Graham S. Timmins

Mycobacterium tuberculosis prevents inflammasome activation.

Mycobacterium tuberculosis (Mtb) parasitizes host macrophages and subverts host innate and adaptive immunity. Several cytokines elicited by Mtb are mediators of mycobacterial clearance or are involved in tuberculosis pathology. Surprisingly, interleukin-1beta (IL-1beta), a major proinflammatory cytokine, has not been implicated in host-Mtb interactions. IL-1beta is activated by processing upon assembly of the inflammasome, a specialized inflammatory caspase-activating protein complex. Here, we show that Mtb prevents inflammasome activation and IL-1beta processing. An Mtb gene, zmp1, which encodes a putative Zn(2+) metalloprotease, is required for this process. Infection of macrophages with zmp1-deleted Mtb triggered activation of the inflammasome, resulting in increased IL-1beta secretion, enhanced maturation of Mtb containing phagosomes, improved mycobacterial clearance by macrophages, and lower bacterial burden in the lungs of aerosol-infected mice. Thus, we uncovered a previously masked role for IL-1beta in the control of Mtb and a mycobacterial system that prevents inflammasome and, therefore, IL-1beta activation.

Autophagy protects against active tuberculosis by suppressing bacterial burden and inflammation

Autophagy is a cell biological pathway affecting immune responses. In vitro, autophagy acts as a cell-autonomous defense against Mycobacterium tuberculosis, but its role in vivo is unknown. Here we show that autophagy plays a dual role against tuberculosis: antibacterial and anti-inflammatory. M. tuberculosis infection of Atg5fl/fl LysM-Cre+ mice relative to autophagy-proficient littermates resulted in increased bacillary burden and excessive pulmonary inflammation characterized by neutrophil infiltration and IL-17 response with increased IL-1α levels. Macrophages from uninfected Atg5fl/fl LysM-Cre+ mice displayed a cell-autonomous IL-1α hypersecretion phenotype, whereas T cells showed propensity toward IL-17 polarization during nonspecific activation or upon restimulation with mycobacterial antigens. Thus, autophagy acts in vivo by suppressing both M. tuberculosis growth and damaging inflammation.

Mechanisms of action of isoniazid

For decades after its introduction, the mechanisms of action of the front‐line antituberculosis therapeutic agent isoniazid (INH) remained unclear. Recent developments have shown that peroxidative activation of isoniazid by the mycobacterial enzyme KatG generates reactive species that form adducts with NAD+ and NADP+ that are potent inhibitors of lipid and nucleic acid biosynthetic enzymes. A direct role for some isoniazid‐derived reactive species, such as nitric oxide, in inhibiting mycobacterial metabolic enzymes has also been shown. The concerted effects of these activities – inhibition of cell wall lipid synthesis, depletion of nucleic acid pools and metabolic depression – drive the exquisite potency and selectivity of this agent. To understand INH action and resistance fully, a synthesis of knowledge is required from multiple separate lines of research – including molecular genetic approaches, in vitro biochemical studies and free radical chemistry – which is the intent of this review.

Confinement-induced quorum sensing of individual Staphylococcus aureus bacteria

It is postulated that in addition to cell density, other factors such as the dimensions and diffusional characteristics of the environment could influence quorum sensing (QS) and induction of genetic reprogramming. Modeling studies predict that QS may operate at the level of a single cell, but, owing to experimental challenges, the potential benefits of QS by individual cells remain virtually unexplored. Here we report a physical system that mimics isolation of a bacterium, such as within an endosome or phagosome during infection, and maintains cell viability under conditions of complete chemical and physical isolation. For Staphylococcus aureus, we show that quorum sensing and genetic reprogramming can occur in a single isolated organism. Quorum sensing allows S. aureus to sense confinement and to activate virulence and metabolic pathways needed for survival. To demonstrate the benefit of confinement-induced quorum sensing to individuals, we showed that quorum-sensing bacteria have significantly greater viability over non-QS bacteria.

Comparison of cross-linked polyethylene materials for orthopaedic applications.

Cross-linked polyethylenes are being marketed by orthopaedic manufacturers to address the problem of osteolysis caused by polyethylene particulate wear debris. Wear testing of these cross-linked polyethylenes in hip simulators has shown dramatic reduction in wear rate compared with standard ultrahigh molecular weight polyethylene, either gamma irradiated in air or nitrogen - or ethylene oxide-sterilized. However, this reduction in wear rate is not without cost. The cross-linking processes can result in materials with lower mechanical properties than standard ultrahigh molecular weight polyethylene. To evaluate the effect of the various cross-linking processes on physical and mechanical properties of ultrahigh molecular weight polyethylene, commercially available cross-linked polyethylenes from six orthopaedic manufacturers were tested. This study was the culmination of collaboration with these manufacturers, who provided cross-linked polyethylene for this study, wear characteristics of the material they provided, and review of the physical and mechanical properties measure for their polyethylene. Cross-linked materials were evaluated as received and after an accelerated aging protocol. Free radical identity and concentration, oxidation, crystallinity, melt temperature, ultimate tensile strength, elongation at break, tensile stress at yield, and toughness are reported for each material. By comparing these physical and mechanical properties, surgeons can evaluate the trade-off that results from developing materials with substantially lower wear rates.

Interstitial pO2 in Ischemic Penumbra and Core are Differentially Affected following Transient Focal Cerebral Ischemia in Rats

Stroke causes heterogeneous changes in tissue oxygenation, with a region of decreased blood flow, the penumbra, surrounding a severely damaged ischemic core. Treatment of acute ischemic stroke aims to save this penumbra before its irreversible damage by continued ischemia. However, effective treatment remains elusive due to incomplete understanding of processes leading to penumbral death. While oxygenation is central in ischemic neuronal death, it is unclear exactly what actual changes occur in interstitial oxygen tension (pO2) in ischemic regions during stroke, particularly the penumbra. Using the unique capability of in vivo electron paramagnetic resonance (EPR) oximetry to measure localized interstitial pO2, we measured both absolute values, and temporal changes of pO2 in ischemic penumbra and core during ischemia and reperfusion in a rat model. Ischemia rapidly decreased interstitial pO2 to 32% ± 7.6% and 4% ± 0.6% of pre-ischemic values in penumbra and core, respectively 1 hour after ischemia. Importantly, whilst reperfusion restored core pO2 close to its pre-ischemic value, penumbral pO2 only partially recovered. Hyperoxic treatment significantly increased penumbral pO2 during ischemia, but not in the core, and also increased penumbral pO2 during reperfusion. These divergent, important changes in pO2 in penumbra and core were explained by combined differences in cellular oxygen consumption rates and microcirculation conditions. We therefore demonstrate that interstitial pO2 in penumbra and core is differentially affected during ischemia and reperfusion, providing new insights to the pathophysiology of stroke. The results support normobaric hyperoxia as a potential early intervention to save penumbral tissue in acute ischemic stroke.

Mycobacteria Inhibit Nitric Oxide Synthase Recruitment to Phagosomes during Macrophage Infection

ABSTRACT Inducible nitric oxide synthase (iNOS) is a cytoplasmic protein responsible for the generation of nitric oxide (NO · ) in macrophages. In this work, we hypothesized that the intracellular localization of iNOS is significant for effective delivery of NO ·  to phagosomes containing ingested microorganisms. Using immunofluorescence microscopy and Western blot analysis, iNOS was shown to localize in the vicinity of phagosomes containing latex beads in stimulated macrophages. iNOS also localized to phagosomes containing Escherichia coli. The colocalization of iNOS with ingested latex beads was an actin-dependent process, since treatment with the actin microfilament disrupter cytochalasin D prevented iNOS recruitment to latex bead phagosomes. In contrast to E. coli and inert particle phagosomes, mycobacterial phagosomes did not colocalize with iNOS. This study demonstrates that (i) iNOS can be recruited to phagosomes; (ii) this recruitment is dependent on a functional actin cytoskeleton; (iii) certain microorganisms have the ability to prevent or reduce colocalization with iNOS; and (iv) spatial exclusion of iNOS may play a role in Mycobacterium tuberculosis pathogenesis.

Oxidative stress response genes in Mycobacterium tuberculosis: role of ahpC in resistance to peroxynitrite and stage-specific survival in macrophages.

The Mycobacterium tuberculosis ahpC gene, encoding the mycobacterial orthologue of alkylhydroperoxide reductase, undergoes an unusual regulatory cycle. The levels of AhpC alternate between stages of expression silencing in virulent strains grown as aerated cultures, secondary to a natural loss of the regulatory oxyR function in all strains of the tubercle bacillus, and expression activation in static bacilli by a yet undefined mechanism. The reasons for this unorthodox regulatory cycle controlling expression of an antioxidant factor are currently not known. In this work, M. tuberculosis H37Rv and Mycobacterium smegmatis mc(2)155 ahpC knockout mutants were tested for sensitivity to reactive nitrogen intermediates, in particular peroxynitrite, a highly reactive combinatorial product of reactive nitrogen and oxygen species, and sensitivity to bactericidal mechanisms in resting and activated macrophages. Both M. tuberculosis ahpC::Km(r) and M. smegmatis ahpC::Km(r) showed increased susceptibility to peroxynitrite. In contrast, inactivation of ahpC in M. tuberculosis did not cause increased sensitivity to donors of NO alone. M. tuberculosis ahpC::Km(r) also showed decreased survival in unstimulated macrophages, but the effect was no longer detectable upon IFNgamma activation. These studies establish a specific role for ahpC in antioxidant defences involving peroxynitrite and most likely additional cidal mechanisms in macrophages, with the regulatory cycle likely contributing to survival upon coming out of the stationary phase during dormancy (latent infection) or upon transmission to a new host.

Immunologic manifestations of autophagy

The broad immunologic roles of autophagy span innate and adaptive immunity and are often manifested in inflammatory diseases. The immune effects of autophagy partially overlap with its roles in metabolism and cytoplasmic quality control but typically expand further afield to encompass unique immunologic adaptations. One of the best-appreciated manifestations of autophagy is protection against microbial invasion, but this is by no means limited to direct elimination of intracellular pathogens and includes a stratified array of nearly all principal immunologic processes. This Review summarizes the broad immunologic roles of autophagy. Furthermore, it uses the autophagic control of Mycobacterium tuberculosis as a paradigm to illustrate the breadth and complexity of the immune effects of autophagy.

Microarray analysis and functional characterization of the nitrosative stress response in nonmucoid and mucoid Pseudomonas aeruginosa.

The type strain of Pseudomonas aeruginosa, PAO1, showed great upregulation of many nitrosative defense genes upon treatment with S-nitrosoglutathione, while the mucoid strain PAO578II showed no further upregulation above its constitutive upregulation of nor and fhp. NO* consumption however, showed that both strains mount functional, protein synthesis-dependent NO*-consumptive responses.