Characterization of the tail-specific protease (Tsp) from Legionella

Bacterial tail-specific proteases (Tsps) have been attributed a wide variety of functions including intracellular virulence, cell wall morphology, proteolytic signal cascades and stress response. This study tested the hypothesis that Tsp has a key function for the transmissive form of Legionella pneumophila. A tsp mutant was generated in Legionella pneumophila 130b and the characteristics of this strain and the isogenic wild-type were examined using a range of growth and proteomic analyses. Recombinant Tsp protein was also produced and analyzed. The L. pneumophila tsp mutant showed no defect in growth on rich media or during thermo-osmotic stress conditions. In addition, no defects in cellular morphology were observed when the cells were examined using transmission electron microscopy. Purified recombinant Tsp was found to be an active protease with a narrow substrate range. Proteome analysis using iTRAQ (5% coverage of the proteome) found that, of those proteins detected, only 5 had different levels in the tsp mutant compared to the wild type. ACP (Acyl Carrier Protein), which has a key role for Legionella differentiation to the infectious form, was reduced in the tsp mutant; however, tsp(-) was able to infect and replicate inside macrophages to the same extent as the wild type. Combined, these data demonstrate that Tsp is a protease but is not essential for Legionella growth or cell infection. Thus, Tsp may have functional redundancy in Legionella.

NADPH oxidases as regulators of tumor angiogenesis : current and emerging concepts

Significance Reactive oxygen species (ROS) such as superoxide, hydrogen peroxide, and peroxynitrite are generated ubiquitously by all mammalian cells and have been understood for many decades as inflicting cell damage and as causing cancer by oxidation and nitration of macromolecules, including DNA, RNA, proteins, and lipids. Recent Advances A current concept suggests that ROS can also promote cell signaling pathways triggered by growth factors and transcription factors that ultimately regulate cell proliferation, differentiation, and apoptosis, all of which are important hallmarks of tumor cell proliferation and angiogenesis. Moreover, an emerging concept indicates that ROS regulate the functions of immune cells that infiltrate the tumor environment and stimulate angiogenesis, such as macrophages and specific regulatory T cells. Critical Issues In this article, we highlight that the NADPH oxidase family of ROS-generating enzymes are the key sources of ROS and, thus, play an important role in redox signaling within tumor, endothelial, and immune cells thereby promoting tumor angiogenesis. Future Directions Knowledge of these intricate ROS signaling pathways and identification of the culprit NADPH oxidases is likely to reveal novel therapeutic opportunities to prevent angiogenesis that occurs during cancer and which is responsible for the revascularization after current antiangiogenic treatment.

Obesity and hypertension have differing oxidant handling molecular pathways in age-related chronic kidney disease

Chronic kidney disease (CKD) in ageing is a burden on health systems worldwide. Rat models of age-related CKD linked with obesity and hypertension were used to investigate alterations in oxidant handling and energy metabolism to identify gene targets or markers for age-related CKD. Young adult (3 months) and old (21–24 months) spontaneously-hypertensive (SHR), normotensive Wistar-Kyoto (WKY) and Wistar rats (normotensive, obese in ageing) were compared for renal functional and physiological parameters, renal fibrosis and inflammation, oxidative stress (hemeoxygenase-1/HO-1), apoptosis and cell injury (including Bax:Bcl-2), phosphorylated and non-phosphorylated forms of oxidant and energy sensing proteins (p66Shc, AMPK), signal transduction proteins (ERK1/2, PKB), and transcription factors (NF-κB, FoxO1). All old rats were normoglycemic. Renal fibrosis, tubular epithelial apoptosis, interstitial macrophages and myofibroblasts (all p < 0.05), p66Shc/phospho-p66 (p < 0.05), Bax/Bcl-2 ratio (p < 0.05) and NF-κB expression (p < 0.01) were highest in old obese Wistars. Expression of phospho-FoxO/FoxO was elevated in old Wistars (p < 0.001) and WKYs (p < 0.01). SHRs had high levels in young and old rats. Expression of PKB, phospho-PKB, ERK1/2 and phospho-ERK1/2 were significantly elevated in all aged animals. These results suggest that obesity and hypertension have differing oxidant handling and signalling pathways that act in the pathogenesis of age-related CKD

Anti-Inflammatory and antiosteoclastogenic activities of parthenolide on human periodontal ligament cellsIn vitro

Periodontitis is an inflammatory disease that causes osteolysis and tooth loss. It is known that the nuclear factor kappa B (NF-κB) signalling pathway plays a key role in the progression of inflammation and osteoclastogenesis in periodontitis. Parthenolide (PTL), a sesquiterpene lactone extracted from the shoots of Tanacetum parthenium, has been shown to possess anti-inflammatory properties in various diseases. In the study reported herein, we investigated the effects of PTL on the inflammatory and osteoclastogenic response of human periodontal ligament-derived cells (hPDLCs) and revealed the signalling pathways in this process. Our results showed that PTL decreased NF-κB activation, I-κB degradation, and ERK activation in hPDLCs. PTL significantly reduced the expression of inflammatory (IL-1β, IL-6, and TNF-α) and osteoclastogenic (RANKL, OPG, and M-CSF) genes in LPS-stimulated hPDLCs. In addition, PTL attenuated hPDLC-induced osteoclastogenic differentiation of macrophages (RAW264.7 cells), as well as reducing gene expression of osteoclast-related markers in RAW264.7 cells in an hPDLC-macrophage coculture model. Taken together, these results demonstrate the anti-inflammatory and antiosteoclastogenic activities of PTL in hPDLCs in vitro. These data offer fundamental evidence supporting the potential use of PTL in periodontitis treatment.

Olfactory ensheathing cells are the main phagocytic cells that remove axon debris during early development of the olfactory system

During development of the primary olfactory system, axon targeting is inaccurate and axons inappropriately project within the target layer or overproject into the deeper layers of the olfactory bulb. As a consequence there is considerable apoptosis of primary olfactory neurons during embryonic and postnatal development and axons of the degraded neurons need to be removed. Olfactory ensheathing cells (OECs) are the glia of the primary olfactory nerve and are known to phagocytose axon debris in the adult and postnatal animal. However, it is unclear when phagocytosis by OECs first commences. We investigated the onset of phagocytosis by OECs in the developing mouse olfactory system by utilizing two transgenic reporter lines: OMP-ZsGreen mice which express bright green fluorescent protein in primary olfactory neurons, and S100β-DsRed mice which express red fluorescent protein in OECs. In crosses of these mice, the fate of the degraded axon debris is easily visualized. We found evidence of axon degradation at embryonic day (E)13.5. Phagocytosis of the primary olfactory axon debris by OECs was first detected at E14.5. Phagocytosis of axon debris continued into the postnatal animal during the period when there was extensive mistargeting of olfactory axons. Macrophages were often present in close proximity to OECs but they contributed only a minor role to clearing the axon debris, even after widespread degeneration of olfactory neurons by unilateral bulbectomy and methimazole treatment. These results demonstrate that from early in embryonic development OECs are the primary phagocytic cells of the primary olfactory nerve.

Biliverdin modulates the expression of C5aR in response to endotoxin in part via mTOR signaling

Macrophages play a crucial role in the maintenance and resolution of inflammation and express a number of pro- and anti-inflammatory molecules in response to stressors. Among them, the complement receptor 5a (C5aR) plays an integral role in the development of inflammatory disorders. Biliverdin and bilirubin, products of heme catabolism, exert anti-inflammatory effects and inhibit complement activation. Here, we define the effects of biliverdin on C5aR expression in macrophages and the roles of Akt and mammalian target of rapamycin (mTOR) in these responses. Biliverdin administration inhibited lipopolysaccharide (LPS)-induced C5aR expression (without altering basal expression), an effect partially blocked by rapamycin, an inhibitor of mTOR signaling. Biliverdin also reduced LPS-dependent expression of the pro-inflammatory cytokines TNF-alpha and IL-6. Collectively, these data indicate that biliverdin regulates LPS-mediated expression of C5aR via the mTOR pathway, revealing an additional mechanism underlying biliverdin's anti-inflammatory effects.

The effect of osteoimmunomodulation on the osteogenic effects of cobalt incorporated β-tricalcium phosphate

Osteoblast lineage cells are direct effectors of osteogenesis and are, therefore, commonly used to evaluate the in vitro osteogenic capacity of bone substitute materials. This method has served its purposes when testing novel bone biomaterials; however, inconsistent results between in vitro and in vivo studies suggest the mechanisms that govern a material's capacity to mediate osteogenesis are not well understood. The emerging field of osteoimmunology and immunomodulation has informed a paradigm shift in our view of bone biomaterials–from one of an inert to an osteoimmunomodulatory material–highlighting the importance of immune cells in materials-mediated osteogenesis. Neglecting the importance of the immune response during this process is a major shortcoming of the current evaluation protocol. In this study we evaluated a potential angiogenic bone substitute material cobalt incorporated with β-tricalcium phosphate (CCP), comparing the traditional “one cell type” approach with a “multiple cell types” approach to assess osteogenesis, the latter including the use of immune cells. We found that CCP extract by itself was sufficient to enhance osteogenic differentiation of bone marrow stem cells (BMSCs), whereas this effect was cancelled out when macrophages were involved. In response to CCP, the macrophage phenotype switched to the M1 extreme, releasing pro-inflammatory cytokines and bone catabolic factors. When the CCP materials were implanted into a rat femur condyle defect model, there was a significant increase of inflammatory markers and bone destruction, coupled with fibrous encapsulation rather than new bone formation. These findings demonstrated that the inclusion of immune cells (macrophages) in the in vitro assessment matched the in vivo tissue response, and that this method provides a more accurate indication of the essential role of immune cells when assessing materials-stimulated osteogenesis in vitro.

Molecular and pathological insights into Chlamydia pecorum-associated sporadic bovine encephalomyelitis (SBE) in Western Australia

Background Despite its global recognition as a ruminant pathogen, cases of Chlamydia pecorum infection in Australian livestock are poorly documented. In this report, a C. pecorum specific Multi Locus Sequence Analysis scheme was used to characterise the C. pecorum strains implicated in two cases of sporadic bovine encephalomyelitis confirmed by necropsy, histopathology and immunohistochemistry. This report provides the first molecular evidence for the presence of mixed infections of C. pecorum strains in Australian cattle. Case presentation Affected animals were two markedly depressed, dehydrated and blind calves, 12 and 16 weeks old. The calves were euthanized and necropsied. In one calf, a severe fibrinous polyserositis was noted with excess joint fluid in all joints whereas in the other, no significant lesions were seen. No gross abnormalities were noted in the brain of either calf. Histopathological lesions seen in both calves included: multifocal, severe, subacute meningoencephalitis with vasculitis, fibrinocellular thrombosis and malacia; diffuse, mild, acute interstitial pneumonia; and diffuse, subacute epicarditis, severe in the calf with gross serositis. Immunohistochemical labelling of chlamydial antigen in brain, spleen and lung from the two affected calves and brain from two archived cases, localised the antigen to the cytoplasm of endothelium, mesothelium and macrophages. C. pecorum specific qPCR, showed dissemination of the pathogen to multiple organs. Phylogenetic comparisons with other C. pecorum bovine strains from Australia, Europe and the USA revealed the presence of two genetically distinct sequence types (ST). The predominant ST detected in the brain, heart, lung and liver of both calves was identical to the C. pecorum ST previously described in cases of SBE. A second ST detected in an ileal tissue sample from one of the calves, clustered with previously typed faecal bovine isolates. Conclusion This report provides the first data to suggest that identical C. pecorum STs may be associated with SBE in geographically separated countries and that these may be distinct from those found in the gastrointestinal tract. This report provides a platform for further investigations into SBE and for understanding the genetic relationships that exist between C. pecorum strains detected in association with other infectious diseases in livestock.

Osteoimmunomodulation: A new aspect for the development and evaluation of bone biomaterials

This thesis successfully introduced the intellectual framework of immunology in the development of bone biomaterials. The project identified the regulatory role of biomaterials to the immune-response in terms of bone formation and healing of bone defects. The novel methods developed in the project will significantly change the ways of biomaterials assessment and evaluation.

Fine mapping of the MHC Class III region demonstrates association of AIF1 and rheumatoid arthritis

Objective. The heritability of RA has been estimated to be ∼55%, of which the MHC contributes about one-third. HLA-DRB1 alleles are strongly associated with RA, but it is likely that significant non-DRB1 MHC genetic susceptibility factors are involved. Previously, we identified two three-marker haplotypes in a 106-kb region in the MHC class III region immediately centromeric to TNF, which are strongly associated with RA on HLA-DRB1*0404 haplotypes. In the present study, we aimed to refine these associations further using a combination of genotyping and gene expression studies. Methods. Thirty-nine nucleotide polymorphisms (SNPs) were genotyped in 95 DRB1*0404 carrying unrelated RA cases, 125 DRB1*0404 - carrying healthy controls and 87 parent-case trio RA families in which the affected child carried HLA-DRB1*04. Quantitative RT-PCR was used to assess the expression of the positional candidate MHC class III genes APOM, BAT2, BAT3, BAT4, BAT5, AIF1, C6orf47, CSNK2β and LY6G5C, and the housekeeper genes, hypoxanthine-guanine phosphoribosyltransferase (HPRT) and β2-microglobulin (B2M) in 31 RA cases and 21 ethnically, age- and sex-matched healthy controls. Synovial membrane specimens from RA, PsA and OA cases were stained by an indirect immunoperoxidase technique using a mouse-anti-human AIF1 monoclonal antibody. Results. Association was observed between RA and single markers or two marker haplotypes involving AIF1, BAT3 and CSNK. AIF1 was also significantly overexpressed in RA mononuclear cells (1.5- to 1.9-fold difference, P = 0.02 vs HPRT, P = 0.002 vs B2M). AIF1 protein was clearly expressed by synovial macrophages in all the inflammatory synovial samples in contrast to the non-inflammatory OA samples. Conclusions. The results of the genotyping and expression studies presented here suggest a role for AIF1 in both the aetiology and pathogenesis of RA.

Temporal dependence of in vivo USPIO-enhanced MRI signal changes in human carotid atheromatous plaques

Introduction: Ultrasmall superparamagnetic iron oxide (USPIO)-enhanced MRI has been shown to be a useful modality to image activated macrophages in vivo, which are principally responsible for plaque inflammation. This study determined the optimum imaging time-window to detect maximal signal change post-USPIO infusion using T1-weighted (T1w), T2*- weighted (T2*w) and quantitative T2*(qT 2*) imaging. Methods: Six patients with an asymptomatic carotid stenosis underwent high resolution T1w, T2*w and qT2*MR imaging of their carotid arteries at 1.5 T. Imaging was performed before and at 24, 36, 48, 72 and 96 h after USPIO (Sinerem™, Guerbet, France) infusion. Each slice showing atherosclerotic plaque was manually segmented into quadrants and signal changes in each quadrant were fitted to an exponential power function to model the optimum time for post-infusion imaging. Results: The power function determining the mean time to convergence for all patients was 46, 41 and 39 h for the T1w, T 2*w and qT2*sequences, respectively. When modelling each patient individually, 90% of the maximum signal intensity change was observed at 36 h for three, four and six patients on T1w, T 2*w and qT2*, respectively. The rates of signal change decrease after this period but signal change was still evident up to 96 h. Conclusion: This study showed that a suitable imaging window for T 1w, T2*w and qT2*signal changes post-USPIO infusion was between 36 and 48 h. Logistically, this would be convenient in bringing patients back for one post-contrast MRI, but validation is required in a larger cohort of patients.

Non-invasive MR imaging of inflammation in a patient with both asymptomatic carotid atheroma and an abdominal aortic aneurysm: A case report

Inflammation is a recognized risk factor for the vulnerable atherosclerotic plaque. USPIO-enhanced MRI imaging is a promising non-i nvasive method to identify high-risk atheromatous plaque inflammation in vivo in humans, in which areas of focal signal loss on MR images have been shown to correspond to the location of activated macrophages, typically at the shoulder regions of the plaque. This is the first report in humans describing simultaneous USPIO uptake within atheroma in two different arterial territories and again emphasises that atherosclerosis is a truly systemic disease. With further work, USPIO-enhanced MR imaging may be useful in identifying inflamed vulnerable atheromatous plaques in vivo, so refining patient selection for intervention and allowing appropriate early aggressive pharmacotherapy to prevent plaque rupture.

Correlation of macrophage location and plaque stress distribution using USPIO-enhanced MRI in a patient with symptomatic severe carotid stenosis: A new insight into risk stratification

High resolution, USPIO-enhanced MR imaging can be used to identify inflamed atherosclerotic plaque. We report a case of a 79-year-old man with a symptomatic carotid stenosis of 82%. The plaque was retrieved for histology and finite element analysis (FEA) based on the preoperative MR imaging was used to predict maximal Von Mises stress on the plaque. Macrophage location correlated with maximal predicted stresses on the plaque. This supports the hypothesis that macrophages thin the fibrous cap at points of highest stress, leading to an increased risk of plaque rupture and subsequent stroke.

The role of H4 flagella in Escherichia coli ST131 virulence

Escherichia coli sequence type 131 (ST131) is a globally dominant multidrug resistant clone associated with urinary tract and bloodstream infections. Most ST131 strains exhibit resistance to multiple antibiotics and cause infections associated with limited treatment options. The largest sub-clonal ST131 lineage is resistant to fluoroquinolones, contains the type 1 fimbriae fimH30 allele and expresses an H4 flagella antigen. Flagella are motility organelles that contribute to UPEC colonisation of the upper urinary tract. In this study, we examined the specific role of H4 flagella in ST131 motility and interaction with host epithelial and immune cells. We show that the majority of H4-positive ST131 strains are motile and are enriched for flagella expression during static pellicle growth. We also tested the role of H4 flagella in ST131 through the construction of specific mutants, over-expression strains and isogenic mutants that expressed alternative H1 and H7 flagellar subtypes. Overall, our results revealed that H4, H1 and H7 flagella possess conserved phenotypes with regards to motility, epithelial cell adhesion, invasion and uptake by macrophages. In contrast, H4 flagella trigger enhanced induction of the anti-inflammatory cytokine IL-10 compared to H1 and H7 flagella, a property that may contribute to ST131 fitness in the urinary tract.

Uropathogenic Escherichia Coli engages CD14-dependent signaling to enable bladder-macrophage-dependent control of acute urinary tract infection

Background CD14, a coreceptor for several pattern recognition receptors and a widely used monocyte/macrophage marker, plays a key role in host responses to gram-negative bacteria. Despite the central role of CD14 in the inflammatory response to lipopolysaccharide and other microbial products and in the dissemination of bacteria in some infections, the signaling networks controlled by CD14 during urinary tract infection (UTI) are unknown. Methods We used uropathogenic Escherichia coli (UPEC) infection of wild-type (WT) C57BL/6 and Cd14−/− mice and RNA sequencing to define the CD14-dependent transcriptional signature and the role of CD14 in host defense against UTI in the bladder. Results UPEC induced the upregulation of Cd14 and the monocyte/macrophage-related genes Emr1/F4/80 and Csf1r/c-fms, which was associated with lower UPEC burdens in WT mice, compared with Cd14−/− mice. Exacerbation of infection in Cd14−/− mice was associated with the absence of a 491-gene transcriptional signature in the bladder that encompassed multiple host networks not previously associated with this receptor. CD14-dependent pathways included immune cell trafficking, differential cytokine production in macrophages, and interleukin 17 signaling. Depletion of monocytes/macrophages in the bladder by administration of liposomal clodronate led to higher UPEC burdens. Conclusions This study identifies new host protective and signaling roles for CD14 in the bladder during UPEC UTI.