Hepcidin deficiency and iron deficiency do not alter tuberculosis susceptibility in a murine M.tb infection model.

Tuberculosis (TB), caused by the macrophage-tropic pathogen Mycobacterium tuberculosis (M.tb) is a highly prevalent infectious disease. Since an immune correlate of protection or effective vaccine have yet to be found, continued research into host-pathogen interactions is important. Previous literature reports links between host iron status and disease outcome for many infections, including TB. For some extracellular bacteria, the iron regulatory hormone hepcidin is essential for protection against infection. Here, we investigated hepcidin (encoded by Hamp1) in the context of murine M.tb infection. Female C57BL/6 mice were infected with M.tb Erdman via aerosol. Hepatic expression of iron-responsive genes was measured by qRT-PCR and bacterial burden determined in organ homogenates. We found that hepatic Hamp1 mRNA levels decreased post-infection, and correlated with a marker of BMP/SMAD signalling pathways. Next, we tested the effect of Hamp1 deletion, and low iron diets, on M.tb infection. Hamp1 knockout mice did not have a significantly altered M.tb mycobacterial load in either the lungs or spleen. Up to 10 weeks of dietary iron restriction did not robustly affect disease outcome despite causing iron deficiency anaemia. Taken together, our data indicate that unlike with many other infections, hepcidin is decreased following M.tb infection, and show that hepcidin ablation does not influence M.tb growth in vivo. Furthermore, because even severe iron deficiency did not affect M.tb mycobacterial load, we suggest that the mechanisms M.tb uses to scavenge iron from the host must be extremely efficient, and may therefore represent potential targets for drugs and vaccines

Antiviral activity of bone morphogenetic proteins and activins

Understanding the control of viral infections is of broad importance. Chronic hepatitis C virus (HCV) infection causes decreased expression of the iron hormone hepcidin, which is regulated by hepatic bone morphogenetic protein (BMP)/SMAD signalling. We found that HCV infection and the BMP/SMAD pathway are mutually antagonistic. HCV blunted induction of hepcidin expression by BMP6, probably via tumour necrosis factor (TNF)-mediated downregulation of the BMP co-receptor haemojuvelin. In HCV-infected patients, disruption of the BMP6/hepcidin axis and genetic variation associated with the BMP/SMAD pathway predicted the outcome of infection, suggesting that BMP/SMAD activity influences antiviral immunity. Correspondingly, BMP6 regulated a gene repertoire reminiscent of type I interferon (IFN) signalling, including upregulating interferon regulatory factors (IRFs) and downregulating an inhibitor of IFN signalling, USP18. Moreover, in BMP-stimulated cells, SMAD1 occupied loci across the genome, similar to those bound by IRF1 in IFN-stimulated cells. Functionally, BMP6 enhanced the transcriptional and antiviral response to IFN, but BMP6 and related activin proteins also potently blocked HCV replication independently of IFN. Furthermore, BMP6 and activin A suppressed growth of HBV in cell culture, and activin A inhibited Zika virus replication alone and in combination with IFN. The data establish an unappreciated important role for BMPs and activins in cellular antiviral immunity, which acts independently of, and modulates, IFN

Interactions of HIV-1 and Hepatitis C virus with iron metabolism

Iron is essential for almost all biological systems and its metabolism is perturbed during infection. Clinical evidence suggests that elevated iron status correlates with a poor prognosis in HIV-1 infection. Four steps of the HIV-1 life cycle are thought to be iron dependent: reverse transcription, transcription, nuclear export of mRNA and virion assembly. The HIV protein Nef downregulates the haemochromatosis protein HFE in vitro, and iron chelators can inhibit HIV-1 replication. Given the evidence that iron can affect HIV -1 disease course and replication we investigated HIV -1 replication in monocyte-derived macrophages (MDMs) from both HFE wild type and C282Y-haemochromatosis patients. Our data from multiple patients suggests that the extent of HIV -1 replication is independent of HFE genotype in MDMs. Supplementation with the iron chelator DFO and the iron salt F AC both inhibited HIV-1 replication. Infecting differentiated U937 cells as a model system to avoid person to person variation gave inconsistent results when different viral strains and-experimental conditions were used. Changes in iron distribution during HIV-1 infection may be mediated by hepcidin. Hepcidin can be produced as part of the innate immune response which is mediated by the recognition of pathogen- associated molecular patterns (PAMPs) by a family of proteins including the Toll-like receptors (TLRs). We found that HIV-derived TLR7/8 ligands that stimulate the production ofTNFa. and IL6 in PBMCs and neutrophils does not result in a significant change in hepcidin expression whereas flagellin, a TLR5 ligand, induces TNFa., IL6 and hepcidin expression. This suggests that the changes in iron distribution may be more strongly influenced by IL6-mediated hepcidin induction in the liver or more directly by bacterial products that can cross the compromised gut mucosa during HIV-1 infection. Iron accumulation is an important eo-morbidity factor in Hepatitis C virus (HCV) infection. Chronic HCV patients have inappropriately low hepcidin expression which may explain their iron overload. Reduced hepcidin also causes the iron loading disorder hereditary haemochromatosis (HH). Genetic mutations underlying HH disrupt bone morphogenetic protein (BMP) dependent signalling pathways that control hepcidin synthesis. We investigated whether HCV was affecting the BMP/SMAD pathway, dampening down the signal for hepcidin induction, using an infectious HCV in vitro model. We found hepcidin induction by BMP6 but not BMP9 to be impaired by HCV infection. HJV, the BMP6 coreceptor, is downregulated and both SMAD6 and SMAD7 which suppress BMP signalling are upregulated in HCV infected cultures. HCV also increases expression of TNFa., and in vitro the inhibitory effect of HCV on BMP signalling can be mimicked by TNFa. In addition, supplementing cultures with an anti- TNFa. antibody restored hepcidin expression in response to BMP6. These observations were followed up in HCV patient liver biopsies from two separate cohorts. Both cohorts had lower hepcidin mRNA expression compared to a control group, consistent with other published clinical studies. HJV and ID1 mRNA was suppressed in biopsies from non-responders in the cohort with more severe disease whereas SMAD6 mRNA expression was raised in the biopsies from the cohort with mild disease. This demonstrates that the BMP/SMAD pathway can be disrupted by HCV infection and may explain the inappropriately low hepcidin expression found in vivo. Understanding the mechanism behind the hepcidin suppression found in HCV patients may suggest new therapies to prevent their iron overload thereby slowing their progression to cirrhosis and hepatocellular carcinoma.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Investigating zero transmission of HIV in the MSM population: a UK modelling case study

Abstract Background UNAIDS 90-90-90 goals for HIV have been surpassed in the UK, with focus now moving to ending transmission by 2030. The concept of zero transmission is complex and many factors can influence transmission. We aimed to investigate how the target of zero transmission might be reached in the UK. Methods We developed a de novo Markov state transition open cohort model of HIV with a 50-year time horizon, which models six key screening, treatment and prevention parameters, including treatment-as-prevention (TasP) and pre-exposure prophylaxis (PrEP). We studied the anticipated HIV epidemic trajectory over time in men who have sex with men (MSM), with and without changing the six key parameters, defining zero transmission as a 60% reduction in incidence compared with 2010 incidence. Results Zero transmission in the MSM population was not achieved within the model’s time horizon in our base case scenario, when the six key parameters were set to their 2019 values. Several future scenarios were explored, including a combination approach to preventing HIV transmission through increasing five key parameter values and considering three different TasP values; zero transmission was achieved by 2030 in the scenario where TasP was increased from its current level of 97–99%, avoiding 48,969 new HIV cases over the time horizon and reducing the lifetime risk of acquiring HIV for HIV-negative MSM not using PrEP from 13.65 to 7.53%. Conclusions Zero transmission in the UK MSM population can be reached by the target year of 2030 with bold changes to HIV policy. A combination approach such as the UK Government’s ‘Towards Zero’ Action plan, impacting multiple policies and including an increase in TasP, has the potential to achieve meaningful reductions in HIV transmission and meet this ambitious goal

The effect of more severe iron deficiency on susceptibility to <i>M</i>.<i>tb</i>.

<p>Female 4–5 week old C57BL/6 mice were fed an iron deficient (2–6ppm) or control (200ppm) diet for 6 weeks prior to infection with 50–100 CFU of aerosolised <i>M</i>.<i>tb</i> Erdman (A). Mice remained on the respective diet until 4 weeks post-infection, when animals were sacrificed (a total of 10 weeks on their respective diets). Lungs and spleen were harvested for enumeration of CFU (B+C), and livers for gene expression analyses for <i>Hamp1</i> (D), <i>Fpn1</i> (E), <i>Id1</i> (F), <i>Bmp6</i> (G) and <i>Tfrc</i> (H). Mann-Whitney tests were performed to compare groups where *, **, *** and **** indicate p = <0.05, p = <0.01, p = <0.001 and p = <0.0001, respectively. N = 8 animals in infected groups and n = 6 in uninfected groups. Black symbols represent uninfected animals, red points infected animals except for CFU graphs where all animals are infected. Closed circles represent control animals and open circles, iron deficient animals.</p

The effect of <i>M</i>.<i>tb</i> infection on the expression of genes involved in the regulation of iron homeostasis.

<p>Female 6–10 week old C57BL/6 mice were infected with 100–200 CFU of <i>M</i>.<i>tb</i> Erdman via aerosol. Animals were sacrificed at day (D) 0 (baseline), D1, D7, D14, D28 and D56 post-infection (outlined in <b>A</b>). Hepatic gene expression analyses over the time course are shown for <i>Hamp1</i> (<b>B</b>), <i>Fpn1</i> (<b>D</b>), <i>Fga</i> (<b>F</b>) and <i>Id1</i> (<b>H</b>). Comparisons of <i>Hamp1</i> and <i>Fpn1</i> gene expression respective to uninfected controls are depicted in <b>C</b> and <b>E</b> respectively. Correlations between <i>Hamp1</i> and <i>Id1</i>, and <i>Hamp1</i> and <i>Fga</i> are shown in <b>G</b> and <b>I</b>, respectively. Expression of immune genes <i>Ifng</i> and <i>Tnfa</i> are shown in figures <b>J</b> and <b>K,</b> respectively. Kruskal-Wallis tests with Dunn’s post-tests for multiple comparisons were done for time course studies, and Mann-Whitney tests for comparisons to uninfected controls. Correlations were Spearman’s rank correlations. In all cases *, **, *** and **** indicate p = <0.05, p = <0.01, p = <0.001 and p = <0.0001, respectively. Baseline values (D0) were not included in correlations. In panels C and E, black symbols represent uninfected animals and red symbols infected animals. In all other panels, animals are infected. N = 5 per group.</p

The effect of iron deficiency on susceptibility to <i>M</i>.<i>tb</i>.

<p>Female 6–10 week old C57BL/6 mice were fed an iron deficient (2–6ppm) or control (200ppm) diet for 2 weeks prior to infection with 50–100 CFU of aerosolised <i>M</i>.<i>tb</i> Erdman (<b>A</b>). Mice remained on the respective diet until 4 weeks or 8 weeks post-infection, when animals were sacrificed. Lungs and spleen were harvested for enumeration of CFU (<b>B-E</b>) and livers (from 4-week infected animals) for gene expression analyses. Gene expression data is shown for <i>Hamp1</i> (<b>F</b>), <i>Fpn1</i> (<b>G</b>), <i>Id1</i> (<b>H</b>), <i>Bmp6</i> (<b>I</b>) and <i>Tfrc</i> (<b>J</b>). Mann-Whitney tests were performed to compare groups where *, **, *** and **** indicate p = <0.05, p = <0.01, p = <0.001 and p = <0.0001, respectively. N = 8 per group for <b>B</b> and <b>C</b>, n = 10 per group for <b>D</b> and <b>E</b>. Gene expression data is representative of the two experiments where n = 10 for infected groups and 6 for uninfected controls. In Panels F-J, black symbols represent uninfected animals, red symbols infected animals except for CFU graphs where all animals are infected. Closed circles represent control animals and open circles represent iron deficient animals in all panels.</p

Characterisation of the iron deficient mouse model.

<p>Female 6–10 week old C57BL/6 mice were fed an iron deficient (2–6ppm) or control (200ppm) diet for a total of 2 weeks. Animals were sacrificed and hepatic gene expression analyses are shown for <i>Hamp1</i> (A), <i>Fpn</i> (B), <i>Id1</i> (C), and <i>Bmp6</i> (D). Mann-Whitney tests were performed to compare groups where *, **, *** and **** indicate p = <0.05, p = <0.01, p = <0.001 and p = <0.0001 respectively. N = 6 animals per group. Closed circles represent control animals and open circles represent iron deficient animals.</p

Characterisation of more severe iron deficiency in younger mice.

<p>Female 4–5 week old mice were fed an iron deficient (2–6ppm iron) or control (200ppm iron) diet for a total of 6 or 10 weeks. Animals were bled via cardiac puncture under terminal anaesthesia and liver and spleen were removed for quantification of tissue iron. Haematological parameters are shown in panels A-F, and liver and spleen non-heme iron content shown in G-H, respectively. Mann-Whitney tests were performed to compare groups where *, **, *** and **** indicate p = <0.05, p = <0.01, p = <0.001 and p = <0.0001, respectively. N = 6 per group.</p