Lysosomal Disorders Drive Susceptibility to Tuberculosis by Compromising Macrophage Migration.

A zebrafish genetic screen for determinants of susceptibility to Mycobacterium marinum identified a hypersusceptible mutant deficient in lysosomal cysteine cathepsins that manifests hallmarks of human lysosomal storage diseases. Under homeostatic conditions, mutant macrophages accumulate undigested lysosomal material, which disrupts endocytic recycling and impairs their migration to, and thus engulfment of, dying cells. This causes a buildup of unengulfed cell debris. During mycobacterial infection, macrophages with lysosomal storage cannot migrate toward infected macrophages undergoing apoptosis in the tuberculous granuloma. The unengulfed apoptotic macrophages undergo secondary necrosis, causing granuloma breakdown and increased mycobacterial growth. Macrophage lysosomal storage similarly impairs migration to newly infecting mycobacteria. This phenotype is recapitulated in human smokers, who are at increased risk for tuberculosis. A majority of their alveolar macrophages exhibit lysosomal accumulations of tobacco smoke particulates and do not migrate to Mycobacterium tuberculosis. The incapacitation of highly microbicidal first-responding macrophages may contribute to smokers' susceptibility to tuberculosis.

Use of Galleria mellonella as a model organism to study Legionella pneumophila infection.

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires' disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae's immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Inhalable antitubercular therapy mediated by locust bean gum microparticles

Tuberculosis remains a major global health problem and alternative therapeutic approaches are needed. Considering the high prevalence of lung tuberculosis (80% of cases), the pulmonary delivery of antitubercular drugs in a carrier system capable of reaching the alveoli, being recognised and phagocytosed by alveolar macrophages (mycobacterium hosts), would be a significant improvement to current oral drug regimens. Locust bean gum (LBG) is a polysaccharide composed of galactose and mannose residues, which may favour specific recognition by macrophages and potentiate phagocytosis. LBG microparticles produced by spray-drying are reported herein for the first time, incorporating either isoniazid or rifabutin, first-line antitubercular drugs (association efficiencies >82%). Microparticles have adequate theoretical properties for deep lung delivery (aerodynamic diameters between 1.15 and 1.67 μm). The cytotoxic evaluation in lung epithelial cells (A549 cells) and macrophages (THP-1 cells) revealed a toxic effect from rifabutin-loaded microparticles at the highest concentrations, but we may consider that these were very high comparing with in vivo conditions. LBG microparticles further evidenced strong ability to be captured by macrophages (percentage of phagocytosis >94%). Overall, the obtained data indicated the potential of the proposed system for tuberculosis therapy.

GM-CSF Priming Drives Bone Marrow-Derived Macrophages to a Pro-Inflammatory Pattern and Downmodulates PGE(2) in Response to TLR2 Ligands

In response to pathogen recognition by Toll-like receptors (TLRs) on their cell surface, macrophages release lipid mediators and cytokines that are widely distributed throughout the body and play essential roles in host responses. Granulocyte macrophage colony-stimulating factor (GM-CSF) is important for the immune response during infections to improve the clearance of microorganisms. In this study, we examined the release of mediators in response to TLR2 ligands by bone marrow-derived macrophages (BMDMs) primed with GM-CSF. We demonstrated that when stimulated with TLR2 ligands, non-primed BMDMs preferentially produced PGE(2) in greater amounts than LTB4. However, GM-CSF priming shifted the release of lipid mediators by BMDMs, resulting in a significant decrease of PGE(2) production in response to the same stimuli. The decrease of PGE(2) production from primed BMDMs was accompanied by a decrease in PGE-synthase mRNA expression and an increase in TNF-alpha and nitric oxide (NO) production. Moreover, some GM-CSF effects were potentiated by the addition of IFN-gamma. Using a variety of TLR2 ligands, we established that PGE(2) release by GM-CSF-primed BMDMs was dependent on TLR2 co-receptors (TLR1, TLR6), CD14, MyD88 and the nuclear translocation of NF kappa B but was not dependent on peroxisome proliferator-activated receptor-gamma (PPAR-gamma) activation. Indeed, GM-CSF priming enhanced TLR2, TLR4 and MyD88 mRNA expression and phospho-I kappa B alpha formation. These findings demonstrate that GM-CSF drives BMDMs to present a profile relevant to the host during infections.

An in vitro triple cell co-culture model with primary cells mimicking the human alveolar epithelial barrier

A triple cell co-culture model was recently established by the authors, consisting of either A549 or 16HBE14o- epithelial cells, human blood monocyte-derived macrophages and dendritic cells, which offers the possibility to study the interaction of xenobiotics with those cells. The 16HBE14o- containing co-culture model mimics the airway epithelial barrier, whereas the A549 co-cultures mimic the alveolar type II-like epithelial barrier. The goal of the present work was to establish a new triple cell co-culture model composed of primary alveolar type I-like cells isolated from human lung biopsies (hAEpC) representing a more realistic alveolar epithelial barrier wall, since type I epithelial cells cover >93% of the alveolar surface. Monocultures of A549 and 16HBE14o- were morphologically and functionally compared with the hAEpC using laser scanning microscopy, as well as transmission electron microscopy, and by determining the epithelial integrity. The triple cell co-cultures were characterized using the same methods. It could be shown that the epithelial integrity of hAEpC (mean ± SD, 1180 ± 188 Ω cm(2)) was higher than in A549 (172 ± 59 Ω cm(2)) but similar to 16HBE14o- cells (1469 ± 156 Ω cm(2)). The triple cell co-culture model with hAEpC (1113 ± 30 Ω cm(2)) showed the highest integrity compared to the ones with A549 (93 ± 14 Ω cm(2)) and 16HBE14o- (558 ± 267 Ω cm(2)). The tight junction protein zonula occludens-1 in hAEpC and 16HBE14o- were more regularly expressed but not in A549. The epithelial alveolar model with hAEpC combined with two immune cells (i.e. macrophages and dendritic cells) will offer a novel and more realistic cell co-culture system to study possible cell interactions of inhaled xenobiotics and their toxic potential on the human alveolar type I epithelial wall.

Intraperitoneal Echinococcus multilocularis infection in C57BL/6 mice affects CD40 and B7 costimulator expression on peritoneal macrophages and impairs peritoneal T cell activation

One of the most important immunopathological consequence of intraperitoneal alveolar echinococcosis (AE) in the mouse is suppression of T cell-mediated immune responses. We investigated whether and how intraperitoneal macrophages (MØs) are, respectively, implicated as antigen-presenting cells (APCs). In a first step we showed that peritoneal MØs from infected mice (AE-MØs) exhibited a reduced ability to present a conventional antigen (chicken ovalbumin, C-Ova) to specific responder lymph node T cells. In a subsequent step, AE-MØs as well as naïve MØs (positive control) proved their ability to uptake and process C-Ova fluorescein isthiocyanate (FITC). Furthermore, in comparison with naïve MØs, the surface expression of Ia molecules was up-regulated on AE-MØs at the early stage of infection, suggesting that AE-MØs provide the first signal via the antigen-Ia complex. To study the accessory activity of MØs, AE-MØs obtained at the early and late stages of infection were found to decrease Con A-induced proliferation of peritoneal naïve T cells as well as of AE-sensitized peritoneal T cells, in contrast to stimulation with naïve MØs. The status of accessory molecules was assessed by analysing the expression level of costimulatory molecules on AE-MØs, with naïve MØs as controls. It was found that B7-1 (CD80) and B7-2 (CD86) expression remained unchanged, whereas CD40 was down-regulated and CD54 (= ICAM-1) was slightly up-regulated. In a leucocyte reaction of AE-MØs with naïve or AE-T cells, both types of T cells increased their proliferative response when CD28 - the ligand of B7 receptors - was exposed to anti-CD28 in cultures. Conversely to naïve MØs, pulsing of AE-MØs with agonistic anti-CD40 did not even partially restore their costimulatory activity and failed to increase naïve or AE-T cell proliferation. Neutralizing anti-B7-1, in combination with anti-B7-2, reduced naïve and AE-T cell proliferation, whereas anti-CD40 treatment of naïve MØs increased their proliferative response to Con A. These results point at the key role of B7 receptors as accessory molecules and the necessity of the integrity of CD40-expression by naïve MØs to improve their accessory activity. Taken together, the obstructed presenting-activity of AE-MØs appeared to trigger an unresponsiveness of T cells, contributing to the suppression of their clonal expansion during the chronic phase of AE-infection.

Different endocytotic uptake mechanisms for nanoparticles in epithelial cells and macrophages.

Precise knowledge regarding cellular uptake of nanoparticles is of great importance for future biomedical applications. Four different endocytotic uptake mechanisms, that is, phagocytosis, macropinocytosis, clathrin- and caveolin-mediated endocytosis, were investigated using a mouse macrophage (J774A.1) and a human alveolar epithelial type II cell line (A549). In order to deduce the involved pathway in nanoparticle uptake, selected inhibitors specific for one of the endocytotic pathways were optimized regarding concentration and incubation time in combination with fluorescently tagged marker proteins. Qualitative immunolocalization showed that J774A.1 cells highly expressed the lipid raft-related protein flotillin-1 and clathrin heavy chain, however, no caveolin-1. A549 cells expressed clathrin heavy chain and caveolin-1, but no flotillin-1 uptake-related proteins. Our data revealed an impeded uptake of 40 nm polystyrene nanoparticles by J774A.1 macrophages when actin polymerization and clathrin-coated pit formation was blocked. From this result, it is suggested that macropinocytosis and phagocytosis, as well as clathrin-mediated endocytosis, play a crucial role. The uptake of 40 nm nanoparticles in alveolar epithelial A549 cells was inhibited after depletion of cholesterol in the plasma membrane (preventing caveolin-mediated endocytosis) and inhibition of clathrin-coated vesicles (preventing clathrin-mediated endocytosis). Our data showed that a combination of several distinguishable endocytotic uptake mechanisms are involved in the uptake of 40 nm polystyrene nanoparticles in both the macrophage and epithelial cell line.

Virulence and genotype-associated infectivity of interferon-treated macrophages by porcine reproductive and respiratory syndrome viruses.

The polarization into M1 and M2 macrophages (MΦ) is essential to understand MΦ function. Consequently, the aim of this study was to determine the impact of IFN-γ (M1), IL-4 (M2) and IFN-β activation of MΦ on the susceptibility to genotype 1 and 2 porcine reproductive respiratory syndrome (PRRS) virus (PRRSV) strains varying in virulence. To this end, monocyte-derived MΦ were generated by culture during 72h and polarization was induced for another 24h by addition of IFN-γ, IL-4 or IFN-β. MΦ were infected with a collection of PRRSV isolates belonging to genotype 1 and genotype 2. Undifferentiated and M2 MΦ were highly susceptible to all PRRSV isolates. In contrast, M1 and IFN-β activated MΦ were resistant to low pathogenic genotype 1 PRRSV but not or only partially to genotype 2 PRRSV strains. Interestingly, highly virulent PRRSV isolates of both genotypes showed particularly high levels of infection compared with the prototype viruses in both M1 and IFN-β-treated MΦ (P<0.05). This was seen at the level of nucleocapsid expression, viral titres and virus-induced cell death. In conclusion, by using IFN-γ and IFN-β stimulated MΦ it is possible to discriminate between PRRSV varying in genotype and virulence. Genotype 2 PRRSV strains are more efficient at escaping the intrinsic antiviral effects induced by type I and II IFNs. Our in vitro model will help to identify viral genetic elements responsible for virulence, an information important not only to understand PRRS pathogenesis but also for a rational vaccine design. Our results also suggest that monocyte-derived MΦ can be used as a PRRSV infection model instead of alveolar MΦ, avoiding the killing of pigs.

An Asp49 Phospholipase A2 From Snake Venom Induces Cyclooxygenase-2 Expression And Prostaglandin E2 Production Via Activation Of Nf-κb, P38mapk, And Pkc In Macrophages.

Phospholipases A2 (PLA2) are key enzymes for production of lipid mediators. We previously demonstrated that a snake venom sPLA2 named MT-III leads to prostaglandin (PG)E2 biosynthesis in macrophages by inducing the expression of cyclooxygenase-2 (COX-2). Herein, we explored the molecular mechanisms and signaling pathways leading to these MT-III-induced effects. Results demonstrated that MT-III induced activation of the transcription factor NF-κB in isolated macrophages. By using NF-κB selective inhibitors, the involvement of this factor in MT-III-induced COX-2 expression and PGE2 production was demonstrated. Moreover, MT-III-induced COX-2 protein expression and PGE2 release were attenuated by pretreatment of macrophages with SB202190, and Ly294002, and H-7-dihydro compounds, indicating the involvement of p38MAPK, PI3K, and PKC pathways, respectively. Consistent with this, MT-III triggered early phosphorylation of p38MAPK, PI3K, and PKC. Furthermore, SB202190, H-7-dihydro, but not Ly294002 treatment, abrogated activation of NF-κB induced by MT-III. Altogether, these results show for the first time that the induction of COX-2 protein expression and PGE2 release, which occur via NF-κB activation induced by the sPLA2-MT-III in macrophages, are modulated by p38MAPK and PKC, but not by PI3K signaling proteins.

Proinflammatory Activity Of Primarily Infected Endodontic Content Against Macrophages After Different Phases Of The Root Canal Therapy.

This study investigated the presence of target bacterial species and the levels of endotoxins in teeth with apical periodontitis. Levels of inflammatory mediators (interleukin [IL]-1β and tumor necrosis factor [TNF]-α) were determined after macrophage stimulation with endodontic content after different phases of endodontic therapy using different irrigants. Thirty primarily infected root canals were randomly assigned into 3 groups according to the irrigant used for root canal preparation (n = 10 per group): GI: 2.5% sodium hypochlorite, GII: 2% chlorhexidine gel, and GIII (control group): saline solution. Root canal samples were taken by using paper points before (s1) and after root canal instrumentation (s2), subsequently to 17% EDTA (s3), after 30 days of intracanal medication (Ca[OH]2 + saline solution) (s4), and before root canal obturation (s5). Polymerase chain reaction (16S recombinant DNA) and limulus amebocyte lysate assay were used for bacterial and endotoxin detection, respectively. Macrophages were stimulated with the root canal contents for IL-1β/TNF-α measurement using enzyme-linked immunosorbent assay. Porphyromonas gingivalis (17/30), Porphyromonas endodontalis (15/30), and Prevotella nigrescens (11/30) were the most prevalent bacterial species. At s1, endotoxins were detected in 100% of the root canals (median = 32.43 EU/mL). In parallel, substantial amounts of IL-1β and TNF-α were produced by endodontic content-stimulated macrophages. At s2, a significant reduction in endotoxin levels was observed in all groups, with GI presenting the greatest reduction (P < .05). After a root canal rinse with EDTA (s3), intracanal medication (s4), and before root canal obturation (s5), endotoxin levels reduced without differences between groups (P < .05). IL-1β and TNF-α release decreased proportionally to the levels of residual endotoxin (P < .05). Regardless of the use of sodium hypochlorite or CHX, the greatest endotoxin reduction occurs after chemomechanical preparation. Increasing steps of root canal therapy associated with intracanal medication enhances endotoxin reduction, leading to a progressively lower activation of proinflammatory cells such as macrophages.

Morfologia alveolar sob a perspectiva da tomografia computadorizada: definindo os limites biológicos para a movimentação dentária

INTRODUÇÃO: a tomografia computadorizada (TC) permite a visualização do osso alveolar que recobre os dentes por vestibular e lingual. OBJETIVO: o propósito deste estudo foi expor e discutir as implicações da morfologia do osso alveolar, visualizado por meio da TC, sobre o diagnóstico e plano de tratamento ortodôntico. MÉTODOS: foram descritas as evidências sobre a inter-relação entre características dentofaciais e a morfologia das tábuas ósseas vestibular e lingual, assim como evidências sobre a repercussão da movimentação ortodôntica sobre o nível e espessura dessas estruturas periodontais. RESULTADOS: pacientes adultos podem apresentar deiscências ósseas previamente ao tratamento ortodôntico, principalmente na região dos incisivos inferiores. Os pacientes com padrão de crescimento vertical parecem apresentar menor espessura das tábuas ósseas vestibular e lingual no nível do ápice dos dentes permanentes, comparados a pacientes com padrão de crescimento horizontal. O movimento dentário vestibulolingual descentraliza os dentes do rebordo alveolar e ocasiona deiscências ósseas. CONCLUSÃO: a morfologia do rebordo alveolar constitui um fator limitante para a movimentação dentária e deve ser considerada, de forma individual, na realização do plano de tratamento ortodôntico.

Vertical alveolar crest bone maintenance around implants in two-stage surgery: an in situ study in dogs

The aim of this study was to evaluate in situ changes in the alveolar crest bone height around immediate implant-supported crowns in comparison to tooth-supported crowns (control) with the cervical margins located at the bone crest level, without occlusal load. In Group I, after extraction of 12 mandibular premolars from 4 adult dogs, implants from Branemark System (MK III TiU RP 4.0 x 11.5 mm) were placed to retain complete acrylic crowns. In Group II, premolars were prepared to receive complete metal crowns. Sixteen weeks after placement of the crowns (38 weeks after tooth extraction), the height of the alveolar bone crest was measured with a digital caliper. Data were analyzed statistically by the Mann-Whitney test at 5% significance level. The in situ analysis showed no statistically significant difference (p=0.880) between the implant-supported and the tooth-supported groups (1.528 + 0.459 mm and 1.570 + 0.263 mm, respectively). Based on the findings of the present study, it may be concluded that initial peri-implant bone loss may result from the remodeling process necessary to establish the biological space, similar to which occurs with tooth-supported crowns.

Treatment with paracetamol, ketorolac or etoricoxib did not hinder alveolar bone healing: a histometric study in rats

Prostaglandins control osteoblastic and osteoclastic function under physiological or pathological conditions and are important modulators of the bone healing process. The non-steroidal anti-inflammatory drugs (NSAIDs) inhibit cyclooxygenase (COX) activity and consequently prostaglandins synthesis. Experimental and clinical evidence has indicated a risk for reparative bone formation related to the use of non-selective (COX-1 and COX-2) and COX-2 selective NSAIDs. Ketorolac is a non-selective NSAID which, at low doses, has a preferential COX-1 inhibitory effect and etoricoxib is a new selective COX-2 inhibitor. Although literature data have suggested that ketorolac can interfere negatively with long bone fracture healing, there seems to be no study associating etoricoxib with reparative bone formation. Paracetamol/acetaminophen, one of the first choices for pain control in clinical dentistry, has been considered a weak anti-inflammatory drug, although supposedly capable of inhibiting COX-2 activity in inflammatory sites. OBJECTIVE: The purpose of the present study was to investigate whether paracetamol, ketorolac and etoricoxib can hinder alveolar bone formation, taking the filling of rat extraction socket with newly formed bone as experimental model. MATERIAL AND METHODS: The degree of new bone formation inside the alveolar socket was estimated two weeks after tooth extraction by a differential point-counting method, using an optical microscopy with a digital camera for image capture and histometry software. Differences between groups were analyzed by ANOVA after confirming a normal distribution of sample data. RESULTS AND CONCLUSIONS: Histometric results confirmed that none of the tested drugs had a detrimental effect in the volume fraction of bone trabeculae formed inside the alveolar socket.

Cola beverage consumption delays alveolar bone healing: a histometric study in rats

Epidemiological studies have suggested that cola beverage consumption may affect bone metabolism and increase bone fracture risk. Experimental evidence linking cola beverage consumption to deleterious effects on bone is lacking. Herein, we investigated whether cola beverage consumption from weaning to early puberty delays the rate of reparative bone formation inside the socket of an extracted tooth in rats. Twenty male Wistar rats received cola beverage (cola group) or tap water (control group) ad libitum from the age of 23 days until tooth extraction at 42 days and euthanasia 2 and 3 weeks later. The neoformed bone volume inside the alveolar socket was estimated in semi-serial longitudinal sections using a quantitative differential point-counting method. Histological examination suggested a decrease in the osteogenic process within the tooth sockets of rats from both cola groups, which had thinner and sparser new bone trabeculae. Histometric data confirmed that alveolar bone healing was significantly delayed in cola-fed rats at three weeks after tooth extraction (ANOVA, p = 0.0006, followed by Tukey's test, p < 0.01). Although the results of studies in rats cannot be extrapolated directly to human clinical dentistry, the present study provides evidence that cola beverage consumption negatively affect maxillary bone formation.

Morfometria e distribuição de leite alveolar e cisternal na glândula mamária de vacas Holandesa e Girolanda

Estudaram-se a distribuição do leite nas porções alveolar e cisternal e a morfometria da glândula mamária de 14 vacas, sete da raça Holandesa, grupo H, e sete da Girolanda, grupo G, com ordem e dias de lactação semelhantes, ordenhadas duas vezes ao dia. A produção de leite foi mensurada diariamente, enquanto as frações de leite cisternal e alveolar foram medidas uma vez por semana. Durante o experimento, também foram realizadas medidas de ultrassonografia e morfometria da glândula mamária. As vacas Girolandas apresentaram úberes e tetos maiores que as vacas Holandesas. Diâmetro, espessura da parede e comprimento do canal do teto, medidos por imagens de ultrassom, também foram maiores nas Girolandas, mas a área de cisterna da glândula mamária foi maior nas Holandesas. Não houve diferença na produção de leite e nas frações de leite cisternal e alveolar entre as raças.