Circulatory Neutrophils Exhibit Enhanced Neutrophil Extracellular Trap Formation in Early Puerperium: NETs at the Nexus of Thrombosis and Immunity.

Pregnancy is associated with elevated maternal levels of cell-free DNA of neutrophil extracellular trap (NET) origin, as circulatory neutrophils exhibit increased spontaneous NET formation, mainly driven by G-CSF and finely modulated by sex hormones. The postpartum period, on the other hand, involves physiological alterations consistent with the need for protection against infections and fatal haemorrhage. Our findings indicate that all relevant serum markers of neutrophil degranulation and NET release are substantially augmented postpartum. Neutrophil pro-NETotic activity in vitro is also upregulated particularly in post-delivery neutrophils. Moreover, maternal puerperal neutrophils exhibit a strong pro-NETotic phenotype, associated with increased levels of all key players in the generation of NETs, namely citH3, MPO, NE, and ROS, compared to non-pregnant and pregnant controls. Intriguingly, post-delivery NET formation is independent of G-CSF in contrast to late gestation and complemented by the presence of TF on the NETs, alterations in the platelet activity status, and activation of the coagulation cascade, triggered by circulating microparticles. Taken together, our results reveal the highly pro-NETotic and potentially procoagulant nature of postpartum neutrophils, bridging an overt immune activation with possible harmful thrombotic incidence.

High-intensity interval training modulates retinal microvascular phenotype and DNA methylation of p66Shc gene: a randomized controlled trial (EXAMIN AGE).

AIMS: Impairments of retinal vessel diameter are associated with major adverse cardiovascular (CV) events. Promoter DNA methylation is a repressor of the mitochondrial adaptor p66Shc gene transcription, a key driver of ageing-induced reactive oxygen species. The study aimed to investigate whether high-intensity interval training (HIIT) affects retinal microvascular phenotype as well as p66Shc expression and oxidative stress in ageing subjects with increased CV risk from the EXAMIN AGE cohort. METHODS AND RESULTS: Eighty-four sedentary subjects (mean age 59.4 ± 7.0 years) with ≥2 CV risk factors were randomized into either a 12-week HIIT or standard physical activity recommendations. Retinal arteriolar and venular diameters were measured by use of a retinal vessel analyser. As a marker of oxidative stress plasma 3-nitrotyrosine (3-NT) level was determined by ELISA. Gene expression of p66Shc and DNA methylation were assessed in mononuclear cells by RT-qPCR and methylated-DNA capture (MethylMiner Enrichment Kit) coupled with qPCR, respectively. High-intensity interval training reduced body mass index, fat mass, low-density lipoprotein and increased muscle mass, as well as maximal oxygen uptake (VO2max). Moreover, HIIT restored microvascular phenotype by inducing retinal arteriolar widening (pre: 175 ± 14 µm vs. post: 181 ± 13 µm, P = 0.001) and venular narrowing (pre: 222 ± 14 µm vs. post: 220 ± 14 µm, P = 0.007). After HIIT, restoration of p66Shc promoter methylation (P = 0.034) reduced p66Shc gene expression (P = 0.037) and, in turn, blunted 3-NT plasma levels (P = 0.002). CONCLUSION: High-intensity interval training rescues microvascular dysfunction in ageing subjects at increased CV risk. Exercise-induced reprogramming of DNA methylation of p66Shc gene may represent a putative mechanistic link whereby exercise protects against age-related oxidative stress. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov: NCT02796976 (https://clinicaltrials.gov/ct2/show/NCT02796976).

Physical activity may drive healthy microvascular ageing via downregulation of p66Shc.

BACKGROUND: Narrower retinal arterioles and wider venules are linked to adverse cardiovascular outcomes. The mitochondrial adaptor p66Shc is a major source of ageing-induced generation of reactive oxygen species. Promoter DNA methylation inhibits p66Shc gene transcription. This cross-sectional study was designed to investigate the link between physical activity, retinal vessel diameters and p66Shc expression in active and sedentary ageing subjects. DESIGN/METHODS: Altogether 158 subjects were included in the study (mean age 59.4 ± 7.0 years). Thirty-eight subjects were healthy active, 36 were healthy sedentary and 84 were sedentary with ≥2 cardiovascular risk factors. Retinal arteriolar and venular diameters were measured by means of a retinal vessel analyser. As a marker of oxidative stress, plasma 3-nitrotyrosine was determined by enzyme-linked immunosorbent assay. Gene expression of p66Shc and DNA methylation were assessed in mononuclear cells by real-time quantitative polymerase chain reaction and methylated-DNA capture (MethylMiner Enrichment kit) coupled with quantitative polymerase chain reaction, respectively. RESULTS: Wider retinal arterioles (179 ± 14 vs 172 ± 11 and 171 ± 14 µm; p < 0.05 and narrower venules (204 ± 17 vs 209 ± 11 and 218 ± 16 µm; p < 0.001) were observed in healthy active subjects compared with healthy sedentary subjects and sedentary subjects with ≥2 cardiovascular risk factors, respectively. Furthermore, healthy active subjects had blunted p66Shc expression and lower 3-nitrotyrosine plasma levels compared with healthy sedentary and sedentary subjects with ≥2 cardiovascular risk factors. Accordingly, hypomethylation of p66Shc promoter observed in healthy sedentary and sedentary subjects with ≥2 cardiovascular risk factors was not found in healthy active subjects. CONCLUSION: Long-term physical activity-induced DNA methylation of p66Shc may represent a putative mechanistic link whereby active lifestyle promotes healthy microvascular ageing.

Differential effects of specific cathepsin S inhibition in biocompartments from patients with primary Sjögren syndrome.

OBJECTIVE: Primary Sjögren syndrome (pSS) is characterized by T and B cell infiltration of exocrine glands. The cysteine protease cathepsin S (CatS) is crucially involved in MHCII processing and T cell stimulation, and elevated levels have been found in patients with RA, psoriasis and pSS. However, little is known about the functional characteristics and mechanisms of SS-A- and SS-B-specific T cells in pSS patients. We herein investigated the inhibition of CatS activity in different biocompartments of pSS patients including antigen-specific T cell responses. METHODS: Ex vivo CatS activity was assessed in tears, plasma and saliva of 15 pSS patients and 13 healthy controls (HC) and in the presence or absence of the specific CatS inhibitor RO5459072. In addition, antigen (SS-A (60kD), SS-B, influenza H3N2, tetanus toxoid and SEB)-specific T cell responses were examined using ex vivo IFN-γ/IL-17 Dual ELISPOT and Bromdesoxyuridin (BrdU) proliferation assays in the presence or absence of RO5459072. Supernatants were analysed for IL-1ß, IL-6, IL-10, TNF-α, IL-21, IL-22 and IL-23, using conventional ELISA. RESULTS: CatS activity was significantly elevated in tear fluid, but not other biocompartments, was inversely associated with exocrinic function in pSS patients and could significantly be suppressed by RO5459072. Moreover, CatS inhibition by RO5459072 led to strong and dose-dependent suppression of SS-A/SS-B-specific T cell effector functions and cytokine secretion by CD14+ monocytes. However, RO5459072 was incapable of suppressing SS-A/SS-B-induced secretion of cytokines in CD14+ monocytes when T cells were absent, confirming a CatS/MHCII-mediated mechanism of suppression. CONCLUSION: CatS activity in tear fluid seems to be a relevant biomarker for pSS disease activity. Conversely, CatS inhibition diminishes T cell and associated monokine responses towards relevant autoantigens in pSS. Thus, CatS inhibition may represent a promising novel treatment strategy in pSS.

miR-221-3p Drives the Shift of M2-Macrophages to a Pro-Inflammatory Function by Suppressing JAK3/STAT3 Activation.

Objectives: Macrophages are conventionally classified as pro-inflammatory (M1) and anti-inflammatory (M2) functional types. There is evidence for a predominance of macrophages with an inflammatory phenotype (M1) in the rheumatoid arthritis (RA) synovium. MicroRNAs (miRs) play a pivotal role in regulating the inflammatory response in innate immune cells and are found at dysregulated levels in RA patients. Here we explored miRs that tune the inflammatory function of M2-macrophages. Methods: Expression profiles of miR-221-3p and miR-155-5p were analyzed in clinical samples from RA, other inflammatory arthritis (OIA), osteoarthritis (OA), and healthy donors (HD) by qPCR. In vitro generated macrophages were transfected with miR-mimics and inhibitors. Transcriptome profiling through RNA-sequencing was performed on M2-macrophages overexpressing miR-221-3p mimic with or without LPS treatment. Secretion of IL-6, IL-10, IL-12, IL-8, and CXCL13 was measured in M1- and M2-macrophages upon TLR2/TLR3/TLR4-stimulation using ELISA. Inflammatory pathways including NF-κB, IRF3, MAPKs, and JAK3/STAT3 were evaluated by immunoblotting. Direct target interaction of miR-221-3p and predicted target sites in 3'UTR of JAK3 were examined by luciferase reporter gene assay. Results: miR-221-3p in synovial tissue and fluid was increased in RA vs. OA or OIA. Endogenous expression levels of miR-221-3p and miR-155-5p were higher in M1- than M2-macrophages derived from RA patients or HD. TLR4-stimulation of M1- and M2-macrophages resulted in downregulation of miR-221-3p, but upregulation of miR-155-5p. M2-macrophages transfected with miR-221-3p mimics secreted less IL-10 and CXCL13 but more IL-6 and IL-8, exhibited downregulation of JAK3 protein and decreased pSTAT3 activation. JAK3 was identified as new direct target of miR-221-3p in macrophages. Co-transfection of miR-221-3p/miR-155-5p mimics in M2-macrophages increased M1-specific IL-12 secretion. Conclusions: miR-221-3p acts as a regulator of TLR4-induced inflammatory M2-macrophage function by directly targeting JAK3. Dysregulated miR-221-3p expression, as seen in synovium of RA patients, leads to a diminished anti-inflammatory response and drives M2-macrophages to exhibit a M1-cytokine profile.

TLR2 stimulation impairs anti-inflammatory activity of M2-like macrophages, generating a chimeric M1/M2 phenotype.

BACKGROUND: Toll-like receptors (TLRs) and macrophages play an important role in rheumatoid arthritis (RA). Currently, it is not clear whether inflammatory M1 or anti-inflammatory M2 predominate among the resident macrophages in the synovium. In the present study, we set out to investigate the impact of TLR stimulation on monocyte-derived M1 and M2 macrophage function and phenotype by mimicking the exposure to abundant TLR agonists as occurs in the context of RA. The response of macrophage subsets to TLR2 and TLR4 activation was evaluated on cluster of differentiation (CD) marker profile; cytokine secretion; gene expression; and NF-κB, interferon regulatory factors 3 and 7 (IRF3/7), and mitogen-activated protein kinase (MAPK) activation. METHODS: Human monocytes were isolated from peripheral blood of healthy individuals and patients with RA and differentiated into M1-like and M2-like macrophages by granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF), respectively. Cells were either (1) stimulated with TLR ligands Pam3 or lipopolysaccharide (LPS) or (2) classically activated via interferon (IFN)-γ/LPS. Cytokine production was measured by enzyme-linked immunosorbent assay, and gene expression was measured by qPCR. Cells were stained for CD markers and analyzed by fluorescence-activated cell sorting. NF-κB, IRF3/7, and MAPKs were detected by Western blotting. RESULTS: Monocyte-derived macrophages of healthy donors (HD) or patients with RA displayed comparable subset-specific phenotypes upon exposure to TLR agonists. CD14 and CD163 marker expression on M2 macrophages did not change upon TLR2 and TLR4 engagement. By contrast, M2 gene markers HMOX1, FOLR2, and SLC40A1 were decreased. Importantly, M2 macrophages derived from HD or patients with RA showed both a decreased ratio of interleukin (IL)-10/IL-6 and IL-10/IL-8 upon stimulation with TLR2 ligand Pam3 compared with TLR4 ligand LPS. Gene expression of TLR2 was increased, whereas TLR4 expression was decreased, by TLR ligand stimulation. MAPKs p38, extracellular signal-regulated kinase 1/2, and c-Jun N-terminal kinase were activated more strongly in M2 than in M1 macrophages by Pam3 or LPS. CONCLUSIONS: We show that the anti-inflammatory activity of M2 macrophages is reduced in the presence of abundant TLR2 ligands without significant changes in cell surface markers. Thus, the classical M1/M2 paradigm based on cellular markers does not apply to macrophage functions in inflammatory conditions such as RA.

Role of HTRA1 in bone formation and regeneration: In vitro and in vivo evaluation.

The role of mammalian high temperature requirement protease A1 (HTRA1) in somatic stem cell differentiation and mineralized matrix formation remains controversial, having been demonstrated to impart either anti- or pro-osteogenic effects, depending on the in vitro cell model used. The aim of this study was therefore to further evaluate the role of HTRA1 in regulating the differentiation potential and lineage commitment of murine mesenchymal stem cells in vitro, and to assess its influence on bone structure and regeneration in vivo. Our results demonstrated that short hairpin RNA-mediated ablation of Htra1 in the murine mesenchymal cell line C3H10T1/2 increased the expression of several osteogenic gene markers, and significantly enhanced matrix mineralization in response to BMP-2 stimulation. These effects were concomitant with decreases in the expression of chondrogenic gene markers, and increases in adipogenic gene expression and lipid accrual. Despite the profound effects of loss-of-function of HTRA1 on this in vitro osteochondral model, these were not reproduced in vivo, where bone microarchitecture and regeneration in 16-week-old Htra1-knockout mice remained unaltered as compared to wild-type controls. By comparison, analysis of femurs from 52-week-old mice revealed that bone structure was better preserved in Htra1-knockout mice than age-matched wild-type controls. These findings therefore provide additional insights into the role played by HTRA1 in regulating mesenchymal stem cell differentiation, and offer opportunities for improving our understanding of how this multifunctional protease may act to influence bone quality.

Prostaglandin E2 inhibits matrix mineralization by human bone marrow stromal cell-derived osteoblasts via Epac-dependent cAMP signaling.

The osteoinductive properties of prostaglandin E2 (PGE2) and its signaling pathways have led to suggestions that it may serve as a potential therapeutic strategy for bone loss. However, the prominence of PGE2 as an inducer of bone formation is attributed primarily to findings from studies using rodent models. In the current study, we investigated the effects of PGE2 on human bone marrow stromal cell (hBMSC) lineage commitment and determined its mode of action. We demonstrated that PGE2 treatment of hBMSCs significantly altered the expression profile of several genes associated with osteoblast differentiation (RUNX2 and ALP) and maturation (BGLAP and MGP). This was attributed to the activation of specific PGE2 receptors, and was associated with increases in cAMP production and sustained AKT phosphorylation. Pharmacological inhibition of exchange protein directly activated by cAMP (Epac), but not protein kinase A (PKA), recovered the mineralization functions of hBMSC-derived osteoblasts treated with PGE2 and restored AKT phosphorylation, along with the expression levels of RUNX2, ALP, BGLAP and MGP. Our findings therefore provide insights into how PGE2 influences hBMSC-mediated matrix mineralization, and should be taken into account when evaluating the role of PGE2 in human bone metabolism.

Tailored Ahp-cyclodepsipeptides as Potent Non-covalent Serine Protease Inhibitors.

The S1 serine protease family is one of the largest and most biologically important protease families. Despite their biomedical significance, generic approaches to generate potent, class-specific, bioactive non-covalent inhibitors for these enzymes are still limited. In this work, we demonstrate that Ahp-cyclodepsipeptides represent a suitable scaffold for generating target-tailored inhibitors of serine proteases. For efficient synthetic access, we developed a practical mixed solid- and solution-phase synthesis that we validated through performing the first chemical synthesis of the two natural products Tasipeptin A and B. The suitability of the Ahp-cyclodepsipeptide scaffold for tailored inhibitor synthesis is showcased by the generation of the most potent human HTRA protease inhibitors to date. We anticipate that our approach may also be applied to other serine proteases, thus opening new avenues for a systematic discovery of serine protease inhibitors.

Loss-of-Function of HtrA1 Abrogates All-Trans Retinoic Acid-Induced Osteogenic Differentiation of Mouse Adipose-Derived Stromal Cells Through Deficiencies in p70S6K Activation.

All-trans retinoic acid (ATRA) is a potent inducer of osteogenic differentiation in mouse adipose-derived stromal cells (mASCs), although the underlying mechanisms responsible for its mode of action have yet to be completely elucidated. High temperature requirement protease A1 (HtrA1) is a newly recognized modulator of human multipotent stromal cell (MSC) osteogenesis and as such, may play a role in regulating ATRA-dependent osteogenic differentiation of mASCs. In this study, we assessed the influence of small interfering RNA (siRNA)-induced repression of HtrA1 production on mASC osteogenesis and examined its effects on ATRA-mediated mammalian target of rapamycin (mTOR) signaling. Inhibition of HtrA1 production in osteogenic mASCs resulted in a significant reduction of alkaline phosphatase activity and mineralized matrix formation. Western blot analyses revealed the rapid activation of Akt (Ser473) and p70S6K (Thr389) in ATRA-treated mASCs, and that levels of phosphorylated p70S6K were noticeably reduced in HtrA1-deficient mASCs. Further studies using mTOR inhibitor rapamycin and siRNA specific for the p70S6K gene Rps6kb1 confirmed ATRA-mediated mASC osteogenesis as being dependent on p70S6K activation. Finally, transfection of cells with a constitutively active rapamycin-resistant p70S6K mutant could restore the mineralizing capacity of HtrA1-deficient mASCs. These findings therefore lend further support for HtrA1 as a positive mediator of MSC osteogenesis and provide new insights into the molecular mode of action of ATRA in regulating mASC lineage commitment.

Novel Function of Serine Protease HTRA1 in Inhibiting Adipogenic Differentiation of Human Mesenchymal Stem Cells via MAP Kinase-Mediated MMP Upregulation.

Adipogenesis is the process by which mesenchymal stem cells (MSCs) develop into lipid-laden adipocytes. Being the dominant cell type within adipose tissue, adipocytes play a central role in regulating circulating fatty acid levels, which is considered to be of critical importance in maintaining insulin sensitivity. High temperature requirement protease A1 (HTRA1) is a newly recognized regulator of MSC differentiation, although its role as a mediator of adipogenesis has not yet been defined. The aim of this work was therefore to evaluate HTRA1's influence on human MSC (hMSC) adipogenesis and to establish a potential mode of action. We report that the addition of exogenous HTRA1 to hMSCs undergoing adipogenesis suppressed their ability to develop into lipid laden adipocytes. These effects were demonstrated as being reliant on both its protease and PDZ domain, and were mediated through the actions of c-Jun N-terminal kinase and matrix metalloproteinases (MMPs). The relevance of such findings with regards to HTRA1's potential influence on adipocyte function in vivo is made evident by the fact that HTRA1 and MMP-13 were readily identifiable within crown-like structures present in visceral adipose tissue samples from insulin resistant obese human subjects. These data therefore implicate HTRA1 as a negative regulator of MSC adipogenesis and are suggestive of its potential involvement in adipose tissue remodeling under pathological conditions. Stem Cells 2016;34:1601-1614.

Use of biomimetic microtissue spheroids and specific growth factor supplementation to improve tenocyte differentiation and adaptation to a collagen-based scaffold in vitro.

Tenocytes represent a valuable source of cells for the purposes of tendon tissue engineering and regenerative medicine and as such, should possess a high degree of tenogenic differentiation prior to their use in vivo in order to achieve maximal efficacy. In the current report, we identify an efficient means by which to maintain differentiated tenocytes in vitro by employing the hanging drop technique in combination with defined growth media supplements. Equine tenocytes retained a more differentiated state when cultured as scaffold-free microtissue spheroids in low serum-containing medium supplemented with L-ascorbic acid 2-phosphate, insulin and transforming growth factor (TGF)-ß1. This was made evident by significant increases in the expression levels of pro-tenogenic markers collagen type I (COL1A2), collagen type III (COL3A1), scleraxis (SCX) and tenomodulin (TNMD), as well as by enhanced levels of collagen type I and tenomodulin protein. Furthermore, tenocytes cultured under these conditions demonstrated a typical spindle-like morphology and when embedded in collagen gels, became highly aligned with respect to the orientation of the collagen structure following their migration out from the microtissue spheroids. Our findings therefore provide evidence to support the use of a biomimetic microtissue approach to culturing tenocytes and that in combination with the defined growth media described, can improve their differentiation status and functional repopulation of collagen matrix.

Hyaluronic acid fragments enhance the inflammatory and catabolic response in human intervertebral disc cells through modulation of toll-like receptor 2 signalling pathways.

INTRODUCTION: Intervertebral disc (IVD) degeneration is characterized by extracellular matrix breakdown and is considered to be a primary cause of discogenic back pain. Although increases in pro-inflammatory cytokine levels within degenerating discs are associated with discogenic back pain, the mechanisms leading to their overproduction have not yet been elucidated. As fragmentation of matrix components occurs during IVD degeneration, we assessed the potential involvement of hyaluronic acid fragments (fHAs) in the induction of inflammatory and catabolic mediators. METHODS: Human IVD cells isolated from patient biopsies were stimulated with fHAs (6 to 12 disaccharides) and their effect on cytokine and matrix degrading enzyme production was assessed using quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). The involvement of specific cell surface receptors and signal transduction pathways in mediating the effects of fHAs was tested using small interfering RNA (siRNA) approaches and kinase inhibition assays. RESULTS: Treatment of IVD cells with fHAs significantly increased mRNA expression levels of interleukin (IL)-1ß, IL-6, IL-8, cyclooxygenase (COX)-2, matrix metalloproteinase (MMP)-1 and -13. The stimulatory effects of fHAs on IL-6 protein production were significantly impaired when added to IVD cells in combination with either Toll-like receptor (TLR)-2 siRNA or a TLR2 neutralizing antibody. Furthermore, the ability of fHAs to enhance IL-6 and MMP-3 protein production was found to be dependent on the mitogen-activated protein (MAP) kinase signaling pathway. CONCLUSIONS: These findings suggest that fHAs may have the potential to mediate IVD degeneration and discogenic back pain through activation of the TLR2 signaling pathway in resident IVD cells.

The emerging roles of HTRA1 in musculoskeletal disease.

High-temperature requirement serine protease A1 (HTRA1) is one of four known proteases belonging to the broadly conserved family of HTRA proteins. Although it was originally considered as representing an important modulator of tumorigenesis, an increasing number of reports have suggested that its influence on human disease may extend beyond cancer. HTRA1 has the capacity to degrade numerous extracellular matrix proteins, and as such, its potential involvement in diseases of the musculoskeletal system has been gaining increased attention. Musculoskeletal disease constitutes a wide variety of degenerative conditions that can manifest themselves in different ways such as joint and back pain, as well as deficiencies in skeletal bone quality, and ultimately result in significant suffering and reduced quality of life. Convincing data now exist to support a detrimental role for HTRA1 in the pathogenesis of joint and intervertebral disk degeneration. However, the function of HTRA1 in other closely related musculoskeletal diseases affecting bone and muscle remains unclear and largely unexplored. To help set the stage for future research, we discuss here some of the recent advances in our understanding of the role played by HTRA1 in musculoskeletal pathology.

Preparation and osteogenic differentiation of scaffold-free mouse adipose-derived stromal cell microtissue spheroids (ASC-MT).

In this unit, previously described methods are expanded upon, where procedures relating to the preparation, culturing, and osteogenic differentiation of scaffold-free mouse adipose-derived stromal cell microtissue spheroids (ASC-MT) are outlined. Not only is a detailed methodology of how to engineer such spheroids are presented, but a full account of how to induce and analyze osteogenesis in these ASC-MT constructs is given along with relevant figures to help better illustrate the methods described.

The Legionella pneumophila orphan sensor kinase LqsT regulates competence and pathogen-host interactions as a component of the LAI-1 circuit.

Legionella pneumophila is an amoeba-resistant opportunistic pathogen that performs cell-cell communication through the signalling molecule 3-hydroxypentadecane-4-one (LAI-1, Legionella autoinducer-1). The lqs (Legionella quorum sensing) gene cluster encodes the LAI-1 autoinducer synthase LqsA, the cognate sensor kinase LqsS and the response regulator LqsR. Here we show that the Lqs system includes an 'orphan' homologue of LqsS termed LqsT. Compared with wild-type L. pneumophila, strains lacking lqsT or both lqsS and lqsT show increased salt resistance, greatly enhanced natural competence for DNA acquisition and impaired uptake by phagocytes. Sensitive novel single round growth assays and competition experiments using Acanthamoeba castellanii revealed that ΔlqsT and ΔlqsS-ΔlqsT, as well as ΔlqsA and other lqs mutant strains are impaired for intracellular growth and cannot compete against wild-type bacteria upon co-infection. In contrast to the ΔlqsS strain, ΔlqsT does not produce extracellular filaments. The phenotypes of the ΔlqsS-ΔlqsT strain are partially complemented by either lqsT or lqsS, but are not reversed by overexpression of lqsA, suggesting that LqsT and LqsS are the sole LAI-1-responsive sensor kinases in L. pneumophila. In agreement with the different phenotypes of the ΔlqsT and ΔlqsS strains, lqsT and lqsS are differentially expressed in the post-exponential growth phase, and transcriptome studies indicated that 90% of the genes, which are downregulated in absence of lqsT, are upregulated in absence of lqsS. Reciprocally regulated genes encode components of a 133 kb genomic 'fitness island' or translocated effector proteins implicated in virulence. Together, these results reveal a unique organization of the L. pneumophila Lqs system comprising two partially antagonistic LAI-1-responsive sensor kinases, LqsT and LqsS, which regulate distinct pools of genes implicated in pathogen-host cell interactions, competence, expression of a genomic island or production of extracellular filaments.

Analysis of legionella infection by flow cytometry.

Legionella pneumophila infects and replicates in environmental protozoa and metazoan macrophages within a specific vacuole. The infection of phagocytes by L. pneumophila can be assessed by an agar plating assay or by fluorescence microscopy. Here, we describe the analysis of Legionella infection by automated flow cytometry using wild-type and mutant bacteria that constitutively produce the green fluorescent protein (GFP). Advantages of the flow cytometry technique include (1) a software-assisted multiple parameter analysis of Legionella infections in real-time at distinct stages of the infection cycle, (2) the simultaneous and fast acquisition of a high number of data points, and (3) a characterization of the infecting bacteria in parallel with the infected host cells.

The dynamics of natural Plasmodium falciparum infections.

BACKGROUND: Natural immunity to Plasmodium falciparum has been widely studied, but its effects on parasite dynamics are poorly understood. Acquisition and clearance rates of untreated infections are key elements of the dynamics of malaria, but estimating these parameters is challenging because of frequent super-infection and imperfect detectability of parasites. Consequently, information on effects of host immune status or age on infection dynamics is fragmentary. METHODS: An age-stratified cohort of 347 individuals from Northern Ghana was sampled six times at 2 month intervals. High-throughput capillary electrophoresis was used to genotype the msp-2 locus of all P. falciparum infections detected by PCR. Force of infection (FOI) and duration were estimated for each age group using an immigration-death model that allows for imperfect detection of circulating parasites. RESULTS: Allowing for imperfect detection substantially increased estimates of FOI and duration. Effects of naturally acquired immunity on the FOI and duration would be reflected in age dependence in these indices, but in our cohort data FOI tended to increase with age in children. Persistence of individual parasite clones was characteristic of all age-groups. Duration peaked in 5-9 year old children (average duration 319 days, 95% confidence interval 318;320). CONCLUSIONS: The main age-dependence is on parasite densities, with only small age-variations in the FOI and persistence of infections. This supports the hypothesis that acquired immunity controls transmission mainly by limiting blood-stage parasite densities rather than changing rates of acquisition or clearance of infections.

Human serine protease HTRA1 positively regulates osteogenesis of human bone marrow-derived mesenchymal stem cells and mineralization of differentiating bone-forming cells through the modulation of extracellular matrix protein.

Mammalian high-temperature requirement serine protease A1 (HTRA1) is a secreted member of the trypsin family of serine proteases which can degrade a variety of bone matrix proteins and as such has been implicated in musculoskeletal development. In this study, we have investigated the role of HTRA1 in mesenchymal stem cell (MSC) osteogenesis and suggest a potential mechanism through which it controls matrix mineralization by differentiating bone-forming cells. Osteogenic induction resulted in a significant elevation in the expression and secretion of HTRA1 in MSCs isolated from human bone marrow-derived MSCs (hBMSCs), mouse adipose-derived stromal cells (mASCs), and mouse embryonic stem cells. Recombinant HTRA1 enhanced the osteogenesis of hBMSCs as evidenced by significant changes in several osteogenic markers including integrin-binding sialoprotein (IBSP), bone morphogenetic protein 5 (BMP5), and sclerostin, and promoted matrix mineralization in differentiating bone-forming osteoblasts. These stimulatory effects were not observed with proteolytically inactive HTRA1 and were abolished by small interfering RNA against HTRA1. Moreover, loss of HTRA1 function resulted in enhanced adipogenesis of hBMSCs. HTRA1 Immunofluorescence studies showed colocalization of HTRA1 with IBSP protein in osteogenic mASC spheroid cultures and was confirmed as being a newly identified HTRA1 substrate in cell cultures and in proteolytic enzyme assays. A role for HTRA1 in bone regeneration in vivo was also alluded to in bone fracture repair studies where HTRA1 was found localized predominantly to areas of new bone formation in association with IBSP. These data therefore implicate HTRA1 as having a central role in osteogenesis through modification of proteins within the extracellular matrix.

α-Hydroxyketone synthesis and sensing by Legionella and Vibrio.

Bacteria synthesize and sense low molecular weight signaling molecules, termed autoinducers, to measure their population density and community complexity. One class of autoinducers, the α-hydroxyketones (AHKs), is produced and detected by the water-borne opportunistic pathogens Legionella pneumophila and Vibrio cholerae, which cause Legionnaires' disease and cholera, respectively. The "Legionella quorum sensing" (lqs) or "cholera quorum sensing" (cqs) genes encode enzymes that produce and sense the AHK molecules "Legionella autoinducer-1" (LAI-1; 3-hydroxypentadecane-4-one) or cholera autoinducer-1 (CAI-1; 3-hydroxytridecane-4-one). AHK signaling regulates the virulence of L. pneumophila and V. cholerae, pathogen-host cell interactions, formation of biofilms or extracellular filaments, expression of a genomic "fitness island" and competence. Here, we outline the processes, wherein AHK signaling plays a role, and review recent insights into the function of proteins encoded by the lqs and cqs gene clusters. To this end, we will focus on the autoinducer synthases catalysing the biosynthesis of AHKs, on the cognate trans-membrane sensor kinases detecting the signals, and on components of the down-stream phosphorelay cascade that promote the transmission and integration of signaling events regulating gene expression.