How do protozoan parasites survive inside macrophages?

During infections with intracellular microbes, macrophages have two roles. On the one hand, they are important effector cells for the control and killing of intracellular bacteria and protozoan parasites by oxidative and non-oxidative mechanisms. On the other hand, macrophages may also serve as long-term host cells that facilitate the replication and survival of the pathogens, for example, by protecting them against toxic components of the extracellular milieu. In this review, Christian Bogdan and Martin R?llinghoff summarize some of the more recently discovered mechanisms by which intracellular protozoan parasites, such as Leishmania spp, Trypanosoma cruzi and Toxoplasma gondii, manage to exploit macrophages as safe target cells.     

TGF-β: upregulator of HIV replication in macrophages

TGF-β at physiological concentrations, when added to monocyte-derived macrophages following HIV1 infection, has an enhancing effect upon the rate of virus production. This effect is observed with the monocytotropic isolate ADA, as well as with HIV1 IIIB, which poorly replicates in macrophages.     

Heterogeneity in 5′-nucleotidase activity of mouse peritoneal macrophages

Summary After stimulation of the mouse peritoneal cavity with newborn calf serum (NBCS), four types of monocyte and macrophage were distinguished on the basis of peroxidase (PO) patterns. Cytochemically, these cells showed strong heterogeneity in 5-nucleotidase (5N) activity. Monocytes and monocyte-derived macrophages with PO activity in granules lacked 5N activity. Resident macrophages (with PO activity in RER and nuclear envelope) generally had significant 5N activity on the plasma membrane, the pattern showing close correlation with the biochemical findings. The group of PO-negative macrophages comprised both 5N-negative and 5N-positive cells. These findings suggest two possibilities, i.e., that monocytes (5N-)transform via PO-negative cells (5N-/+) into resident macrophages (5N+), or that the monocytes and monocyte-derived macrophages and the resident macrophages represent separate lineages. The fourth type of macrophage, the exudate-resident cell (with PO activity both in granules and in the RER and nuclear envelope), occurred only in low numbers and very late after NBCS stimulation, and is therefore considered not to be a transitional cell between monocytes and resident macrophages.     

Defective pro-IL-1β responses in macrophages from aged mice

Background

Cytokines regulated by the inflammasome pathway have been extensively implicated in various age-related immune pathologies. We set out to elucidate the contribution of the nod-like receptor protein 3 (NLRP3) inflammasome pathway to the previously described deficiencies in IL-1β production by macrophages from aged mice. We examined the production of pro-IL-1β and its conversion into IL-1β as two separate steps and compared these cytokine responses in bone marrow derived macrophages from young (6–8 weeks) and aged (18–24 months) C57BL/6 mice.

Findings

Relative to macrophages from young mice, macrophages from aged mice produced less pro-IL-1β after TLR4 stimulation with LPS. However upon activation of the NLRP3 inflammasome with ATP, macrophages from young and aged mice were able to efficiently convert and secrete intracellular pro-cytokines as functional cytokines.

Conclusions

Lower levels of IL-1β production are a result of slower and lower overall production of pro-IL-1β in macrophages from aged mice.

     

Occurrence of histone H1° protein in rat alveolar macrophages

Histone H1 of rat alveolar macrophages, neutrophilic granulocytes and monocytes extracted with 5% (v/v) perchloric acid was studied in order to see whether a protein similar to histone H1° of rat liver exists in these specialized cells. The biochemical methods used involved SDS and acid-urea polyacrylamide gel electrophoresis, gel filtration on BioGel P100 and raising antisera against chromatographically purified rat liver H1° and histone H1. The antiserum was applied for further characterization of the presumptive H1° fraction using ELISA and Western blot analysis.The results from our studies showed that histone H1° protein is present in rat alveolar macrophages, monocytes and neutrophilic granulocytes, but its quantity in neutrophilic granulocytes is very much less than macrophages and monocytes.     

NF-κB signaling in fetal lung macrophages disrupts airway morphogenesis

Bronchopulmonary dysplasia is a common pulmonary complication of extreme prematurity. Arrested lung development leads to bronchopulmonary dysplasia, but the molecular pathways that cause this arrest are unclear. Lung injury and inflammation increase disease risk, but the cellular site of the inflammatory response and the potential role of localized inflammatory signaling in inhibiting lung morphogenesis are not known. In this study, we show that tissue macrophages present in the fetal mouse lung mediate the inflammatory response to LPS and that macrophage activation inhibits airway morphogenesis. Macrophage depletion or targeted inactivation of the NF-κB signaling pathway protected airway branching in cultured lung explants from the effects of LPS. Macrophages also appear to be the primary cellular site of IL-1β production following LPS exposure. Conversely, targeted NF-κB activation in transgenic macrophages was sufficient to inhibit airway morphogenesis. Macrophage activation in vivo inhibited expression of multiple genes critical for normal lung development, leading to thickened lung interstitium, reduced airway branching, and perinatal death. We propose that fetal lung macrophage activation contributes to bronchopulmonary dysplasia by generating a localized inflammatory response that disrupts developmental signals critical for lung formation.     

Sustained anti-inflammatory effects of TGF-β1 on microglia/macrophages

Ischemic brain injuries caused release of damage-associated molecular patterns (DAMPs) that activate microglia/macrophages (MG/MPs) by binding to Toll-like receptors. Using middle cerebral artery transiently occluded rats, we confirmed that MG/MPs expressed inducible nitric oxide synthase (iNOS) on 3 days after reperfusion (dpr) in ischemic rat brain. iNOS expression almost disappeared on 7 dpr when transforming growth factor-β1 (TGF-β1) expression was robustly increased. After transient incubation with TGF-β1 for 24 h, rat primary microglial cells were incubated with lipopolysaccharide (LPS) and released NO level was measured. The NO release was persistently suppressed even 72 h after removal of TGF-β1. The sustained TGF-β1 effects were not attributable to microglia-derived endogenous TGF-β1, as revealed by TGF-β1 knockdown and in vitro quantification studies. Then, boiled supernatants prepared from ischemic brain tissues showed the similar sustained inhibitory effects on LPS-treated microglial cells that were prevented by the TGF-β1 receptor-selective blocker SB525334. After incubation with TGF-β1 for 24 h and its subsequent removal, LPS-induced phosphorylation of IκB kinases (IKKs), IκB degradation, and NFκB nuclear translocation were inhibited in a sustained manner. SB525334 abolished all these effects of TGF-β1. In consistent with the in vitro results, phosphorylated IKK-immunoreactivity was abundant in MG/MPs in ischemic brain lesion on 3 dpr, whereas it was almost disappeared on 7 dpr. The findings suggest that abundantly produced TGF-β1 in ischemic brain displays sustained anti-inflammatory effects on microglial cells by persistently inhibiting endogenous Toll-like receptor ligand-induced IκB degradation.     

Cl-IB-MECA enhances TNF-α release in peritoneal macrophages stimulated with LPS

Adenosine receptor A3 (A3R) belongs to the Gi/Gq-coupled receptor family, that leads to the intracellular cAMP reduction and intracellular calcium increase, respectively. A3R is widely expressed and it can play a crucial role in many patho-physiological conditions, including inflammation. Here we investigate the effect of Cl-IB-MECA, A3R agonist, on the production of TNF-α. We found that Cl-IB-MECA enhances LPS-induced TNF-α release in peritoneal macrophages. This effect is reduced by MRS1191, A3R antagonist and by forskolin, activator of adenylyl cyclase. pIκBα increased in LPS+Cl-IB-MECA-treated macrophages, while total IκB kinase-β (IKKβ) reduced. Indeed, p65NF-κB nuclear translocation increased in cells treated with LPS+Cl-IB-MECA. Moreover, IMD 0354, IKKβ inhibitor, significantly abrogated the effect of Cl-IB-MECA on TNF-α release. Inhibition of protein kinase C (PKC) significantly reduced Cl-IB-MECA-induced TNF-α release in LPS-stimulated macrophages. Furthermore, LY-294002, PI3K inhibitor, reduced the TNF-α production enhanced by Cl-IB-MECA, although the phosphorylation status of Akt did not change in cells treated with LPS+Cl-IB-MECA than LPS alone. In summary, these data show that Cl-IB-MECA is able to enhance TNF-α production in LPS-treated macrophages in an NF-κB- dependent manner.     

Secretion of IL-1β triggered by dynasore in murine peritoneal macrophages

The interaction of lipopolysaccharide-primed murine peritoneal macrophages with ivermectin, an antiparasite drug which potentiates P2X(4) receptors and dynasore which inhibits the GTPase activity of dynamin, a protein contributing to the internalization of plasma membrane proteins, was tested. Murine peritoneal macrophages express P2X(4) receptors which are mostly intracellular. In cells from P2X(7)-knockout mice (KO mice), 10 μm adenosine triphosphate (ATP) provoked a transient increase of the intracellular concentration of calcium. Ivermectin had no effect by itself but potentiated the increase of the intracellular concentration of calcium by ATP. The combination of ATP plus ivermectin also decreased the intracellular concentration of potassium and promoted the secretion of IL-1β. Concentrations of dynasore above 50?μm affected the integrity of mitochondria (MTT test) and of the plasma membrane (release of lactate dehydrogenase, LDH). At a 10 μm concentration, dynasore had no effect on the responses to ATP and on the internalization of P2X(4) receptors. By itself dynasore promoted the release of potassium and the secretion of IL-1β after activation of caspase-1. In conclusion, our results confirm that ivermectin potentiates the responses coupled to P2X(4) receptors probably by interaction with an allosteric site. We also show that this potentiation triggers the release of IL-1β by macrophages. As opposed to ivermectin, dynasore has no effect on P2X(4) receptors. This drug triggers a potassium efflux via a mechanism which does not involve purinergic receptors and generates, in consequence, the activation of caspase-1 and the secretion of IL-1β.     

High salt induces anti-inflammatory MΦ2-like phenotype in peripheral macrophages

Macrophages play a critical role in inflammation and antigen-presentation. Abnormal macrophage function has been attributed in autoimmune diseases and cancer progression. Recent evidence suggests that high salt tissue micro-environment causes changes in macrophage activation. In our current report, we studied the role of extracellular sodium chloride on phenotype changes in peripheral circulating monocyte/macrophages collected from healthy donors. High salt (0.2 M NaCl vs basal 0.1 M NaCl) treatment resulted in a decrease in MΦ1 macrophage phenotype (CD11b⁺CD14^highCD16^low) from 77.4±6.2% (0.1 M) to 29.3±5.7% (0.2 M, p<0.05), while there was an increase in MΦ2 macrophage phenotype (CD11b⁺ CD14^lowCD16^high) from 17.2±5.9% (0.1 M) to 67.4±9.4% (0.2 M, p<0.05). ELISA-based cytokine analysis demonstrated that high salt treatment induced decreased expression of in the MΦ1 phenotype specific pro-inflammatory cytokine, TNFα (3.3 fold), IL-12 (2.3 fold), CCL-10 (2 fold) and CCL-5 (3.8 fold), but conversely induced an enhanced expression MΦ2-like phenotype specific anti-inflammatory cytokine, IL-10, TGFβ, CCL-17 (3.7 fold) and CCR-2 (4.3 fold). Further high salt treatment significantly decreased phagocytic efficiency of macrophages and inducible nitric oxide synthetase expression. Taken together, these data suggest that high salt extracellular environment induces an anti-inflammatory MΦ2-like macrophage phenotype with poor phagocytic and potentially reduced antigen presentation capacity commonly found in tumor microenvironment.     

Metallophilic macrophages are lacking in the thymus of lymphotoxin-β receptor-deficient mice

Lymphotoxin-β receptor (LTβR) axis plays a crucial role in development and compartmentalization of peripheral lymphatic organs. But, it is also required for the appropriate function and maintenance of structural integrity of the thymus: in LTβR-deficient animals the clonal deletion of autoreactive lymphocytes is impaired and differentiation of thymic medullary epithelial cells is disturbed. In this study, using several markers, we showed that thymic metallophilic macrophages were lacking in LTβR-deficient mice. In tumor necrosis factor receptor-I (p55)-deficient mice (which we used as positive control) thymic metallophilic cells were located, similarly as in normal mice, in the thymic cortico-medullary zone at the junction of cortex and medulla. These findings show that LTβR is necessary for maintenance of metallophilic macrophages in the thymus and provide further evidence that these cells may represent a factor involved in thymic negative selection.     

Modulation of cisPlatin cytotoxicity by interleukin-1α and resident tumor macrophages

The modulation of cisPlatin cytotoxicity by interleukin-1 (IL-1α) was studied in cultures of SCC-7 tumor cells with and without tumor macrophages to examine potential mechanisms for the synergistic antitumor activity of cisPlatin and IL-1α in SCC-7 solid tumors. Neither IL-1α nor tumor macrophages affected the survival of clonogenic tumor cells and IL-1α had no direct effect on tumor cell growthin vitro. Macrophages had no direct effect on cisPlatin sensitivity (IC₉₀=6.0 μM), but, the addition of IL-1α (500–2000U/ml) to co-cultures of cisPlatin pretreated tumor cells and resident tumor macrophages increased cell killing (IC₉₀=3.1 μM). Similar responses were seen in primary cultures treated with cisPlatin before IL-1α. The modulation of cisPlatin cytotoxicity by IL-1α exhibited a biphasic dose response that paralleled the IL-1α dose dependent release of H₂O₂by resident tumor macrophages. Further, IL-1α modification of cisPlatin cytotoxicity was prompt and inhibited by catalase. CisPlatin and exogenous H₂O₂ (50 μM) produced more than additive SCC-7 clonogenic cell kill and hydroxyl radicals played an important role in the response. Interleukin-1 modulation of cisPlatin cytotoxicity was schedule dependent. IL-1α treatment for 24 hrs, before cisPlatin, produced drug resistance (IC₉₀=11.1 μM). Our study shows that IL-1α can stimulate tumor macrophages to release pro-oxidants that modify cellular chemosensitivity in a schedule and dose dependent fashion. Our findings may also provide a mechanistic explanation for the synergistic antitumor activity of cisPlatin and IL-1αin vivo.     

Interactions between naïve and infected macrophages reduce Mycobacterium tuberculosis viability

A high intracellular bacillary load of Mycobacterium tuberculosis in macrophages induces an atypical lysosomal cell death with early features of apoptosis that progress to necrosis within hours. Unlike classical apoptosis, this cell death mode does not appear to diminish M. tuberculosis viability. We previously reported that culturing heavily infected macrophages with na?ve macrophages produced an antimicrobial effect, but only if na?ve macrophages were added during the pre-necrotic phase of M. tuberculosis-induced cell death. In the present study we investigated the mechanism of antimicrobial activity in co-cultures, anticipating that efferocytosis of bacilli in apoptotic bodies would be required. Confocal microscopy revealed frustrated phagocytosis of M. tuberculosis-infected macrophages with no evidence that significant numbers of bacilli were transferred to the na?ve macrophages. The antimicrobial effect of na?ve macrophages was retained when they were separated from infected macrophages in transwells, and conditioned co-culture supernatants transferred antimicrobial activity to cultures of infected macrophages alone. Antimicrobial activity in macrophage co-cultures was abrogated when the na?ve population was deficient in IL-1 receptor or when the infected population was deficient in inducible nitric oxide synthase. The participation of nitric oxide suggested a conventional antimicrobial mechanism requiring delivery of bacilli to a late endosomal compartment. Using macrophages expressing GFP-LC3 we observed the induction of autophagy specifically by a high intracellular load of M. tuberculosis. Bacilli were identified in LC3-positive compartments and LC3-positive compartments were confirmed to be acidified and LAMP1 positive. Thus, the antimicrobial effect of na?ve macrophages acting on M. tuberculosis in heavily-infected macrophages is contact-independent. Interleukin-1 provides an afferent signal that induces an as yet unidentified small molecule which promotes nitric oxide-dependent antimicrobial activity against bacilli in autolysosomes of heavily infected macrophages. This cooperative, innate antimicrobial interaction may limit the maximal growth rate of M. tuberculosis prior to the expression of adaptive immunity in pulmonary tuberculosis.     

IFN-λ3 inhibits HIV infection of macrophages through the JAK-STAT pathway

Background

Interferon lambda 3 (IFN-λ3) is a newly identified cytokine with antiviral activity, and its single nucleotide polymorphisms are strongly associated with the treatment effectiveness and development of chronic hepatitis C virus infection. We thus examined the potential of IFN-λ3 to inhibit HIV replication and the possible mechanisms of the anti-HIV action by IFN-λ3 in human macrophages.

Principal Findings

Under different conditions (before, during, and after HIV infection), IFN-λ3 significantly inhibited viral replication in macrophages, which was associated with the induction of multiple antiviral cellular factors (ISG56, MxA, OAS-1, A3G/F and tetherin) and IFN regulatory factors (IRF-1, 3, 5, 7 and 9). This anti-HIV action of IFN-λ3 could be compromised by the JAK-STAT inhibitor. In addition, IFN-λ3 treatment of macrophages induced the expression of toll-like receptor 3 (TLR3) and two key adaptors (MyD88 and TRIF) in type I IFN pathway activation. However, HIV infection compromised IFN-λ3-mediated induction of the key elements in JAK-STAT signaling pathway.

Conclusions

These data indicate that IFN-λ3 exerts its anti-HIV function by activating JAK-STAT pathway-mediated innate immunity in macrophages. Future in vivo studies are necessary in order to explore the potential for developing IFN-λ3-based therapy for HIV disease.