215 research outputs found
TLR4-Mediated Pathway Triggers Interferon-Independent G0 Arrest and Antiviral SAMHD1 Activity in Macrophages
Macrophages exist predominantly in two distinct states, G0 and a G1-like state that is accompanied by phosphorylation of SAMHD1 at T592. Here we demonstrate that TLR4 activation mediated by LPS or ingestion of whole E. Coli can potently induce G0 arrest and SAMHD1 antiretroviral activity by a novel IFN-independent pathway. This pathway is not sensitive to TBK1 depletion or to block of IRF3 translocation, but is sensitive to TRIF depletion by siRNA, indicating that the pathway requires TLR4 engagement with TRIF but not involvement of TBK1 dependent IRF3 translocation. Exclusive Myd88 activators, such as Tenascin C, a product of tissue breakdown that activates TLR4, and Flagellin, a bacterial protein recognized by TLR5, are unable to trigger G0 arrest or SAMHD1 dephosphorylation, demonstrating this arrest is also Myd88- and thus NFkB-independent. The G0 arrest is accompanied by p21 upregulation and CDK1 depletion, consistent with the observed SAMHD1 dephosphorylation at T592. Furthermore, we show by siRNA knockdown of SAMHD1 that this TLR4 activated pathway is able to potently block HIV-1 infection in macrophages specifically via SAMHD1. Together, these data demonstrate that macrophages can mobilize an intrinsic cell arrest and anti-viral state by activating TLR4 prior to IFN secretion, thereby highlighting the importance of cell cycle regulation as a response to pathogen associated danger signals in macrophages
Cell Cycle Regulation in Macrophages and Susceptibility to HIV-1.
Macrophages are the first line of defence against invading pathogens. They play a crucial role in immunity but also in regeneration and homeostasis. Their remarkable plasticity in their phenotypes and function provides them with the ability to quickly respond to environmental changes and infection. Recent work shows that macrophages undergo cell cycle transition from a G0/terminally differentiated state to a G1 state. This G0-to-G1 transition presents a window of opportunity for HIV-1 infection. Macrophages are an important target for HIV-1 but express high levels of the deoxynucleotide-triphosphate hydrolase SAMHD1, which restricts viral DNA synthesis by decreasing levels of dNTPs. While the G0 state is non-permissive to HIV-1 infection, a G1 state is very permissive to HIV-1 infection. This is because macrophages in a G1 state switch off the antiviral restriction factor SAMHD1 by phosphorylation, thereby allowing productive HIV-1 infection. Here, we explore the macrophage cell cycle and the interplay between its regulation and permissivity to HIV-1 infection
Sequential CCR5-Tropic HIV-1 Reactivation from Distinct Cellular Reservoirs following Perturbation of Elite Control
Background
HIV Elite Controllers may reveal insights into virus persistence given they harbour small reservoir sizes, akin to HIV non-controllers treated early with combination antiretroviral therapy. Both groups of patients represent the most promising candidates for interventions aimed at sustained remission or ‘cure’. Analytic treatment interruption (ATI) in the latter group leads to stochastic rebound of virus, though it is unclear whether loss of elite control is also associated with similar rebound characteristics.
Methods
We studied three discrete periods of virus rebound during myeloma related immune disruption over 2.5 years in an elite controller who previously underwent autologous stem cell transplantation (ASCT) in the absence of any antiretroviral therapy. Single genome sequencing of the V1-V4 region of env in PBMC and plasma was performed and phylogenies reconstructed. Average pairwise distance (APD) was calculated and non-parametric methods used to assess compartmentalisation. Coreceptor usage was predicted based on genotypic algorithms.
Results
122 single genome sequences were obtained (median 26 sequences per rebound). The initial rebounding plasma env sequences following ASCT represented two distinct lineages, and clustered with proviral DNA sequences isolated prior to ASCT. One of the lineages was monophyletic, possibly indicating reactivation from clonally expanded cells. The second rebound occurred 470 days after spontaneous control of the first rebound and was phylogenetically distinct from the first, confirmed by compartmentalisation analysis, with a different cellular origin rather than ongoing replication. By contrast, third rebound viruses clustered with second rebound viruses, with evidence for ongoing evolution that was associated with lymphopenia and myeloma progression. Following ASCT a shift in tropism from CXCR4-tropic viruses to a CCR5-tropic population was observed to persist through to the third rebound.
Conclusions
Our data highlight similarities in the viral reservoir between elite and non-controllers undergoing ATI following allogeneic transplantation. The lack of propagation of CXCR4 using viruses following transplantation warrants further study
Epithelial-mesenchymal transition-associated microRNA/mRNA signature is linked to metastasis and prognosis in clear-cell renal cell carcinoma
Clear-cell renal cell carcinomas (ccRCCs) are genetically heterogeneous tumors
presenting diverse clinical courses. Epithelial-mesenchymal transition (EMT)
is a crucial process involved in initiation of metastatic cascade. The aim of
our study was to identify an integrated miRNA/mRNA signature associated with
metastasis and prognosis in ccRCC through targeted approach based on analysis
of miRNAs/mRNAs associated with EMT. A cohort of 230 ccRCC was included in our
study and further divided into discovery, training and validation cohorts. EMT
markers were evaluated in ccRCC tumor samples, which were grouped accordingly
to EMT status. By use of large-scale miRNA/mRNA expression profiling, we
identified miRNA/mRNA with significantly different expression in EMT-positive
tumors and selected 41 miRNAs/mRNAs for training phase of the study to
evaluate their diagnostic and prognostic potential. Fifteen miRNAs/mRNAs were
analyzed in the validation phase, where all evaluated miRNA/mRNA candidates
were confirmed to be significantly deregulated in tumor tissue. Some of them
significantly differed in metastatic tumors, correlated with clinical stage,
with Fuhrman grade and with overall survival. Further, we established an EMT-
based stage-independent prognostic scoring system enabling identification of
ccRCC patients at high-risk of cancer-related death. Finally, we confirmed
involvement of miR-429 in EMT regulation in RCC cells in vitro
DNA damage induced by topoisomerase inhibitors activates SAMHD1 and blocks HIV-1 infection of macrophages
We report that DNA damage induced by topoisomerase inhibitors, including etoposide (ETO), results in a potent block to HIV-1 infection in human monocyte-derived macrophages (MDM). SAMHD1 suppresses viral reverse transcription (RT) through depletion of cellular dNTPs but is naturally switched off by phosphorylation in a subpopulation of MDM found in a G1-like state. We report that SAMHD1 was activated by dephosphorylation following ETO treatment, along with loss of expression of MCM2 and CDK1, and reduction in dNTP levels. Suppression of infection occurred after completion of viral DNA synthesis, at the step of 2LTR circle and provirus formation. The ETO-induced block was completely rescued by depletion of SAMHD1 in MDM Concordantly, infection by HIV-2 and SIVsm encoding the SAMHD1 antagonist Vpx was insensitive to ETO treatment. The mechanism of DNA damage-induced blockade of HIV-1 infection involved activation of p53, p21, decrease in CDK1 expression, and SAMHD1 dephosphorylation. Therefore, topoisomerase inhibitors regulate SAMHD1 and HIV permissivity at a post-RT step, revealing a mechanism by which the HIV-1 reservoir may be limited by chemotherapeutic drugs
Vpx complementation of 'non-macrophage tropic' R5 viruses reveals robust entry of infectious HIV-1 cores into macrophages.
Background
It is now known that clinically derived viruses are most commonly R5 tropic with very low infectivity in macrophages. As these viruses utilize CD4 inefficiently, defective entry has been assumed to be the dominant restriction. The implication is that macrophages are not an important reservoir for the majority of circulating viruses.
Results
Macrophage infection by clinical transmitted/founder isolates was 10-100 and 30-450 fold less efficient as compared to YU-2 and BaL respectively. Vpx complementation augmented macrophage infection by non-macrophage tropic viruses to the level of infectivity observed for YU-2 in the absence of Vpx. Augmentation was evident even when Vpx was provided 24 hours post-infection. The entry defect was measured as 2.5-5 fold, with a further 3.5-10 fold block at strong stop and subsequent stages of reverse transcription as compared to YU-2. The overall block to infection was critically dependent on the mechanism of entry as demonstrated by rescue of infection after pseudotyping with VSV-G envelope. Reverse transcription in macrophages could not be enhanced using a panel of cytokines or lipopolysaccharide (LPS).
Conclusions
Although the predominant block to clinical transmitted/founder viruses is post-entry, infectivity is determined by Env-CD4 interactions and can be rescued with VSV-G pseudotyping. This suggests a functional link between the optimal entry pathway taken by macrophage tropic viruses and downstream events required for reverse transcription. Consistent with a predominantly post-entry block, replication of R5 using viruses can be greatly enhanced by Vpx. We conclude therefore that entry is not the limiting step and that macrophages represent clinically relevant reservoirs for ‘non-macrophage tropic’ viruses
Comparison of mobility shift affinity capillary electrophoresis and capillary electrophoresis frontal analysis for binding constant determination between human serum albumin and small drugs
In this study, two capillary electrophoresis–based ligand binding assays,
namely, mobility shift affinity capillary electrophoresis (ms-ACE) and capil-
lary electrophoresis-frontal analysis (CE-FA), were applied to determine binding
parameters of human serum albumin toward small drugs under similar exper-
imental conditions. The substances S-amlodipine (S-AML), lidocaine (LDC),
l-tryptophan (l-TRP), carbamazepine (CBZ), ibuprofen (IBU), and R-verapamil
(R-VPM) were used as the main binding partners. The scope of this comparative
study was to estimate and compare both the assays in terms of their primary mea-
sure’s precision and the reproducibility of the derived binding parameters. The
effective mobility could be measured with pooled CV values between 0.55% and
7.6%. The precision of the r values was found in the range between 1.5% and 10%.
Both assays were not universally applicable. The CE-FA assay could successfully
be applied to measure the drugs IBU, CBZ, and LDC, and the interaction toward
CBZ, S-AML, l-TRP, and R-VPM could be determined using ms-ACE. The aver-
age variabilities of the estimated binding constants were 64% and 67% for CE-FA
and ms-ACE, respectively
The HUSH complex is a gatekeeper of type I interferon through epigenetic regulation of LINE-1s
The Human Silencing Hub (HUSH) complex is necessary for epigenetic repression of LINE-1 elements. We show that HUSH-depletion in human cell lines and primary fibroblasts leads to induction of interferon-stimulated genes (ISGs) through JAK/STAT signaling. This effect is mainly attributed to MDA5 and RIG-I sensing of double-stranded RNAs (dsRNAs). This coincides with upregulation of primate-conserved LINE-1s, as well as increased expression of full-length hominid-specific LINE-1s that produce bidirectional RNAs, which may form dsRNA. Notably, LTRs nearby ISGs are derepressed likely rendering these genes more responsive to interferon. LINE-1 shRNAs can abrogate the HUSH-dependent response, while overexpression of an engineered LINE-1 construct activates interferon signaling. Finally, we show that the HUSH component, MPP8 is frequently downregulated in diverse cancers and that its depletion leads to DNA damage. These results suggest that LINE-1s may drive physiological or autoinflammatory responses through dsRNA sensing and gene-regulatory roles and are controlled by the HUSH complex
Human primary macrophages derived in vitro from circulating monocytes comprise adherent and non-adherent subsets with differential expression of Siglec-1 and CD4 and permissiveness to HIV-1 infection
Urinary microRNA can be concentrated, dried on membranes and stored at room temperature in vacuum bags
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