which produces large amounts of nitric oxide (NO) in response to inflammatory stimuli. Several factors affect the synthesis and catalytic activity of iNOS. Particularly, dimerization of NOS monomers is promoted by heme, whereas an intracellular depletion of heme and/or L-arginine considerably decreases NOS resistance to proteolysis. In this study, we found that oxalomalate (OMA, oxalomalic acid, alpha-hydroxy-beta-oxalosuccinic acid), an inhibitor of both aconitase and NADP-dependent isocitrate dehydrogenase, inhibited nitrite production and iNOS protein expression in lipopolysaccharide (LPS)-activated J774 macrophages, without affecting iNOS mRNA content. Furthermore, injection of OMA precursors to LPS-stimulated rats also decreased nitrite production and iNOS expression in isolated peritoneal macrophages. Interestingly, alpha-ketoglutarate or succinyl-CoA administration reversed OMA effect on NO production, thus correlating NO biosynthesis with the anabolic capacity of Krebs cycle. When protein synthesis was blocked by cycloheximide in LPS-activated J774 cells treated with OMA, iNOS protein levels, evaluated by Western blot analysis and (35)S-metabolic labelling, were decreased, suggesting that OMA reduces iNOS biosynthesis and induces an increase in the degradation rate of iNOS protein. Moreover, we showed that OMA inhibits the activity of the iNOS from lung of LPS-treated rats by enzymatic assay. Our results, demonstrating that OMA acts regulating synthesis, catalytic activity and degradation of iNOS, suggest that this compound might have a potential role in reducing the NO overproduction occurring in some pathological conditions.     

Effect of the microtubule stabilising agent taxol on leishmanial protozoan parasites in vitro

Taxol, a mitotic spindle toxin, was found to selectively inhibit the proliferation of Leishmania donovani in vitro at nanomolar concentrations with an IC50 of 35 nM. Concentrations of taxol as high as 50 nM, however, did not affect J774A.1 murine macrophages. Taxol (30 nM) also inhibited amastigote multiplication within a J774A.1 macrophage cell line when used in a 10-day experiment. It resulted in the in vitro assembly of L. donovani microtubules in a dose-dependent manner. When promastigotes were exposed to different concentrations of taxol for 24 h, cells were largely blocked in the G2-M phase of the cell cycle and there was a marked reduction in the percentage of cells in the S phase. The selective nature of taxol action against the parasite and its effectiveness in controlling amastigote multiplication emphasise its use as a promising chemotherapeutic against kala-azar.     

Functional alterations in macrophages after hypoxia selection

Regions of low oxygen tension are common features of inflamed and infected tissues and provide physiologic selective pressure for the expansion of cells with enhanced hypoxia tolerance. The aim of this study was to investigate whether macrophages resistant to death induced by hypoxia were accompanied by functional alterations. A mouse macrophage cell line (J774 cells) was used to obtain subpopulations of death-resistant macrophages induced by long-term exposure to severe hypoxia (<1% O(2)). The results indicated that exposing J774 macrophages to periods of severe hypoxia results in the selection of cells with phenotypes associated with the modulation of heat-shock protein 70 kDa (HSP70) expression, tumor necrosis factor-alpha (TNF-alpha), and nitric oxide (NO) production and reduced susceptibility to parasite Leishmania infection. Thus, we suggest that hypoxia-selected macrophages may influence the outcome of inflammation and infection.     

Neutrophils activate macrophages for intracellular killing of Leishmania major through recruitment of TLR4 by neutrophil elastase

We investigated the role of neutrophil elastase (NE) in interactions between murine inflammatory neutrophils and macrophages infected with the parasite Leishmania major. A blocker peptide specific for NE prevented the neutrophils from inducing microbicidal activity in macrophages. Inflammatory neutrophils from mutant pallid mice were defective in the spontaneous release of NE, failed to induce microbicidal activity in wild-type macrophages, and failed to reduce parasite loads upon transfer in vivo. Conversely, purified NE activated macrophages and induced microbicidal activity dependent on secretion of TNF-alpha. Induction of macrophage microbicidal activity by either neutrophils or purified NE required TLR4 expression by macrophages. Injection of purified NE shortly after infection in vivo reduced the burden of L. major in draining lymph nodes of TLR4-sufficient, but not TLR4-deficient mice. These results indicate that NE plays a previously unrecognized protective role in host responses to L. major infection.     

Effect of the anti-microtubule compound tubulozole on Leishmania protozoan parasites in vitro

Abstract For the cis and trans stereoisomers of the synthetic anti-microtubule compound tubulozole, at micromolar concentrations, tubulozole-C is cytotoxic to mammalian cells whereas tubulozole-T is not. The effect of tubulozoles on the parasitic protozoan Leishmania was tested. For the promastigote stage of L. mexicana amazonensis , both isomers inhibited parasite growth. For the amastigote stage of L. mexicana amazonensis and L. major , within murine J774 macrophage line as host cells in vitro, tubulozole-T reduced the infective index. Despite the observation of macrophage cytotoxicity of tubulozole-T, this compound may be a potentially useful and novel anti-leishmanial drug.