Hepcidin induces HIV-1 transcription inhibited by ferroportin

<p>Abstract</p> <p>Background</p> <p>Physiological regulation of cellular iron involves iron export by the membrane protein, ferroportin, the expression of which is induced by iron and negatively modulated by hepcidin. We previously showed that iron chelation is associated with decreased HIV-1 transcription. We hypothesized that increased iron export by ferroportin might be associated with decreased HIV-1 transcription, and degradation of ferroportin by hepcidin might in turn induce HIV-1 transcription and replication. Here, we analyzed the effect of ferroportin and hepcidin on HIV-1 transcription.</p> <p>Results</p> <p>Expression of ferroportin was associated with reduced HIV-1 transcription in 293T cells and addition of hepcidin to ferroportin-expressing cells counteracted this effect. Furthermore, exposure of promonocytic THP-1 cells to hepcidin was associated with decreased ferroportin expression, increased intracellular iron and induction of reporter luciferase gene expression. Finally, exposure of human primary macrophages and CD4⁺T cells to hepcidin and iron was also associated with induction of viral production.</p> <p>Conclusion</p> <p>Our results suggest that the interplay between ferroportin-mediated iron export and hepcidin-mediated degradation of ferroportin might play a role in the regulation of HIV-1 transcription and may be important for understanding of HIV-1 pathogenesis.</p

Diversity of virulence phenotypes among type III secretion negative Pseudomonas aeruginosa clinical isolates.

Pseudomonas aeruginosa is a frequent cause of acute infections. The primary virulence factor that has been linked to clinical disease is the type III secretion system, a molecular syringe that delivers effector proteins directly into host cells. Despite the importance of type III secretion in dictating clinical outcomes and promoting disease in animal models of infections, clinical isolates often do not express the type III secretion system in vitro. Here we screened 81 clinical P. aeruginosa isolates for secretion of type III secretion system substrates by western blot. Non-expressing strains were also subjected to a functional test assaying the ability to intoxicate epithelial cells in vitro, and to survive and cause disease in a murine model of corneal infection. 26 of 81 clinical isolates were found to be type III secretion negative by western blot. 17 of these 26 non-expressing strains were tested for their ability to cause epithelial cell rounding. Of these, three isolates caused epithelial cell rounding in a type III secretion system dependent manner, and one strain was cytotoxic in a T3SS-independent manner. Five T3SS-negative isolates were also tested for their ability to cause disease in a murine model of corneal infection. Of these isolates, two strains caused severe corneal disease in a T3SS-independent manner. Interestingly, one of these strains caused significant disease (inflammation) despite being cleared. Our data therefore show that P. aeruginosa clinical isolates can cause disease in a T3SS-independent manner, demonstrating the existence of novel modifiers of clinical disease

Diversity of virulence phenotypes among type III secretion negative Pseudomonas aeruginosa clinical isolates.

Pseudomonas aeruginosa is a frequent cause of acute infections. The primary virulence factor that has been linked to clinical disease is the type III secretion system, a molecular syringe that delivers effector proteins directly into host cells. Despite the importance of type III secretion in dictating clinical outcomes and promoting disease in animal models of infections, clinical isolates often do not express the type III secretion system in vitro. Here we screened 81 clinical P. aeruginosa isolates for secretion of type III secretion system substrates by western blot. Non-expressing strains were also subjected to a functional test assaying the ability to intoxicate epithelial cells in vitro, and to survive and cause disease in a murine model of corneal infection. 26 of 81 clinical isolates were found to be type III secretion negative by western blot. 17 of these 26 non-expressing strains were tested for their ability to cause epithelial cell rounding. Of these, three isolates caused epithelial cell rounding in a type III secretion system dependent manner, and one strain was cytotoxic in a T3SS-independent manner. Five T3SS-negative isolates were also tested for their ability to cause disease in a murine model of corneal infection. Of these isolates, two strains caused severe corneal disease in a T3SS-independent manner. Interestingly, one of these strains caused significant disease (inflammation) despite being cleared. Our data therefore show that P. aeruginosa clinical isolates can cause disease in a T3SS-independent manner, demonstrating the existence of novel modifiers of clinical disease

Reduced sensitivity of the ferroportin Q248H mutant to physiological concentrations of hepcidin

Ferroportin Q248H mutation has an allele frequency of 2.2-13.4% in African populations and is associated with a mild tendency to increased serum ferritin in the general population. Some investigators have reported that ferroportin Q248H is degraded after exposure to hepcidin in exactly the same manner as wild-type ferroportin, but supraphysiological concentrations of hepcidin were used. The aim of our study was to determine whether ferroportin Q248H may have reduced sensitivity to physiological concentrations of hepcidin. The sensitivity of ferroportin Q248H to hepcidin was determined in 293T cells transiently expressing ferroportin using immunoblotting and fluorescence analysis. Ferritin concentrations were measured in these cells and also in human primary monocytes derived from humans with different ferroportin genotypes. The effect of Q248H on serum iron measures was examined in patients with sickle cell anemia. Immunoblotting and fluorescence analysis showed decreased sensitivity of ferroportin Q248H to physiological concentrations of hepcidin. Lower ferritin concentrations were observed after incubation with iron and hepcidin in 293T cells expressing ferroportin Q248H and in primary monocytes from ferroportin Q248H subjects. In sickle cell anemia, ferroportin Q248H heterozygotes had lower serum ferritin concentrations than wild-type subjects, consistent with enhanced iron release by macrophage ferroportin Q248H. A clinical benefit of ferroportin Q248H was suggested by lower echocardiographic estimates of pulmonary artery pressure in patients carrying mutant alleles. In conclusion, our results suggest that ferroportin Q248H protein is resistant to physiological concentrations of hepcidin and that this mutation has discernible effects on iron metabolism-related clinical complications of sickle cell anemia. They provide a mechanistic explanation for the effect of ferroportin Q248H on iron status in individuals of African descent and suggest that these changes in iron metabolism may be beneficial under certain disease-specific circumstances. (ClinicalTrials.gov Identifier:NCT00011648). © 2013 Ferrata Storti Foundation

Effector expression by clinical <i>P. aeruginosa</i> isolates.

<p>A) A representative western blot showing effector (ExoS, ExoT and ExoU) as well as translocator (PopB and PopD) production in clinical isolates of <i>P. aeruginosa</i>. Protein supernatants were isolated from the indicated <i>P. aeruginosa</i> strain under T3SS inducing conditions (low calcium) and concentrated by precipitation with trichloroacetic acid before separating the proteins by SDS-PAGE and probing for the presence of the indicated effector and translocator proteins by western blot. B) Distribution of T3SS effector-positive and negative bacteria by site of isolation. C) Total distribution of produced effectors by T3SS effector-positive strains. D) Distribution of effectors produced by site from which the strains were isolated.</p

Regulation of HIV-1 transcription at 3% versus 21% oxygen concentration

HIV transcription is induced by the HIV-1 Tat protein, in concert with cellular co-factors including CDK9, CDK2, NF-κB, and others. The cells of most of the body\u27s organs are exposed to ∼3-6% oxygen, but most in vitro studies of HIV replication are conducted at 21% oxygen. We hypothesized that activities of host cell factors involved in HIV-1 replication may differ at 3% versus 21% O2, and that such differences may affect HIV-1 replication. Here we show that Tat-induced HIV-1 transcription was reduced at 3% O2 compared to 21% O2. HIV-1 replication was also reduced in acutely or chronically infected cells cultured at 3% O2 compared to 21% O2. This reduction was not due the decreased cell growth or increased cellular toxicity and also not due to the induction of hypoxic response. At 3% O2, the activity of CDK9/cyclin T1 was inhibited and Sp1 activity was reduced, whereas the activity of other host cell factors such as CDK2 or NF-κB was not affected. CDK9-specific inhibitor ARC was much less efficient at 3% compared to 21% O2 and also expression of CDK9/cyclin T1-dependent IκB inhibitor a was repressed. Our results suggest that lower HIV-1 transcription at 3% O2 compared to 21% O2 may be mediated by lower activity of CDK9/cyclin T1 and Sp1 at 3% O2 and that additional host cell factors such as CDK2 and NF-κB might be major regulators of HIV-1 transcription at low O2 concentrations. © 2009 Wiley-Liss, Inc

Intoxication of A549 lung epithelial cells by effector-negative clinical isolates of <i>P. aeruginosa</i>.

<p>Clinical isolates that do not secrete detectable levels of known effectors and translocators <i>in vitro</i> as well as defined T3SS null derivatives (Δ<i>popD</i>, Δ<i>pscD</i>, where the entire open reading frame was removed by an in-frame deletion, or <i>pscC</i>-, where the <i>pscC</i> open reading frame was disrupted by the insertion of a non-replicating plasmid) were tested for their ability to intoxicate A549 epithelial cells in a T3SS-dependent manner. Delivery of effector proteins was measured by assaying rounding of A549 cells by microscopic examination. Data presented is the mean of three independent experiments with standard deviation (error bar). A) Isolates with no significant cytotoxicity. B) Isolates that are cytotoxic in a T3SS-dependent manner. C) Isolate JT87 has no detectable T3SS-related genes by PCR. Representative phase contrast images of A549 cells infected with PAO1, PAO1 Δ<i>pscD</i> or JT87 are shown to the right of the graph in panel C. ** p<0.01, Student’s T-test.</p

Virulence of effector-negative clinical isolates of <i>P. aeruginosa</i>.

<p>Corneas of C57BL/6 mice were scarified and infected with 2*10^∧5 CFU/eye. A) Images of infected eyes were taken at 24 h and 48 h post-infection to assess corneal opacification due to infiltration of neutrophils. Corneal opacification was quantitated digitally using Metamorph software as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086829#pone.0086829-Sun1" target="_blank">[11]</a>. Opacity scores for individual eyes were plotted. The median value with interquartile range is indicated. Representative images of infected corneas are shown to the left of the graph. Statistical significance of differences was determined by Mann-Whitney test: ** p<0.01, * p<0.05 B) Mice were infected with 2*10^∧5 CFU/eye using the scratch model of corneal infection. Bacterial load (CFU/eye) was determined 48 h after infection by euthanizing the mice, removing the infected eye, homogenizing it and plating serial dilutions on BHI agar plates. Bacterial loads for individual eyes were plotted. The median value with interquartile range is indicated. ** p<0.01, * p<0.05, Mann-Whitney test.</p