Macrophage killing is an essential virulence mechanism of Salmonella typhimurium.

Phagocytic cells are a critical line of defense against infection. The ability of a pathogen to survive and even replicate within phagocytic cells is a potent method of evading the defense mechanisms of the host. A number of pathogens survive within macrophages after phagocytosis and this contributes to their virulence. Salmonella is one of these pathogens. Here we report that 6-14 hr after Salmonella enters the macrophage and replicates, it resides in large vacuoles and causes the destruction of these cells. Furthermore, we identified four independently isolated MudJ-lacZ insertion mutants that no longer cause the formation of these vacuoles or kill the macrophages. All four insertions were located in the ompR/envZ regulon. These findings suggest that killing and escape from macrophages may be as important steps in Salmonella pathogenesis as are survival and replication in these host cells.

Hematopoietic and lung abnormalities in mice with a null mutation of the common beta subunit of the receptors for granulocyte-macrophage colony-stimulating factor and interleukins 3 and 5.

Gene targeting was used to create mice with a null mutation of the gene encoding the common beta subunit (beta C) of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3; multi-CSF), and interleukin 5 (IL-5) receptor complexes (beta C-/- mice). High-affinity binding of GM-CSF was abolished in beta C-/- bone marrow cells, while cells from heterozygous animals (beta C+/- mice) showed an intermediate number of high-affinity receptors. Binding of IL-3 was unaffected, confirming that the IL-3-specific beta chain remained intact. Eosinophil numbers in peripheral blood and bone marrow of beta C-/- animals were reduced, while other hematological parameters were normal. In clonal cultures of beta C-/- bone marrow cells, even high concentrations of GM-CSF and IL-5 failed to stimulate colony formation, but the cells exhibited normal quantitative responsiveness to stimulation by IL-3 and other growth factors. beta C-/- mice exhibited normal development and survived to young adult life, although they developed pulmonary peribronchovascular lymphoid infiltrates and areas resembling alveolar proteinosis. There was no detectable difference in the systemic clearance and distribution of GM-CSF between beta C-/- and wild-type littermates. The data establish that beta C is normally limiting for high-affinity binding of GM-CSF and demonstrate that systemic clearance of GM-CSF is not mediated via such high-affinity receptor complexes.

Arginase activity in a murine macrophage cell line (RAW264.7) stimulated with lipopolysaccharide from Actinobacillus actinomycetemcomitans

Aims: The aim of the present study was to determine the role of cyclic adenosine monophosphate (cAMP) on arginase activity in a murine macrophage cell line (RAW264.7 cells) stimulated with lipopolysaccharide (LPS) from Actinobacillus actinomycetemcomitans. Materials and methods: The cells were treated with A. actinomycetemcomitans LPS for 24 h. The effects of SQ22536 (an adenylyl cyclase inhibitor), ODQ (a guanylyl cyclase inhibitor), dibutyryl cAMP (a cAMP analog), 8-bromo cyclic guanosine monophosphate (a cGMP analog), forskolin (an adenylyl cylase activator), and cycloheximide (a protein synthesis inhibitor) on arginase activity in A. actinomycetemcomitans LPS-stimulated RAW264.7 cells were also determined. Arginase activity was assessed in LPS-stimulated cells in the presence of 3-isobutyl-1-methylxanthine (IBMX), siguazodan and rolipram [phosphodiesterase (PDE) inhibitors] as well as KT5720 [a protein kinase A (PKA) inhibitor]. Results: Arginase activity in A. actinomycetemcomitans LPS-stimulated RAW264.7 cells was suppressed by SQ22536 but not ODQ. Enhancement of arginase activity was observed in the presence of cAMP analog or forskolin but not cGMP analog. Cycloheximide blocked arginase activity in the cells in the presence of cAMP analog or forskolin with or without A. actinomycetemcomitans LPS. IBMX augmented arginase activity in A. actinomycetemcomitans LPS-stimulated cells. Rolipram (a PDE4 inhibitor) increased the levels of arginase activity higher than siguazodan (a PDE3 inhibitor) in the antigen-stimulated cells. The effect of cAMP analog or forskolin on arginase activity in the presence or absence of A. actinomycetemcomitans LPS was blocked by the PKA inhibitor (KT5720). Conclusion: The results of the present study suggest that A. actinomycetemcomitans LPS may stimulate arginase activity in murine macrophages (RAW264.7 cells) in a cAMP-PKA-dependent pathway.

Apoptotic cell-derived ICAM-3 promotes both macrophage chemoattraction to and tethering of apoptotic cells

A wide range of molecules acting as apoptotic cell-associated ligands, phagocyte-associated receptors or soluble bridging molecules have been implicated within the complex sequential processes that result in phagocytosis and degradation of apoptotic cells. Intercellular adhesion molecule 3 (ICAM-3, also known as CD50), a human leukocyte-restricted immunoglobulin super-family (IgSF) member, has previously been implicated in apoptotic cell clearance, although its precise role in the clearance process is ill defined. The main objective of this work is to further characterise the function of ICAM-3 in the removal of apoptotic cells. Using a range of novel anti-ICAM-3 monoclonal antibodies (mAbs), including one (MA4) that blocks apoptotic cell clearance by macrophages, alongside apoptotic human leukocytes that are normal or deficient for ICAM-3, we demonstrate that ICAM-3 promotes a domain 1-2-dependent tethering interaction with phagocytes. Furthermore, we demonstrate an apoptosis-associated reduction in ICAM-3 that results from release of ICAM-3 within microparticles that potently attract macrophages to apoptotic cells. Taken together, these data suggest that apoptotic cell-derived microparticles bearing ICAM-3 promote macrophage chemoattraction to sites of leukocyte cell death and that ICAM-3 mediates subsequent cell corpse tethering to macrophages. The defined function of ICAM-3 in these processes and profound defect in chemotaxis noted to ICAM-3-deficient microparticles suggest that ICAM-3 may be an important adhesion molecule involved in chemotaxis to apoptotic human leukocytes. © 2012 Macmillan Publishers Limited All rights reserved.

Macrophage-mediated clearance of cells undergoing caspase-3-independent death

Little is known of the functions of caspases in mediating the surface changes required for phagocytosis of dying cells. Here we investigate the role played by the effector caspase, caspase-3 in this process using the caspase-3-defective MCF-7 breast carcinoma line and derived caspase-3-expressing transfectants. Our results indicate that, while certain typical features of apoptosis induced by etoposide – namely classical morphological changes and the ability to degrade DNA into oligonucleosomal fragments – are caspase-3-dependent, loss of cell adhesion to plastic and the capacity to interact with, and to be phagocytosed by, human monocyte-derived macrophages – both by CD14-dependent and CD14-independent mechanisms – do not require caspase-3. Furthermore, both etoposide-induced caspase-3-positive and -negative MCF-7 cells suppressed proinflammatory cytokine release by macrophages. These results demonstrate directly that cell surface changes that are sufficient for anti-inflammatory clearance by human macrophages can be regulated independently of stereotypical features of the apoptosis programme that require caspase-3.

Regulation of Rab5 GTPase activity during Pseudomonas aeruginosa-macrophage interaction

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen. Several antibiotic resistant strains of P. aeruginosa are commonly found as secondary infection in immune-compromised patients leaving significant mortality and healthcare cost. Pseudomonas aeruginosa successfully avoids the process of phagocytosis, the first line of host defense, by secreting several toxic effectors. Effectors produced from P. aeruginosa Type III secretion system are critical molecules required to disrupt mammalian cell signaling and holds particular interest to the scientists studying host-pathogen interaction. Exoenzyme S (ExoS) is a bi-functional Type III effector that ADP-ribosylates several intracellular Ras (Rat sarcoma) and Rab (Response to abscisic acid) small GTPases in targeted host cells. The Rab5 protein acts as a rate limiting protein during phagocytosis by switching from a GDP- bound inactive form to a GTP-bound active form. Activation and inactivation of Rab5 protein is regulated by several Rab5-GAPs (GTPase Activating Proteins) and Rab5-GEFs (Rab5-Guanine nucleotide Exchange Factors). Some pathogenic bacteria have shown affinity for Rab proteins during infection and make their way inside the cell. This dissertation demonstrated that Rab5 plays a critical role during early steps of P. aeruginosa invasion in J774-Eclone macrophages. It was found that live, but not heat inactivated, P. aeruginosa inhibited phagocytosis that occurred in conjunction with down-regulation of Rab5 activity. Inactivation of Rab5 was dependent on ExoS ADP-ribosyltransferase activity, and more than one arginine sites in Rab5 are possible targets for ADP-ribosylation modification. However, the expression of Rin1, but not other Rab5GEFs (Rabex-5 and Rap6) reversed this down-regulation of Rab5 in vivo. Further studies revealed that the C-terminus of Rin1 carrying Rin1:Vps9 and Rin1:RA domains are required for optimal Rab5 activation in conjunction with active Ras. These observations demonstrate a novel mechanism of Rab5 targeting to phagosome via Rin1 during the phagocytosis of P. aeruginosa. The second part of this dissertation investigated antimicrobial activities of Dehydroleucodine (DhL), a secondary metabolite from Artemisia douglasiana, against P. aeruginosa growth and virulence. Populations of several P. aeruginosa strains were completely susceptible to DhL at a concentration between 0.48~0.96 mg/ml and treatment at a threshold concentration (0.12 mg/ml) inhibited growth and many virulent activities without damaging the integrity of the cell suggesting anti-Pseudomonas activity of DhL.

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.

Comparison of human alveolar osteoblasts cultured on polymer-ceramic composite scaffolds and tissue culture plates

The effects of medical grade polycaprolactone–tricalcium phosphate (mPCL–TCP) (80:20) scaffolds on primary human alveolar osteoblasts (AOs) were compared with standard tissue-culture plates. Of the seeded AOs, 70% adhered to and proliferated on the scaffold surface and within open and interconnected pores; they formed multi-layered sheets and collagen fibers with uniform distribution within 28 days. Elevation of alkaline phosphatase activity occurred in scaffold–cell constructs independent of osteogenic induction. AO proliferation rate increased and significant decrease in calcium concentration of the medium for both scaffolds and plates under induction conditions were seen. mPCL–TCP scaffolds significantly influenced the AO expression pattern of osterix and osteocalcin (OCN). Osteogenic induction down-regulated OCN at both RNA and protein level on scaffolds (3D) by day 7, and up-regulated OCN in cell-culture plates (2D) by day 14, but OCN levels on scaffolds were higher than on cell-culture plates. Immunocytochemical signals for type I collagen, osteopontin and osteocalcin were detected at the outer parts of scaffold–cell constructs. More mineral nodules were found in induced than in non-induced constructs. Only induced 2D cultures showed nodule formation. mPCL–TCP scaffolds appear to stimulate osteogenesis in vitro by activating a cellular response in AO's to form mineralized tissue. There is a fundamental difference between culturing AOs on 2D and 3D environments that should be considered when studying osteogenesis in vitro.