Non-professional Phagocytic Cells Research Articles

The Role of Extracellular Vesicles in Efferocytosis.

Efferocytosis is the physiological process of phagocytic clearance of apoptotic cells by both professional phagocytic cells, such as macrophages, and non-professional phagocytic cells, such as epithelial cells. This process is crucial for maintaining tissue homeostasis in normal physiology. Any defects in efferocytosis can lead to pathological consequences and result in inflammatory diseases. Extracellular vesicles (EVs), including exosomes, microvesicles (MVs), and apoptotic vesicles (ApoVs), play a crucial role in proper efferocytosis. These EVs can significantly impact efferocytosis by affecting the polarization of macrophages and impacting calreticulin (CRT), TAM receptors, and MFG-E8. With further knowledge of these effects, new treatment strategies can be proposed for many inflammatory diseases caused by efferocytosis disorders. This review article aims to investigate the role of EVs during efferocytosis and its potential clinical applications in inflammatory diseases.

The phosphatidylinositol-5' phosphatase synaptojanin1 limits integrin-mediated invasion of Staphylococcus aureus.

The gram-positive bacterium Staphylococcus aureus can invade non-professional phagocytic cells by associating with the plasma protein fibronectin to exploit host cell integrins. Integrin-mediated internalization of these pathogens is facilitated by the local production of phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2) via an integrin-associated isoform of phosphatidylinositol-5' kinase. In this study, we addressed the role of PI-4,5-P2-directed phosphatases on internalization of S. aureus. ShRNA-mediated knockdown of individual phosphoinositide 5-phosphatases revealed that synaptojanin1 (SYNJ1) is counteracting invasion of S. aureus into mammalian cells. Indeed, shRNA-mediated depletion as well as genetic deletion of synaptojanin1 via CRISPR/Cas9 resulted in a gain-of-function phenotype with regard to integrin-mediated uptake. Surprisingly, the surface level of integrins was slightly downregulated in Synj1-KO cells. Nevertheless, these cells showed enhanced local accumulation of PI-4,5-P2 and exhibited increased internalization of S. aureus. While the phosphorylation level of the integrin-associated protein tyrosine kinase FAK was unaltered, the integrin-binding and -activating protein talin was enriched in the vicinity of S. aureus in synaptojanin1 knockout cells. Scanning electron microscopy revealed enlarged membrane invaginations in the absence of synaptojanin1 explaining the increased capability of these cells to internalize integrin-bound microorganisms. Importantly, the enhanced uptake by Synj1-KO cells and the exaggerated morphological features were rescued by the re-expression of the wild-type enzyme but not phosphatase inactive mutants. Accordingly, synaptojanin1 activity limits integrin-mediated invasion of S. aureus, corroborating the important role of PI-4,5-P2 during this process.IMPORTANCEStaphylococcus aureus, an important bacterial pathogen, can invade non-professional phagocytes by capturing host fibronectin and engaging integrin α5β1. Understanding how S. aureus exploits this cell adhesion receptor for efficient cell entry can also shed light on the physiological regulation of integrins by endocytosis. Previous studies have found that a specific membrane lipid, phosphatidylinositol-4,5-bisphosphate (PIP2), supports the internalization process. Here, we extend these findings and report that the local levels of PIP2 are controlled by the activity of the PIP2-directed lipid phosphatase Synaptojanin1. By dephosphorylating PIP2 at bacteria-host cell attachment sites, Synaptojanin1 counteracts the integrin-mediated uptake of the microorganisms. Therefore, our study not only generates new insight into subversion of cellular receptors by pathogenic bacteria but also highlights the role of host cell proteins acting as restriction factors for bacterial invasion at the plasma membrane.

Brucella targets the host ubiquitin-specific protease, Usp8, through the effector protein, TcpB, for facilitating infection of macrophages.

Brucella species are Gram-negative intracellular bacterial pathogens that cause the worldwide zoonotic disease brucellosis. Brucella can infect many mammals, including humans and domestic and wild animals. Brucella manipulates various host cellular processes to invade and multiply in professional and non-professional phagocytic cells. However, the host targets and their modulation by Brucella to facilitate the infection process remain obscure. Here, we report that the host ubiquitin-specific protease, USP8, negatively regulates the invasion of Brucella into macrophages through the plasma membrane receptor, CXCR4. Upon silencing or chemical inhibition of USP8, the membrane localization of the CXCR4 receptor was enriched, which augmented the invasion of Brucella into macrophages. Activation of USP8 through chemical inhibition of 14-3-3 protein affected the invasion of Brucella into macrophages. Brucella suppressed the expression of Usp8 at its early stage of infection in the infected macrophages. Furthermore, we found that only live Brucella could negatively regulate the expression of Usp8, suggesting the role of secreted effector protein of Brucella in modulating the gene expression. Subsequent studies revealed that the Brucella effector protein, TIR-domain containing protein from Brucella, TcpB, plays a significant role in downregulating the expression of Usp8 by targeting the cyclic-AMP response element-binding protein pathway. Treatment of mice with USP8 inhibitor resulted in enhanced survival of B. melitensis, whereas mice treated with CXCR4 or 14-3-3 antagonists showed a diminished bacterial load. Our experimental data demonstrate a novel role of Usp8 in the host defense against microbial intrusion. The present study provides insights into the microbial subversion of host defenses, and this information may ultimately help to develop novel therapeutic interventions for infectious diseases.

Intracellular activity and in vivo efficacy in a mouse model of septic arthritis of the novel pseudopeptide Pep16 against Staphylococcus aureus clinical isolates.

Assessing the therapeutic potential of a novel antimicrobial pseudopeptide, Pep16, both in vitro and in vivo for the treatment of septic arthritis caused by Staphylococcus aureus. Seven clinical isolates of S. aureus (two MRSA and five MSSA) were studied. MICs of Pep16 and comparators (vancomycin, teicoplanin, daptomycin and levofloxacin) were determined through the broth microdilution method. The intracellular activity of Pep16 and levofloxacin was assessed in two models of infection using non-professional (osteoblasts MG-63) or professional (macrophages THP-1) phagocytic cells. A mouse model of septic arthritis was used to evaluate the in vivo efficacy of Pep16 and vancomycin. A preliminary pharmacokinetic (PK) analysis was performed by measuring plasma concentrations using LC-MS/MS following a single subcutaneous injection of Pep16 (10 mg/kg). MICs of Pep16 were consistently at 8 mg/L for all clinical isolates of S. aureus (2- to 32-fold higher to those of comparators) while MBC/MIC ratios confirmed its bactericidal activity. Both Pep16 and levofloxacin (when used at 2 × MIC) significantly reduced the bacterial load of all tested isolates (two MSSA and two MRSA) within both osteoblasts and macrophages. In MSSA-infected mice, Pep16 demonstrated a significant (∼10-fold) reduction on bacterial loads in knee joints. PK analysis following a single subcutaneous administration of Pep16 revealed a gradual increase in plasma concentrations, reaching a peak of 5.6 mg/L at 12 h. Pep16 is a promising option for the treatment of septic arthritis due to S. aureus, particularly owing to its robust intracellular activity.

Analysis of production levels of InlA and InlB invasion factors in Listeria monocytogenes isolates collected in the Russian Federation

Background. Listeria monocytogenes is characterized by the presence of epidemic hypervirulent clones. A key feature of L. monocytogenes is its capacity to invade non-professional phagocytic cells. Hypervirulent clones are strongly associated with the increased production and/or the presence of certain isoforms of invasion factors InlA and InlB.
 The purpose of the study is to create a test system for InlA and InlB detection and to measure the InlA and InlB production levels in L. monocytogenes isolates belonging to clonal groups with different virulence potential.
 Materials and methods. The study was performed using 32 L. monocytogenes strains belonging to epidemic clones ECII, ECIV, ECVII (clonal complexes CC1, CC2, CC7) and hypovirulent clonal complex CC9. Sequencing of inlA and inlB genes was performed. The indirect enzyme-linked immunosorbent assay was used to analyze the production levels of InlA and InlB proteins.
 Results. The variability of InlA was revealed among strains belonging to the same clonal complex: 3 InlA isoforms were identified among strains belonging to CC7; out of 8 strains belonging to CC9, one strain had a stop codon in the inlA gene, leading to the loss of function of the InlA protein. The differences between inlB alleles correlated with the specificity of strains belonging to a certain clonal complex. Differences in production levels of invasion factors were measured. In strains belonging to CC9, the InlA production level was 2.5 times as low compared to strains belonging to CC1, CC2, and CC7. In strains belonging to phylogenetically related CC1 and CC2, the InlB production level was on average 4 times as high compared to strains belonging to CC7 and CC9.
 Conclusion. The obtained results confirm the variability of major invasion factors both among clonal complexes and strains of the same complex. The increased production of invasion factors InlA and InlB correlates with the potential virulence of strains.

A Real-Time Image-Based Efferocytosis Assay for the Discovery of Functionally Inhibitory Anti-MerTK Antibodies.

Efferocytosis is a phagocytic process by which apoptotic cells are cleared by professional and nonprofessional phagocytic cells. In tumors, efferocytosis of apoptotic cancer cells by tumor-associated macrophages prevents Ag presentation and suppresses the host immune response against the tumor. Therefore, reactivating the immune response by blockade of tumor-associated macrophage-mediated efferocytosis is an attractive strategy for cancer immunotherapy. Even though several methods have been developed to monitor efferocytosis, an automated and high-throughput quantitative assay should offer highly desirable advantages for drug discovery. In this study, we describe a real-time efferocytosis assay with an imaging system for live-cell analysis. Using this assay, we successfully discovered potent anti-MerTK Abs that block tumor-associated macrophage-mediated efferocytosis in mice. Furthermore, we used primary human and cynomolgus monkey macrophages to identify and characterize anti-MerTK Abs for potential clinical development. By studying the phagocytic activities of different types of macrophages, we demonstrated that our efferocytosis assay is robust for screening and characterization of drug candidates that inhibit unwanted efferocytosis. Moreover, our assay is also applicable to investigating the kinetics and molecular mechanisms of efferocytosis/phagocytosis.

The cross-talk between soluble "Find me" and "Keep out" signals as an initial step in regulating efferocytosis.

The rapid clearance of apoptotic cells (ACs), known as efferocytosis, prompts the inhibition of inflammatory responses and autoimmunity and maintains homeostatic cell turnover by controlling the release of intracellular contents. The fast clearance of ACs requires professional and nonprofessional phagocytic cells that can accurately and promptly recognize ACs and migrate towards them. Cells undergoing apoptosis alarm their presence by releasing special soluble chemotactic factors, such as lactoferrin, that act as "Find me," "Keep out,"or "Stay away" signals to recruit phagocytic cells, such as macrophages or prevent granulocyte migration. Efferocytosis effectively serves to prevent damage-associated molecular pattern release and secondary necrosis and inhibit inflammation/autoimmunity at the very first step. Since less attention has been given to the cross-talk and balance of "Find me" and "Keep out" signals released from ACs in efferocytosis, we set out to investigate the current knowledge of the roles of "Find me" and "Keep out" signals in the efferocytosis process.

Host F-Box Protein 22 Enhances the Uptake of Brucella by Macrophages and Drives a Sustained Release of Proinflammatory Cytokines through Degradation of the Anti-Inflammatory Effector Proteins of Brucella.

Brucella species are intracellular bacterial pathogens, causing the worldwide zoonotic disease brucellosis. Brucella invades professional and nonprofessional phagocytic cells, followed by resisting intracellular killing and establishing a replication permissive niche. Brucella also modulates the innate and adaptive immune responses of the host for its chronic persistence. The complex intracellular cycle of Brucella depends in a major way on multiple host factors, but limited information is available on host and bacterial proteins that play an essential role in the invasion, intracellular replication, and modulation of host immune responses. By employing a small interfering RNA (siRNA) screening, we identified a role for the host protein FBXO22 in the Brucella-macrophage interaction. FBXO22 is the key element in the SCF E3 ubiquitination complex, where it determines the substrate specificity for ubiquitination and degradation of various host proteins. Downregulation of FBXO22 by siRNA or the CRISPR-Cas9 system resulted in diminished uptake of Brucella into macrophages, which was dependent on NF-κB-mediated regulation of phagocytic receptors. FBXO22 expression was upregulated in Brucella-infected macrophages, which resulted in induction of phagocytic receptors and enhanced production of proinflammatory cytokines through NF-κB. Furthermore, we found that FBXO22 recruits the effector proteins of Brucella, including the anti-inflammatory proteins TcpB and OMP25, for degradation through the SCF complex. We did not observe any role for another F-box-containing protein of the SCF complex, β-TrCP, in the Brucella-macrophage interaction. Our findings unravel novel functions of FBXO22 in host-pathogen interaction and its contribution to pathogenesis of infectious diseases.

Importance of Efferocytosis in COVID-19 Mortality.

COVID-19 is a generally benign coronavirus disease that can spread rapidly, except for those with a group of risk factors. Since the pathogenesis responsible for the severity of the disease has not been clearly revealed, effective treatment alternatives has not been developed. The hallmark of the SARS-CoV-2-infected cells is apoptosis. Apoptotic cells are cleared through a sterile process defined as efferocytosis by professional and nonprofessional phagocytic cells. The disease would be rapidly brought under control in the organism that can achieve effective efferocytosis, which is also a kind of innate immune response. In the risk group, the efferocytic process is defective. With the addition of the apoptotic cell load associated with SARS-COV-2 infection, failure to achieve efferocytosis of dying cells can initiate secondary necrosis, which is a highly destructive process. Uncontrolled inflammation and coagulation abnormalities caused by secondary necrosis reason in various organ failures, lung in particular, which are responsible for the poor prognosis. Following the short and simplified information, this opinion paper aims to present possible treatment options that can control the severity of COVID-19 by detailing the mechanisms that can cause defective efferocytosis.

Biological and Molecular Effects of Trypanosoma cruzi Residence in a LAMP-Deficient Intracellular Environment.

Trypanosoma cruzi invades non-professional phagocytic cells by subverting their membrane repair process, which is dependent on membrane injury and cell signaling, intracellular calcium increase, and lysosome recruitment. Cells lacking lysosome-associated membrane proteins 1 and 2 (LAMP1 and LAMP2) are less permissive to parasite invasion but more prone to parasite intracellular multiplication. Several passages through a different intracellular environment can significantly change T. cruzi’s gene expression profile. Here, we evaluated whether one single passage through LAMP-deficient (KO) or wild-type (WT) fibroblasts, thus different intracellular environments, could influence T. cruzi Y strain trypomastigotes’ ability to invade L6 myoblasts and WT fibroblasts host cells. Parasites released from LAMP2 KO cells (TcY-L2−/−) showed higher invasion, calcium signaling, and membrane injury rates, for the assays in L6 myoblasts, when compared to those released from WT (TcY-WT) or LAMP1/2 KO cells (TcY-L1/2−/−). On the other hand, TcY-L1/2−/− showed higher invasion, calcium signaling, and cell membrane injury rates, for the assays in WT fibroblasts, compared to TcY-WT and TcY-L1/2−/−. Albeit TcY-WT presented an intermediary invasion and calcium signaling rates, compared to the others, in WT fibroblasts, they induced lower levels of injury, which reinforces that signals mediated by surface membrane protein interactions also have a significant contribution to trigger host cell calcium signals. These results clearly show that parasites released from WT or LAMP KO cells are distinct from each other. Additionally, these parasites’ ability to invade the cell may be distinct depending on which cell type they interact with. Since these alterations most likely would reflect differences among parasite surface molecules, we also evaluated their proteome. We identified few protein complexes, membrane, and secreted proteins regulated in our dataset. Among those are some members of MASP, mucins, trans-sialidases, and gp63 proteins family, which are known to play an important role during parasite infection and could correlate to TcY-WT, TcY-L1/2−/−, and TcY-L2−/− biological behavior.

Modulation of the Host Nuclear Compartment by Trypanosoma cruzi Uncovers Effects on Host Transcription and Splicing Machinery.

Host manipulation is a common strategy for invading pathogens. Trypanosoma cruzi, the causative agent of Chagas Disease, lives intracellularly within host cells. During infection, parasite-associated modifications occur to the host cell metabolism and morphology. However, little is known about the effect of T. cruzi infection on the host cell nucleus and nuclear functionality. Here, we show that T. cruzi can modulate host transcription and splicing machinery in non-professional phagocytic cells during infection. We found that T. cruzi regulates host RNA polymerase II (RNAPII) in a time-dependent manner, resulting in a drastic decrease in RNAPII activity. Furthermore, host cell ribonucleoproteins associated with mRNA transcription (hnRNPA1 and AB2) are downregulated concurrently. We reasoned that T. cruzi may hijack the host U2AF35 auxiliary factor, a key regulator for RNA processing, as a strategy to affect the splicing machinery activities directly. In support of our hypothesis, we carried out in vivo splicing assays using an adenovirus E1A pre-mRNA splicing reporter, showing that intracellular T. cruzi directly modulates the host cells by appropriating U2AF35. For the first time, our results provide evidence of a complex and intimate molecular relationship between T. cruzi and the host cell nucleus during infection.

Improved Enzyme Protection Assay to Study <em>Staphylococcus aureus</em> Internalization and Intracellular Efficacy of Antimicrobial Compounds

Staphylococcus aureus expresses virulence factors to trigger its internalization into eukaryote cells and to survive inside different subcellular compartments. This paper describes an enzyme protection assay to study the extent of S. aureus internalization and its intracellular survival in adherent non-professional phagocytic cells (NPPCs) as well as the intracellular efficacy of antimicrobial compounds. NPPCs are grown in a multi-well plate until they reach 100% confluence. S. aureus cultures are grown overnight in cell culture medium. The bacterial suspension is diluted according to the number of cells per well to inoculate the cells at a controlled multiplicity of infection. Inoculated cells are incubated for 2 h to allow the bacteria to be internalized by the NPPCs, following which lysostaphin is added to the culture medium to selectively kill extracellular bacteria. Lysostaphin is present in the culture medium for the rest of the experiment. At this point, the infected cells could be incubated with antimicrobial compounds to assess their intracellular activities against S. aureus. Next, the cells are washed three times to remove the drugs, and intracellular S. aureus load is then quantified by culturing on agar plates. Alternatively, for studying staphylococcal virulence factors involved in intracellular survival and cell toxicity, lysostaphin could be inactivated with proteinase K to eliminate the need for washing steps. This tip improves the reliability of the intracellular bacterial load quantification, especially if cells tend to detach from the culture plate when they become heavily infected because of the multiplication of intracellular S. aureus. These protocols can be used with virtually all types of adherent NPPCs and with 3D cell culture models such as organoids.

Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds.

Staphylococcus aureus expresses virulence factors to trigger its internalization into eukaryote cells and to survive inside different subcellular compartments. This paper describes an enzyme protection assay to study the extent of S. aureus internalization and its intracellular survival in adherent non-professional phagocytic cells (NPPCs) as well as the intracellular efficacy of antimicrobial compounds. NPPCs are grown in a multi-well plate until they reach 100% confluence. S. aureus cultures are grown overnight in cell culture medium. The bacterial suspension is diluted according to the number of cells per well to inoculate the cells at a controlled multiplicity of infection. Inoculated cells are incubated for 2 h to allow the bacteria to be internalized by the NPPCs, following which lysostaphin is added to the culture medium to selectively kill extracellular bacteria. Lysostaphin is present in the culture medium for the rest of the experiment. At this point, the infected cells could be incubated with antimicrobial compounds to assess their intracellular activities against S. aureus. Next, the cells are washed three times to remove the drugs, and intracellular S. aureus load is then quantified by culturing on agar plates. Alternatively, for studying staphylococcal virulence factors involved in intracellular survival and cell toxicity, lysostaphin could be inactivated with proteinase K to eliminate the need for washing steps. This tip improves the reliability of the intracellular bacterial load quantification, especially if cells tend to detach from the culture plate when they become heavily infected because of the multiplication of intracellular S. aureus. These protocols can be used with virtually all types of adherent NPPCs and with 3D cell culture models such as organoids.

In vitro antibiotic activity against intraosteoblastic Staphylococcus aureus: a narrative review of the literature.

Staphylococcus aureus – a major aetiological agent of bone and joint infection (BJI) – is associated with a high risk of relapse and chronicity, in part due to its ability to invade and persist in non-professional phagocytic bone cells such as osteoblasts. This intracellular reservoir protects S. aureus from the action of the immune system and most antibiotics. To date, the choice of antimicrobial strategies for BJI treatment mostly relies on standard susceptibility testing, bone penetration of antibiotics and their ‘antibiofilm’ activity. Despite the role of intracellular persistent S. aureus in the development of chronic infection, the ability of antibiotics to target the S. aureus intraosteoblastic reservoir is not considered in therapeutic choices but might represent a key determinant of treatment outcome. This review provides an overview of the intracellular pharmacokinetics of antistaphylococcal drugs used in the treatment of BJI and of their ability to target intraosteoblastic S. aureus. Thirteen studies focusing on the intraosteoblastic activity of antibiotics against S. aureus were reviewed, all relying on in vitro models of osteoblast infection. Despite varying incubation times, multiplicities of infection, bacterial strains, and the types of infected cell lines, rifamycins and fluoroquinolones remain the two most potent antimicrobial classes for intraosteoblastic S. aureus eradication, consistent with clinical data showing a superiority of this combination therapy in S. aureus orthopaedic device-related infections.

Trypanosoma cruzi extracellular amastigotes engage Rac1 and Cdc42 to invade RAW macrophages

Cell invasion by Trypanosoma cruzi extracellular amastigotes (EAs) relies significantly upon the host cell actin cytoskeleton. In past decades EAs have been established as a reliable model for phagocytosis inducer in non-phagocytic cells. Our current hypothesis is that EAs engage a phagocytosis-like mechanism in non-professional phagocytic cells; however, the molecular mechanisms in professional phagocytes still remain unexplored. In this work, we evaluated the involvement of Rac1 and Cdc42 in the actin-dependent internalization of EAs in RAW 264.7 macrophages. Kinetic assays showed similar internalization of EAs in unstimulated RAW and non-phagocytic HeLa cells but increased in LPS/IFN-γ stimulated RAW cells. However, depletion of Rac1, Cdc42 or RhoA inhibited EA internalization similarly in both unstimulated and stimulated RAW cells. Overexpression of active, but not the dominant-negative, construct of Rac1 increased EA internalization. Remarkably, for Cdc42, both the active and the inactive mutants decreased EA internalization when compared to wild type groups. Despite that, both Rac1 and Cdc42 activation mutants were similarly recruited to and colocalized with actin at the EA-macrophage contact sites when compared to their native isoforms. Altogether, these results corroborate that EAs engage phagocytic processes to invade both professional and non-professional phagocytic cells providing evidences of converging actin mediated mechanisms induced by intracellular pathogens in both cell types.

Different Modulatory Effects of Four Methicillin-Resistant Staphylococcus aureus Clones on MG-63 Osteoblast-Like Cells.

Staphylococcus aureus is a Gram-positive bacterium responsible for a variety of mild to life-threatening infections including bone infections such as osteomyelitis. This bacterium is able to invade and persist within non-professional phagocytic cells such as osteoblasts. In the present study, four different S. aureus strains, namely, 2SA-ST239-III (ST239), 5SA-ST5-II (ST5), 10SA-ST228-I (ST228), and 14SA-ST22-IVh (ST22), were tested for their ability to modulate cell viability in MG-63 osteoblast-like cells following successful invasion and persistence. Methicillin-sensitive S. aureus (MSSA) ATCC-12598-ST30 (ST30) was used as control strain. Despite being proven that ST30, ST239, and ST22 have a similar ability to internalize and persist in MG-63 osteoblast-like cells under our experimental conditions, we demonstrated that the observed decrease in cell viability was due to the different behavior of the considered strains, rather than the number of intracellular bacteria. We focused our attention on different biochemical cell functions related to inflammation, cell metabolism, and oxidative stress during osteoblast infections. We were able to show the following: (1) ST30 and ST239 were the only two clones able to persist and maintain their number in the hostile environment of the cell during the entire period of infection; (2) ST239 was the only clone able to significantly increase gene expression (3 and 24 h post-infection (p.i.)) and protein secretion (24 h p.i.) of both interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) in MG-63 osteoblast-like cells; (3) the same clone determined a significant up-regulation of the transforming growth factorbeta 1 (TGF-β1) and of the metabolic marker glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNAs at 24 h p.i.; and (4) neither the MSSA nor the four methicillin-resistant S. aureus (MRSA) strains induced oxidative stress phenomena in MG-63 cells, although a high degree of variability was observed for the different clones with regard to the expression pattern of nuclear factor E2-related factor 2 (Nrf2) and its downstream gene heme oxygenase 1 (HO-1) activation. Our results may pave the way for an approach to S. aureus-induced damage, moving towards individualized therapeutic strategies that take into account the differences between MSSA and MRSA as well as the distinctive features of the different clones. This approach is based on a change of paradigm in antibiotic therapy involving a case-based use of molecules able to counteract pro-inflammatory cytokines activity such as selective cytokine signaling inhibitors (IL-6, TNF-α).

Role of tumor cell senescence in non-professional phagocytosis and cell-in-cell structure formation

BackgroundNon-professional phagocytosis is usually triggered by stimuli such as necrotic cell death. In tumor therapy, the tumors often disappear slowly and only long time after the end of therapy. Here, tumor therapy inactivates the cells by inducing senescence. Therefore, study focused whether senescence is a stimulus for non-professional phagocytosis or whether senescent cells themselves phagocytize non-professionally.ResultsSenescence was induced in cell lines by camptothecin and a phagocytosis assay was performed. In tissue of a cohort of 192 rectal cancer patients senescence and non-professional phagocytosis was studied by anti-histone H3K9me3 and anti-E-cadherin staining. Senescent fibroblasts and pancreas carcinoma cells phagocytize necrotic cells but are not phagocytized. In the tissue of rectal carcinoma, senescent cells can phagocytize and can be phagocytized. A high number of senescent cells and, at the same time, high numbers of non-professional phagocytizing cells in the rectal carcinoma tissue lead to an extremely unfavorable prognosis regarding overall survival.ConclusionSenescent cells can be non-professionally phagocytized and at the same time they can non-professionally phagocytize in vivo. In vitro experiments indicate that it is unlikely that senescence is a strong trigger for non-professional phagocytosis. Combined high rates of non-professional phagocytosis and high rates of senescence are an extremely poor prognostic factor for overall survival.

Endocytic Rabs Are Recruited to the Trypanosoma cruzi Parasitophorous Vacuole and Contribute to the Process of Infection in Non-professional Phagocytic Cells.

Trypanosoma cruzi is the parasite causative of Chagas disease, a highly disseminated illness endemic in Latin-American countries. T. cruzi has a complex life cycle that involves mammalian hosts and insect vectors both of which exhibits different parasitic forms. Trypomastigotes are the infective forms capable to invade several types of host cells from mammals. T. cruzi infection process comprises two sequential steps, the formation and the maturation of the Trypanosoma cruzi parasitophorous vacuole. Host Rab GTPases are proteins that control the intracellular vesicular traffic by regulating budding, transport, docking, and tethering of vesicles. From over 70 Rab GTPases identified in mammalian cells only two, Rab5 and Rab7 have been found in the T. cruzi vacuole to date. In this work, we have characterized the role of the endocytic, recycling, and secretory routes in the T. cruzi infection process in CHO cells, by studying the most representative Rabs of these pathways. We found that endocytic Rabs are selectively recruited to the vacuole of T. cruzi, among them Rab22a, Rab5, and Rab21 right away after the infection followed by Rab7 and Rab39a at later times. However, neither recycling nor secretory Rabs were present in the vacuole membrane at the times studied. Interestingly loss of function of endocytic Rabs by the use of their dominant-negative mutant forms significantly decreases T. cruzi infection. These data highlight the contribution of these proteins and the endosomal route in the process of T. cruzi infection.

Microautophagy upregulation in cutaneous lymph nodes of dogs naturally infected by Leishmania infantum.

This is the first study showing an in vivo microautophagy upregulation by Leishmania infantum in dogs. Both Leishmania amastigotes and promastigotes were detected in the cytoplasm of many professional and nonprofessional phagocytic cells of popliteal lymph node of three dogs suffering from chronic cutaneous leishmaniasis. Ultrastructurally, parasites appeared to be wrapped by lysosomes and/or multivesicular bodies. Neither phagophores nor double-membraned vacuoles consistent with autophagosomes were observed. Transcription factor EB (TFEB), a key factor involved in lysosome biogenesis, showed a statistically significant increase in the total component when examined by western blot in samples from leishmaniotic dogs compared with samples from healthy dogs. Instead, phosphorylated TFEB showed unmodified expression levels both in leishmaniotic and healthy dogs. Furthermore, Hsc70 and endosomal sorting complex required for transport (ESCRT)-I, which are known to play a role in microautophagy, showed no variation in expression levels both in diseased and healthy animals. Vps4A/B, an evolutionary conserved ATPase responsible for ESCRT-I complex disassembly and MVB maturation, was statistically significantly overexpressed in lymph nodal samples from leishmaniotic dogs. Bag3 was downregulated in diseased dogs whereas CHIP, p62, and LC3-II did not show any variation in expression levels. The altered expression profile of Bag3 suggested an altered interaction of Bag3 with Hsc70 and CHIP, which usually form a molecular complex involved in autophagosome-lysosome pathways. Ultrastructural and molecular findings suggested that the microautophagy pathway is upregulated in lymph nodes of dogs suffering from a chronic natural infection by Leishmania infantum.

The Role of the Flagellar Protein FlgJ in the Virulence of Brucella abortus.

Brucella abortus is a facultative intracellular pathogen that causes a zoonosis called brucellosis. This disease leads to abortion and infertility in cattle, and diverse complications in humans. B. abortus is a successful intracellular bacterium that has developed the ability to evade the host's immune system and it replicates in professional and non-professional phagocytic cells, persisting in the different tissues, and organs of its hosts. It has been described that Brucella expresses a polar flagellum under certain conditions, but its function is still unknown. In this study we evaluated the role of the FlgJ, a protein, presumably a peptidoglycan hydrolase involved in flagellum formation and in the virulence of B. abortus strain 2308. B. abortus 2308 ΔflgJ mutant and complemented strains were constructed to study the function of the FlgJ protein in the context of the virulence of this pathogen in in vitro and in vivo assays. The results showed that the elimination of the flgJ gene delays the growth rate of B. abortus in culture, reduces its intracellular survival capacity in professional and non-professional phagocytic cells, rendering it unable to escape from the endocytic route and not reaching the endoplasmic reticulum. It also negatively affects their persistence in BALB/c mice. Functionally, the B. abortus 2308 flgJ gene restored motility to an E. coli flgJ mutant gene. Furthermore, it was discovered that the production of FlgJ protein is associated with the bacterial adherence by B. abortus. Therefore, although the specific function of the polar flagellum for Brucella is unknown, the data indicates that the flagellar flgJ gene and its product are required for full virulence of B. abortus 2308, since its deletion significantly reduces the fitness of this pathogen in vitro and in vivo.