A Recombinant Protein Based on Trypanosoma cruzi P21 Enhances Phagocytosis

Background

P21 is a secreted protein expressed in all developmental stages of Trypanosoma cruzi. The aim of this study was to determine the effect of the recombinant protein based on P21 (P21-His₆) on inflammatory macrophages during phagocytosis.

Findings

Our results showed that P21-His₆ acts as a phagocytosis inducer by binding to CXCR4 chemokine receptor and activating actin polymerization in a way dependent onthe PI3-kinase signaling pathway.

Conclusions

Thus, our results shed light on the notion that native P21 is a component related to T. cruzi evasion from the immune response and that CXCR4 may be involved in phagocytosis. P21-His₆ represents an important experimental control tool to study phagocytosis signaling pathways of different intracellular parasites and particles.     

Efficient Measurement of Opsonising Antibodies to Plasmodium falciparum Merozoites

Background

Antibodies targeting merozoites are important in protection from malaria. Therefore, merozoite surface proteins are attractive vaccine candidates. There is a need for robust functional assays to investigate mechanisms of acquired immunity and vaccine efficacy. To date, the study of merozoite phagocytosis has been confounded by the complexity and variability of in vitro assays.

Methodology/Principal findings

We have developed a new flow cytometry-based merozoite phagocytosis assay. An optimized merozoite preparation technique produced high yields of merozoites separated from haemozoin. Phagocytosis by the undifferentiated THP-1 monocytic cell line was mediated only by Fc Receptors, and was therefore ideal for studying opsonising antibody responses. The assay showed robust phagocytosis with highly diluted immune sera and strong inter-assay correlation. The assay effectively measured differences in opsonisation-dependent phagocytosis among individuals.

Conclusions/Significance

This highly reproducible assay has potential applications in assessing the role of opsonic phagocytosis in naturally acquired immunity and vaccine trials.     

Oxidized Low Density Lipoprotein Induced Caspase-1 Mediated Pyroptotic Cell Death in Macrophages: Implication in Lesion Instability?

Background

Macrophage death in advanced lesion has been confirmed to play an important role in plaque instability. However, the mechanism underlying lesion macrophage death still remains largely unknown.

Methods and Results

Immunohistochemistry showed that caspase-1 activated in advanced lesion and co-located with macrophages and TUNEL positive reaction. In in-vitro experiments showed that ox-LDL induced caspase-1 activation and this activation was required for ox-LDL induced macrophages lysis, IL-1β and IL-18 production as well as DNA fragmentation. Mechanism experiments showed that CD36 and NLRP3/caspase-1/pathway involved in ox-LDL induced macrophage pyroptosis.

Conclusion

Our study here identified a novel cell death, pyroptosis in ox-LDL induced human macrophage, which may be implicated in lesion macrophages death and play an important role in lesion instability.     

Chronic Household Air Pollution Exposure Is Associated with Impaired Alveolar Macrophage Function in Malawian Non-Smokers

Background

Household air pollution in low income countries is an important cause of mortality from respiratory infection. We hypothesised that chronic smoke exposure is detrimental to alveolar macrophage function, causing failure of innate immunity. We report the relationship between macrophage function and prior smoke exposure in healthy Malawians.

Methods

Healthy subjects exposed daily to cooking smoke at home volunteered for bronchoalveolar lavage. Alveolar macrophage particulate content was measured as a known correlate of smoke exposure. Phagocytosis and intraphagosomal function (oxidative burst and proteolysis) were measured by a flow cytometric assay. Cytokine responses in macrophages were compared following re-exposure in vitro to wood smoke, before and after glutathione depletion.

Results

Volunteers had a range of alveolar macrophage particulate loading. The macrophage capacity for phagosomal oxidative burst was negatively associated with alveolar macrophage particulate content (n = 29, r² = 0.16, p = 0.033), but phagocytosis per se and proteolytic function were unaffected. High particulate content was associated with lower baseline CXCL8 release (ratio 0.51, CI 0.29–0.89) and lower final concentrations on re-exposure to smoke in vitro (ratio 0.58, CI 0.34–0.97). Glutathione depletion augmented CXCL8 responses by 1.49x (CI 1.02–2.17) compared with wood smoke alone. This response was specific to smoke as macrophages response to LPS were not modulated by glutathione.

Conclusion

Chronic smoke exposure is associated with reduced human macrophage oxidative burst, and dampened inflammatory cytokine responses. These are critical processes in lung defence against infection and likely to underpin the relationship between air pollution and pneumonia.     

Signaling pathways required for macrophage scavenger receptor-mediated phagocytosis: analysis by scanning cytometry

Background

Scavenger receptors are important components of the innate immune system in the lung, allowing alveolar macrophages to bind and phagocytose numerous unopsonized targets. Mice with genetic deletions of scavenger receptors, such as SR-A and MARCO, are susceptible to infection or inflammation from inhaled pathogens or dusts. However, the signaling pathways required for scavenger receptor-mediated phagocytosis of unopsonized particles have not been characterized.

Methods

We developed a scanning cytometry-based high-throughput assay of macrophage phagocytosis that quantitates bound and internalized unopsonized latex beads. This assay allowed the testing of a panel of signaling inhibitors which have previously been shown to target opsonin-dependent phagocytosis for their effect on unopsonized bead uptake by human in vitro-derived alveolar macrophage-like cells. The non-selective scavenger receptor inhibitor poly(I) and the actin destabilizer cytochalasin D were used to validate the assay and caused near complete abrogation of bead binding and internalization, respectively.

Results

Microtubule destabilization using nocodazole dramatically inhibited bead internalization. Internalization was also significantly reduced by inhibitors of tyrosine kinases (genistein and herbimycin A), protein kinase C (staurosporine, chelerythrine chloride and Gö 6976), phosphoinositide-3 kinase ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and wortmannin), and the JNK and ERK pathways. In contrast, inhibition of phospholipase C by U-73122 had no effect.

Conclusion

These data indicate the utility of scanning cytometry for the analysis of phagocytosis and that phagocytosis of unopsonized particles has both shared and distinct features when compared to opsonin-mediated phagocytosis.     

Bioinformatics analysis of macrophages exposed to Porphyromonas gingivalis: implications in acute vs. chronic infections

Background

Periodontitis is the most common human infection affecting tooth-supporting structures. It was shown to play a role in aggravating atherosclerosis. To deepen our understanding of the pathogenesis of this disease, we exposed human macrophages to an oral bacteria, Porphyromonas gingivalis (P. gingivalis), either as live bacteria or its LPS or fimbria. Microarray data from treated macrophages or control cells were analyzed to define molecular signatures. Changes in genes identified in relevant pathways were validated by RT-PCR.

Methodology/Principal Findings

We focused our analysis on three important groups of genes. Group PG (genes differentially expressed by live bacteria only); Group LFG (genes differentially expressed in response to exposure to LPS and/or FimA); Group CG (core gene set jointly activated by all 3 stimulants). A total of 842 macrophage genes were differentially expressed in at least one of the three conditions compared to naïve cells. Using pathway analysis, we found that group CG activates the initial phagocytosis process and induces genes relevant to immune response, whereas group PG can de-activate the phagocytosis process associated with phagosome-lysosome fusion. LFG mostly affected RIG-I-like receptor signaling pathway.

Conclusion/Significance

In light of the fact that acute infections involve live bacteria while chronic infections involve a combination of live bacteria and their byproducts, group PG could represent acute P. gingivalis infection while group LFG could represent chronic P. gingivalis infection. Group CG may be associated with core immune pathways, triggered irrespective of the specific stimulants and indispensable to mount an appropriate immune response. Implications in acute vs. chronic infection are discussed.     

Polymorphisms of SP110 Are Associated with both Pulmonary and Extra-Pulmonary Tuberculosis among the Vietnamese

Background

Tuberculosis (TB) is an infectious disease that remains a major cause of morbidity and mortality worldwide, yet the reasons why only 10% of people infected with Mycobacterium tuberculosis go on to develop clinical disease are poorly understood. Genetically determined variation in the host immune response is one factor influencing the response to M. tuberculosis. SP110 is an interferon-responsive nuclear body protein with critical roles in cell cycling, apoptosis and immunity to infection. However association studies of the gene with clinical TB in different populations have produced conflicting results.

Methods

To examine the importance of the SP110 gene in immunity to TB in the Vietnamese we conducted a case-control genetic association study of 24 SP110 variants, in 663 patients with microbiologically proven TB and 566 unaffected control subjects from three tertiary hospitals in northern Vietnam.

Results

Five SNPs within SP110 were associated with all forms of TB, including four SNPs at the C terminus (rs10208770, rs10498244, rs16826860, rs11678451) under a dominant model and one SNP under a recessive model, rs7601176. Two of these SNPs were associated with pulmonary TB (rs10208770 and rs16826860) and one with extra-pulmonary TB (rs10498244).

Conclusion

SP110 variants were associated with increased susceptibility to both pulmonary and extra-pulmonary TB in the Vietnamese. Genetic variants in SP110 may influence macrophage signaling responses and apoptosis during M. tuberculosis infection, however further research is required to establish the mechanism by which SP110 influences immunity to tuberculosis infection.     

RNAi-mediated CD40-CD154 interruption promotes tolerance in autoimmune arthritis

Introduction

We have previously demonstrated that ex vivo inhibition of costimulatory molecules on antigen-pulsed dendritic cells (DCs) can be useful for induction of antigen-specific immune deviation and suppression of autoimmune arthritis in the collagen induced arthritis (CIA) model. The current study evaluated a practical method of immune modulation through temporary systemic inhibition of the costimulatory molecule CD40.

Methods

Mice with collagen II (CII)-induced arthritis (CIA) were administered siRNA targeting the CD40 molecule. Therapeutic effects were evaluated by clinical symptoms, histopathology, Ag-specific T cell and B cell immune responses.

Results

Systemic administration of CD40-targeting siRNA can inhibit antigen-specific T cell response to collagen II, as well as prevent pathogenesis of disease in both a pre- and post-immunization manner in the CIA model. Disease amelioration was associated with suppression of Th1 cytokines, attenuation of antibody production, and upregulation of T regulatory cells.

Conclusions

These studies support the feasibility of transient gene silencing at a systemic level as a mechanism of resetting autoreactive immunity.     

Poractant alfa (Curosurf?) increases phagocytosis of apoptotic neutrophils by alveolar macrophages in vivo

Background

Clearance of apoptotic neutrophils in the lung is an essential process to limit inflammation, since they could become a pro-inflammatory stimulus themselves. The clearance is partially mediated by alveolar macrophages, which phagocytose these apoptotic cells. The phagocytosis of apoptotic immune cells by monocytes in vitro has been shown to be augmented by several constituents of pulmonary surfactant, e.g. phospholipids and hydrophobic surfactant proteins. In this study, we assessed the influence of exogenous poractant alfa (Curosurf^®) instillation on the in vivo phagocytosis of apoptotic neutrophils by alveolar macrophages.

Methods

Poractant alfa (200 mg/kg) was instilled intratracheally in the lungs of three months old adult male C57/Black 6 mice, followed by apoptotic neutrophil instillation. Bronchoalveloar lavage was performed and alveolar macrophages and neutrophils were counted. Phagocytosis of apoptotic neutrophils was quantified by determining the number of apoptotic neutrophils per alveolar macrophages.

Results

Exogenous surfactant increased the number of alveolar macrophages engulfing apoptotic neutrophils 2.6 fold. The phagocytosis of apoptotic neutrophils was increased in the presence of exogenous surfactant by a 4.7 fold increase in phagocytosed apoptotic neutrophils per alveolar macrophage.

Conclusions

We conclude that the anti-inflammatory properties of surfactant therapy may be mediated in part by increased numbers of alveolar macrophages and increased phagocytosis of apoptotic neutrophils by alveolar macrophages.     

Gene expression and biological pathways in tissue of men with prostate cancer in a randomized clinical trial of lycopene and fish oil supplementation

Background

Studies suggest that micronutrients may modify the risk or delay progression of prostate cancer; however, the molecular mechanisms involved are poorly understood. We examined the effects of lycopene and fish oil on prostate gene expression in a double-blind placebo-controlled randomized clinical trial.

Methods

Eighty-four men with low risk prostate cancer were stratified based on self-reported dietary consumption of fish and tomatoes and then randomly assigned to a 3-month intervention of lycopene (n = 29) or fish oil (n = 27) supplementation or placebo (n = 28). Gene expression in morphologically normal prostate tissue was studied at baseline and at 3 months via cDNA microarray analysis. Differential gene expression and pathway analyses were performed to identify genes and pathways modulated by these micronutrients.

Results

Global gene expression analysis revealed no significant individual genes that were associated with high intake of fish or tomato at baseline or after 3 months of supplementation with lycopene or fish oil. However, exploratory pathway analyses of rank-ordered genes (based on p-values not corrected for multiple comparisons) revealed the modulation of androgen and estrogen metabolism in men who routinely consumed more fish (p = 0.029) and tomato (p = 0.008) compared to men who ate less. In addition, modulation of arachidonic acid metabolism (p = 0.01) was observed after 3 months of fish oil supplementation compared with the placebo group; and modulation of nuclear factor (erythroid derived-2) factor 2 or Nrf2-mediated oxidative stress response for either supplement versus placebo (fish oil: p = 0.01, lycopene: p = 0.001).

Conclusions

We did not detect significant individual genes associated with dietary intake and supplementation of lycopene and fish oil. However, exploratory analyses revealed candidate in vivo pathways that may be modulated by these micronutrients.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00402285","term_id":"NCT00402285"}}NCT00402285     

Macrophage Dysfunction Impairs Resolution of Inflammation in the Wounds of Diabetic Mice

Background

Chronic inflammation is a characteristic feature of diabetic cutaneous wounds. We sought to delineate novel mechanisms involved in the impairment of resolution of inflammation in diabetic cutaneous wounds. At the wound-site, efficient dead cell clearance (efferocytosis) is a pre-requisite for the timely resolution of inflammation and successful healing.

Methodology/Principal Findings

Macrophages isolated from wounds of diabetic mice showed significant impairment in efferocytosis. Impaired efferocytosis was associated with significantly higher burden of apoptotic cells in wound tissue as well as higher expression of pro-inflammatory and lower expression of anti-inflammatory cytokines. Observations related to apoptotic cell load at the wound site in mice were validated in the wound tissue of diabetic and non-diabetic patients. Forced Fas ligand driven elevation of apoptotic cell burden at the wound site augmented pro-inflammatory and attenuated anti-inflammatory cytokine response. Furthermore, successful efferocytosis switched wound macrophages from pro-inflammatory to an anti-inflammatory mode.

Conclusions/Significance

Taken together, this study presents first evidence demonstrating that diabetic wounds suffer from dysfunctional macrophage efferocytosis resulting in increased apoptotic cell burden at the wound site. This burden, in turn, prolongs the inflammatory phase and complicates wound healing.     

Altered gene expression patterns of innate and adaptive immunity pathways in transgenic rainbow trout harboring Cecropin P1 transgene

Background

We have recently developed several homozygous families of transgenic rainbow trout harbouring cecropin P1 transgene. These fish exhibit resistance characteristic to infection by Aeromonas salmonicida and infectious hematopoietic necrosis virus (IHNV). In our earlier studies we have reported that treatment of a rainbow trout macrophage cell line (RTS11) with a linear cationic α-helical antimicrobial peptide (e.g., cecropin B) resulted in elevated levels of expression of two pro-inflammatory relevant genes (e.g., IL-1β and COX-2). Therefore, we hypothesized that in addition to the direct antimicrobial activity of cecropin P1 in the disease resistant transgenic rainbow trout, this antimicrobial peptide may also affect the expression of immune relevant genes in the host. To confirm this hypothesis, we launched a study to determine the global gene expression profiles in three immune competent organs of cecropin P1 transgenic rainbow trout by using a 44k salmonid microarray.

Results

From the microarray data, a total of 2480 genes in the spleen, 3022 in the kidney, and 2102 in the liver were determined as differentially expressed genes (DEGs) in the cecropin P1 transgenic rainbow trout when compared to the non-transgenics. There were 478 DEGs in common among three tissues. Enrichment analyses conducted by two different bioinformatics tools revealed a tissue specific profile of functional pathway perturbation. Many of them were directly related to innate immune system such as phagocytosis, lysosomal processing, complement activation, antigen processing/presentation, and leukocyte migration. Perturbation of other biological functions that might contribute indirectly to host immunity was also observed.

Conclusions

The gene product of cecropin P1 transgene produced in the disease resistant transgenic rainbow trout not only can kill the pathogens directly but also exert multifaceted immunomodulatory properties to boost host immunity. The identified genes involved in different pathways related to immune function are valuable indicators associated with enhanced host immunity. These genes may serve as markers for selective breeding of rainbow trout or other aquaculture important fish species bearing traits of disease resistance.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-887) contains supplementary material, which is available to authorized users.     

Transcriptomic analysis reveals tomato genes whose expression is induced specifically during effector-triggered immunity and identifies the Epk1 protein kinase which is required for the host response to three bacterial effector proteins

Background

Plants have two related immune systems to defend themselves against pathogen attack. Initially, pattern-triggered immunity is activated upon recognition of microbe-associated molecular patterns by pattern recognition receptors. Pathogenic bacteria deliver effector proteins into the plant cell that interfere with this immune response and promote disease. However, some plants express resistance proteins that detect the presence of specific effectors leading to a robust defense response referred to as effector-triggered immunity. The interaction of tomato with Pseudomonas syringae pv. tomato is an established model system for understanding the molecular basis of these plant immune responses.

Results

We apply high-throughput RNA sequencing to this pathosystem to identify genes whose expression changes specifically during pattern-triggered or effector-triggered immunity. We then develop reporter genes for each of these responses that will enable characterization of the host response to the large collection of P. s. pv. tomato strains that express different combinations of effectors. Virus-induced gene silencing of 30 of the effector-triggered immunity-specific genes identifies Epk1 which encodes a predicted protein kinase from a family previously unknown to be involved in immunity. Knocked-down expression of Epk1 compromises effector-triggered immunity triggered by three bacterial effectors but not by effectors from non-bacterial pathogens. Epistasis experiments indicate that Epk1 acts upstream of effector-triggered immunity-associated MAP kinase signaling.

Conclusions

Using RNA-seq technology we identify genes involved in specific immune responses. A functional genomics screen led to the discovery of Epk1, a novel predicted protein kinase required for plant defense activation upon recognition of three different bacterial effectors.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0492-1) contains supplementary material, which is available to authorized users.     

The Transient Receptor Potential Vanilloid 2 Cation Channel Is Abundant in Macrophages Accumulating at the Peri-Infarct Zone and May Enhance Their Migration Capacity towards Injured Cardiomyocytes following Myocardial Infarction

Purpose

A novel family of transient receptor potential (TRP) channels, that may hold a role in calcium homeostasis, has recently been described. By employing a GeneChip array analysis we have demonstrated a clear and specific upregulation of the TRP vanilloid 2 (TRPV2) mRNA in the left ventricles (LV) 3–5 days post-acute myocardial infarction (MI) compared to sham-operated controls, both in rats and in mice. We sought to characterize the cardiac cellular subpopulations in which TRPV2 is overexpressed upon acute MI.

Methods

Lewis rats underwent an acute MI by ligation of the left anterior descending artery or chest opening only (sham). The animals were terminated at various time points and an immunohistochemical (IHC) and immunofloerescent (IFC) staining of the LV sections as well as a flow cytometry analysis of LV-derived cells were carried out, using anti-TRPV2 and anti-monocyte/macrophage antibodies. Rat alveolar macrophage cells, NR8383, transiently transfected with TRPV2 siRNA were allowed to migrate towards hypoxic conditioned media of the rat cardiac myoblast line H9C2 using a trans-well migration assay. The macrophage cells migrating to the bottom side of the inserts were counted.

Results

The IHC and IFC staining as well as the flow cytometry data demonstrated a substantial expression of TRPV2 in infiltrating macrophages in the peri-infarct region 3–5 days post-acute MI. The in vitro migration assay data demonstrated that following inhibition of the TRPV2 channel, the number of migrating macrophages towards conditioned medium of hypoxic cardiomyocytes was significantly reduced.

Conclusions

TRPV2 is highly expressed on the peri-infarct infiltrating macrophages and may play an important role in post-MI phagocytosis. Better characterization of this channel may pave the way for identifying a new target for modulating the dramatic post-MI immune reactions.     

Amniotic Fluid Protein Profiles of Intraamniotic Inflammatory Response to Ureaplasma spp. and Other Bacteria

Objective

This study aimed to evaluate the amniotic fluid protein profiles and the intensity of intraamniotic inflammatory response to Ureaplasma spp. and other bacteria, using the multiplex xMAP technology.

Methods

A retrospective cohort study was undertaken in the Department of Obstetrics and Gynecology, University Hospital Hradec Kralove, Czech Republic. A total of 145 pregnant women with preterm prelabor rupture of membranes between gestational age 24+0 and 36+6 weeks were included in the study. Amniocenteses were performed. The presence of Ureaplasma spp. and other bacteria was evaluated using 16S rRNA gene sequencing. The levels of specific proteins were determined using multiplex xMAP technology.

Results

The presence of Ureaplasma spp. and other bacteria in the amniotic fluid was associated with increased levels of interleukin (IL)-6, IL-8, IL-10, brain-derived neurotropic factor, granulocyte macrophage colony stimulating factor, monocyte chemotactic protein-1, macrophage inflammatory protein-1, and matrix metalloproteinasis-9. Ureaplasma spp. were also associated with increased levels of neurotropin-3 and triggering receptor expressed on myeloid cells-1.

Conclusions

The presence of Ureaplasma spp. in the amniotic fluid is associated with a slightly different protein profile of inflammatory response, but the intensity of inflammatory response to Ureaplasma spp. is comparable with the inflammatory response to other bacteria.     

Nasopharyngeal colonization and invasive disease are enhanced by the cell wall hydrolases LytB and LytC of Streptococcus pneumoniae

Background

Streptococcus pneumoniae is a common colonizer of the human nasopharynx and one of the major pathogens causing invasive disease worldwide. Dissection of the molecular pathways responsible for colonization, invasion, and evasion of the immune system will provide new targets for antimicrobial or vaccine therapies for this common pathogen.

Methodology/Principal Findings

We have constructed mutants lacking the pneumococcal cell wall hydrolases (CWHs) LytB and LytC to investigate the role of these proteins in different phases of the pneumococcal pathogenesis. Our results show that LytB and LytC are involved in the attachment of S. pneumoniae to human nasopharyngeal cells both in vitro and in vivo. The interaction of both proteins with phagocytic cells demonstrated that LytB and LytC act in concert avoiding pneumococcal phagocytosis mediated by neutrophils and alveolar macrophages. Furthermore, C3b deposition was increased on the lytC mutant confirming that LytC is involved in complement evasion. As a result, the lytC mutant showed a reduced ability to successfully cause pneumococcal pneumonia and sepsis. Bacterial mutants lacking both LytB and LytC showed a dramatically impaired attachment to nasopharyngeal cells as well as a marked degree of attenuation in a mouse model of colonization. In addition, C3b deposition and phagocytosis was more efficient for the double lytB lytC mutant and its virulence was greatly impaired in both systemic and pulmonary models of infection.

Conclusions/Significance

This study confirms that the CWHs LytB and LytC of S. pneumoniae are essential virulence factors involved in the colonization of the nasopharynx and in the progress of invasive disease by avoiding host immunity.