Myeloid ABCG1 Deficiency Enhances Apoptosis and Initiates Efferocytosis in Bronchoalveolar Lavage Cells of Murine Multi-Walled Carbon Nanotube-Induced Granuloma Model

The use of carbon nanotubes has increased in the past few decades. Carbon nanotubes are implicated in the pathogenesis of pulmonary sarcoidosis, a chronic granulomatous inflammatory condition. We developed a murine model of chronic granulomatous inflammation using multiwall carbon nanotubes (MWCNT) to investigate mechanisms of granuloma formation. Using this model, we demonstrated that myeloid deficiency of ATP-binding cassette (ABC) cholesterol transporter (ABCG1) promotes granuloma formation and fibrosis with MWCNT instillation; however, the mechanism remains unclear. Our previous studies showed that MWCNT induced apoptosis in bronchoalveolar lavage (BAL) cells of wild-type (C57BL/6) mice. Given that continual apoptosis causes persistent severe lung inflammation, we hypothesized that ABCG1 deficiency would increase MWCNT-induced apoptosis thereby promoting granulomatous inflammation and fibrosis. To test our hypothesis, we utilized myeloid-specific ABCG1 knockout (ABCG1 KO) mice. Our results demonstrate that MWCNT instillation enhances pulmonary fibrosis in ABCG1 KO mice compared to wild-type controls. Enhanced fibrosis is indicated by increased trichrome staining and transforming growth factor-beta (TGF-β) expression in lungs, together with an increased expression of TGF-β related signaling molecules, interleukin-13 (IL-13) and Smad-3. MWCNT induced more apoptosis in BAL cells of ABCG1 KO mice. Initiation of apoptosis is most likely mediated by the extrinsic pathway since caspase 8 activity and Fas expression are significantly higher in MWCNT instilled ABCG1 KO mice compared to the wild type. In addition, TUNEL staining shows that ABCG1 KO mice instilled with MWCNT have a higher percentage of TUNEL positive BAL cells and more efferocytosis than the WT control. Furthermore, BAL cells of ABCG1 KO mice instilled with MWCNT exhibit an increase in efferocytosis markers, milk fat globule-EGF factor 8 (MFG-E8) and integrin β3. Therefore, our observations suggest that ABCG1 deficiency promotes pulmonary fibrosis by MWCNT, and this effect may be due to an increase in apoptosis and efferocytosis in BAL cells

Diverse Genetic Regulon of the Virulence-Associated Transcriptional Regulator MucR in Brucella abortus 2308

The Ros-type regulator MucR is one of the few transcriptional regulators that have been linked to virulence in Brucella. Here, we show that a Brucella abortus in-frame mucR deletion strain exhibits a pronounced growth defect during in vitro cultivation and, more importantly, that the mucR mutant is attenuated in cultured macrophages and in mice. The genetic basis for the attenuation of Brucella mucR mutants has not been defined previously, but in the present study the genes regulated by MucR in B. abortus have been elucidated using microarray analysis and real-time reverse transcription-PCR (RT-PCR). In B. abortus 2308, MucR regulates a wide variety of genes whose products may function in establishing and maintaining cell envelope integrity, polysaccharide biosynthesis, iron homeostasis, genome plasticity, and transcriptional regulation. Particularly notable among the MucR-regulated genes identified is arsR6 (nolR), which encodes a transcriptional regulator previously linked to virulence in Brucella melitensis 16 M. Importantly, electrophoretic mobility shift assays (EMSAs) determined that a recombinant MucR protein binds directly to the promoter regions of several genes repressed by MucR (including arsR6 [nolR]), and in Brucella, as in other alphaproteobacteria, MucR binds to its own promoter to repress expression of the gene that encodes it. Overall, these studies have uncovered the diverse genetic regulon of MucR in Brucella, and in doing so this work has begun to define the MucR-controlled genetic circuitry whose misregulation contributes to the virulence defect of Brucella mucR mutants

Characterization of FtrABCD: a ferrous iron-specific transporter that is required for the virulence of Brucella abortus 2308 in mice

Brucella strains are Gram-negative bacteria that cause abortion and infertility in their natural animal hosts resulting in extensive economic losses. Humans can acquire a chronic, debilitating febrile illness known as brucellosis as the result of contact with infected animals or consumption of their products. The brucellae live mainly inside phagosomal compartments in host macrophages where they struggle to acquire enough of the essential micronutrient iron to meet their physiological needs. Brucella spp. have evolved multiple strategies to overcome iron limitation in their mammalian hosts, including siderophore-dependent ferric iron-specific transport and heme acquisition systems. The work presented in this dissertation characterized a ferrous iron (Fe2+) specific transporter encoded by the genes designated bab2_0837-0840 in the Brucella abortus 2308 genome sequence. This transporter is homologous to the FtrABCD transporter recently described in Bordetella. Interestingly, this transporter is required for the virulence of B. abortus 2308 in experimentally infected mice, supporting the proposition that ferrous iron is a critical iron source for these bacteria in the mammalian host. The iron response regulator (Irr), the predominant iron-responsive regulator in Brucella and the other alpha-proteobacteria, activates the expression of the ftrABCD operon in response to iron deprivation by binding a consensus Irr binding site in the promoter region of these genes. These genes are also activated by exposure to acidic pH, which is important because it potentially allows the brucellae to fine-tune the expression of their iron acquisition genes to adapt to the acidic environment they encounter in the phagosomal compartment of host macrophages, where Fe2+ is thought to be a biologically relevant iron source. The ftr locus is also regulated by the Zn finger protein MucR which belongs to the Ros/MucR family of transcriptional regulators that function as global transcriptional regulators in the [alpha]-proteobacteria. MucR has a secondary role in regulating the expression of the ftr genes in B. abortus 2308 by binding to a region very near to the Irr binding site and the acid-responsive regulatory element in the promoter region of these genes. MucR potentially interacts with Irr and the as yet unidentified acid-responsive transcriptional regulator, thus modulating the capacity of these regulators to bind to their target sites in the promoter region of the ftr locus. This type of secondary regulatory function is consistent with what has described for several MucR-regulated genes in other [alpha]-proteobacteria. In conclusion, the work presented in this dissertation showed that the ferrous iron specific transporter FtrABCD is required for Brucella abortus 2308 to survive and replicate in the host macrophage where Fe2+ is an essential and relevant micronutrient. The regulation of the expression of the ftrABCD genes by Irr, the acid-responsive transcriptional regulator and MucR is important because it potentially allows the brucellae to adapt to the environmental niches they inhabit during their residence in host macrophages

Characterization of FtrABCD: a ferrous iron-specific transporter that is required for the virulence of Brucella abortus 2308 in mice

Brucella strains are Gram-negative bacteria that cause abortion and infertility in their natural animal hosts resulting in extensive economic losses. Humans can acquire a chronic, debilitating febrile illness known as brucellosis as the result of contact with infected animals or consumption of their products. The brucellae live mainly inside phagosomal compartments in host macrophages where they struggle to acquire enough of the essential micronutrient iron to meet their physiological needs. Brucella spp. have evolved multiple strategies to overcome iron limitation in their mammalian hosts, including siderophore-dependent ferric iron-specific transport and heme acquisition systems. The work presented in this dissertation characterized a ferrous iron (Fe2+) specific transporter encoded by the genes designated bab2_0837-0840 in the Brucella abortus 2308 genome sequence. This transporter is homologous to the FtrABCD transporter recently described in Bordetella. Interestingly, this transporter is required for the virulence of B. abortus 2308 in experimentally infected mice, supporting the proposition that ferrous iron is a critical iron source for these bacteria in the mammalian host. The iron response regulator (Irr), the predominant iron-responsive regulator in Brucella and the other alpha-proteobacteria, activates the expression of the ftrABCD operon in response to iron deprivation by binding a consensus Irr binding site in the promoter region of these genes. These genes are also activated by exposure to acidic pH, which is important because it potentially allows the brucellae to fine-tune the expression of their iron acquisition genes to adapt to the acidic environment they encounter in the phagosomal compartment of host macrophages, where Fe2+ is thought to be a biologically relevant iron source. The ftr locus is also regulated by the Zn finger protein MucR which belongs to the Ros/MucR family of transcriptional regulators that function as global transcriptional regulators in the [alpha]-proteobacteria. MucR has a secondary role in regulating the expression of the ftr genes in B. abortus 2308 by binding to a region very near to the Irr binding site and the acid-responsive regulatory element in the promoter region of these genes. MucR potentially interacts with Irr and the as yet unidentified acid-responsive transcriptional regulator, thus modulating the capacity of these regulators to bind to their target sites in the promoter region of the ftr locus. This type of secondary regulatory function is consistent with what has described for several MucR-regulated genes in other [alpha]-proteobacteria. In conclusion, the work presented in this dissertation showed that the ferrous iron specific transporter FtrABCD is required for Brucella abortus 2308 to survive and replicate in the host macrophage where Fe2+ is an essential and relevant micronutrient. The regulation of the expression of the ftrABCD genes by Irr, the acid-responsive transcriptional regulator and MucR is important because it potentially allows the brucellae to adapt to the environmental niches they inhabit during their residence in host macrophages

Ultra performance liquid chromatography-tandem mass spectrometeric analysis of ethyl acetate fraction from saudi Lavandula coronopifolia Poir and evaluation of its cytotoxic and antioxidant activities

Introduction: The ethyl acetate fraction of the Saudi Lavandula coronopifolia Poir has been previously reported to have hepatoprotective activity against ethanol-induced oxidative stress. The aim of the current study was to investigate the chemical composition, cytotoxic effect, and antioxidant activities of ethyl acetate fraction of the aerial parts of Saudi L. coronopifolia Poir. Methods: Air dried aerial parts of L. coronopifolia were extracted using 90% ethyl alcohol. The dried extract was suspended in water, defatted with light petroleum and then fractionated with ethyl acetate. The ethyl acetate fraction was subjected to ultra performance liquid chromatography-tandem mass spectrometeric (UPLC-ESI/MS/MS) analysis in a negative ionization mode. The antioxidant activity of the fraction was determined using free radical 2,2-diphyenyl-picrylhydrazyl (DPPH) scavenging assay and its cytotoxic effect against HepG2 (human hepatocarcinoma) and MCF-7 (human breast carcinoma) cells were determined using (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium (MTT) cell viability assay. Results: The major components of the ethyl acetate fraction included carvacrol-O-diglucoside, (34.98%) and trihydroxy ursolic acid (12.07%). Moreover, the DPPH radical scavenging activity of ethyl acetate fraction was measured. The ethyl acetate fraction revealed an antioxidant potential with EC50 17.8 ± 1.3 µg/mL. Additionally, he ethyl acetate fraction showed cytotoxic activity against HepG-2 and MCF-7 cells with IC50=29.3 ± 0.9 µg/mL and 14.6 ± 0.3 µg/mL, respectively. Conclusion: The ethyl acetate fraction of the Saudi L. coronopifolia has antioxidant activity and also cytotoxic activity against breast and liver cancer cells