Human activated macrophages and hypoxia: a comprehensive review of the literature

Macrophages accumulate in poorly vascularised and hypoxic sites including solid tumours, wounds and sites of infection and inflammation where they can be exposed to low levels of oxygen for long periods. Up to date, different studies have shown that a number of transcription factors are activated by hypoxia which in turn activate a broad array of mitogenic, pro-invasive, pro-angiogenic, and pro-metastatic genes. On the other hand, macrophages respond to hypoxia by up-regulating several genes which are chief factors in angiogenesis and tumorigenesis. Therefore, in this review article we focus mainly on the role of macrophages during inflammation and discuss their response to hypoxia by regulating a diverse array of transcription factors. We also review the existing literatures on hypoxia and its cellular and molecular mechanism which mediates macrophages activation

DataSheet_1_High hypoxia status in pancreatic cancer is associated with multiple hallmarks of an immunosuppressive tumor microenvironment.pdf

IntroductionPancreatic ductal adenocarcinoma (PDAC), the most common form of pancreatic cancer, is a particularly lethal disease that is often diagnosed late and is refractory to most forms of treatment. Tumour hypoxia is a key hallmark of PDAC and is purported to contribute to multiple facets of disease progression such as treatment resistance, increased invasiveness, metabolic reprogramming, and immunosuppression.MethodsWe used the Buffa gene signature as a hypoxia score to profile transcriptomics datasets from PDAC cases. We performed cell-type deconvolution and gene expression profiling approaches to compare the immunological phenotypes of cases with low and high hypoxia scores. We further supported our findings by qPCR analyses in PDAC cell lines cultured in hypoxic conditions.ResultsFirst, we demonstrated that this hypoxia score is associated with increased tumour grade and reduced survival suggesting that this score is correlated to disease progression. Subsequently, we compared the immune phenotypes of cases with high versus low hypoxia score expression (HypoxiaHI vs. HypoxiaLOW) to show that high hypoxia is associated with reduced levels of T cells, NK cells and dendritic cells (DC), including the crucial cDC1 subset. Concomitantly, immune-related gene expression profiling revealed that compared to HypoxiaLOW tumours, mRNA levels for multiple immunosuppressive molecules were notably elevated in HypoxiaHI cases. Using a Random Forest machine learning approach for variable selection, we identified LGALS3 (Galectin-3) as the top gene associated with high hypoxia status and confirmed its expression in hypoxic PDAC cell lines.DiscussionIn summary, we demonstrated novel associations between hypoxia and multiple immunosuppressive mediators in PDAC, highlighting avenues for improving PDAC immunotherapy by targeting these immune molecules in combination with hypoxia-targeted drugs.</p

Activity of versican promoter luciferase reporter constructs in normoxia and hypoxia in HMDM.

<p><b>(A)</b> Putative transcription binding sites for hypoxia inducible factor (HIF), cAMP responsive element binding (CREB), activator Protein 1 (AP1), SP1, nuclear factor 1 (NF-1) and E2F within the 240 bp (-56+184) versican promoter sequence. The -56 to -26 and +54 to +104 sequences which are important for high level expression are in bold. <b>(B)</b> Schematic diagram of versican promoter or random 26mer luciferase reporter constructs used. <b>(C)</b> activity of versican promoter or random 26mer luciferase reporter constructs in HMDM after 5d incubation in normoxia (20.9% O₂), or hypoxia (0.2% O₂). Data from an average of 6 independent experiments with each construct, minimum n = 3 for each construct, are expressed as means ± SEM. Luciferase data were normalized to protein levels. Data assessed for significant increase in hypoxia compared to random construct control using two-tailed t tests, *** = p <0.001 ** p < 0.01,* = p <0.05, ns = not significant.</p

Real Time RT-PCR analysis of the effect of PI3K inhibitors on induction of versican and GLUT-1 mRNAs by 18h of exposure to hypoxia (0.2% O₂).

<p>LY290042 was used at 2μM and wortmannin at 300μM. ^^; p<0.05 compared to DMSO control, *; p<0.05 compared to untreated control, ratio paired t test, one tailed. Data from 5 independent experiments using HMDM from different donors, expressed as means ± SEM.</p

Quantitation of versican protein expression in monocytes / macrophages by flow cytometry.

<p><b>(A and D)</b> Dot plot analysis of PBMC after 5 days in normoxia (20.9% O₂; A) and hypoxia (0.2% O₂; D). Monocyte/macrophages are subdivided into 3 regions R3-R1 in respect of increasing cell size (forward scatter). Lymphocytes are included in region 4. Region 5 encompasses all monocyte macrophages in Regions 1, 2, and 3. A representative example of 5 independent experiments is shown. <b>(B and E)</b> Percentage of the total monocyte/macrophage population (R5) present in regions R1, R2, and R3 in normoxia (B) and hypoxia (E). Data from 5 independent experiments are expressed as means ± SEM. <b>(C and F)</b> Versican mean fluorescent intensity in regions 1, 2, 3 and 4 in Normoxia (C) and Hypoxia (F). Data from 5 independent experiments are expressed as means ± SEM. <b>(G)</b> Histogram of the fluorescent intensity with a versican specific antibody (black fill) compared to the isotype control antibody (white line) in region R1 cells in Normoxia and Hypoxia. A representative example of 5 independent experiments is shown. <b>(H)</b> Histogram analysis of the versican fluorescent intensity in region R1 cells in Normoxia (shaded) and Hypoxia (clear). A representative example of 5 independent experiments is shown. <b>(I)</b> Versican protein fold induction in cells region R1 in normoxia and hypoxia. Data from 5 independent experiments are expressed as means ± SEM. For panels B, E, C, F, and I, the normoxic value in each experiment was assigned an arbitrary value of 1. Data were further analyzed using two-tailed, paired t-tests. ** p < 0.01,* = p <0.05.</p

Assessment of versican mRNA decay in HMDM in normoxia and hypoxia by real-time RT-PCR after addition of Actinomycin D.

<p>N: normoxia 20.9% O₂, H: hypoxia 0.2% O₂. Data were normalized to 2MG mRNA levels. Data from 5 independent experiments are expressed as means ± SEM.</p

Up-regulation of versican gene expression by hypoxia in primary human macrophages.

<p><b>(A)</b> Real Time RT-PCR quantification of the effect of 18hrs hypoxia (0.2% O₂) on versican mRNA in 5-day differentiated HMDM from 13 different donors. Values are hypoxic fold induction relative to normoxia. <b>(B)</b> Changes in versican mRNA fold induction levels in response to 18hrs of hypoxia (0.2% O₂) were quantified by real-time RT-PCR in HMDM, CD14⁺ magnetic bead purified monocyte-macrophages and CD14^- cells, all incubated for 5d after isolation before being exposed to a further 18h of either normoxia or hypoxia, in 3 independent experiments using different donors. Values are hypoxic fold induction relative to normoxia. <b>(C)</b> Real-time RT-PCR quantification of versican mRNA isoforms in HMDM after differentiation either 5d in normoxia (20.9% O₂), 4d in normoxia followed by 1d in hypoxia, or 5d in hypoxia (0.2% O₂), in 4 independent experiments using different donors. All data were normalized to 2MG mRNA levels determined by separate PCRs, and are expressed as mean fold induction (relative to the equivalent normoxic culture) ± SEM, and were analyzed for significance using paired t-tests. **** = p <0.0001, *** = p <0.001, * = p <0.05.</p

Immunoblotting shows lack of correlation between HIF-1α protein level and versican mRNA up-regulation by hypoxia.

<p><b>(A)</b> After incubation under the conditions indicated, cell lysates were prepared from HMDM and immunoblotted for HIF-1α and actin. A blot representative of 3 independent experiments is shown. <b>(B and C)</b> Real time-PCR analyses show versican and GLUT-1 mRNA fold induction after 5 days of normoxia, 4 days of normoxia followed by 1 day of hypoxia, or 5 days of hypoxia. N: normoxia (20.9% O₂), H: hypoxia (0.2% O₂). Data were normalized to 2MG mRNA levels. Data from 3 independent experiments are expressed as means ± SEM. The normoxic value in each experiment was assigned an arbitrary value of 1. Data were further analyzed using two-tailed, paired t-tests. *** p< 0.001</p

Investigation of the role of Hypoxia Inducible Factor 1 (HIF-1) in versican up-regulation.

<p><b>(A)</b> Effect of over-expression of HIF-1α on the 240 bp (-56+184) versican promoter construct in HMDM. PGK was used as a positive control and pGL4 basic as a negative control. <b>(B and C)</b> Real time-PCR analyses showing VEGF and versican mRNA fold induction after treatment with two different preparations of LPS (MINN LPS, Salmonella Minnesota LPS; SAE LPS, Salmonella abortus equii LPS) in comparison with hypoxia. <b>(D)</b> Real time-PCR analyses show versican, VEGF, and GLUT-1 mRNA fold induction in hypoxic and normoxic HMDM. <b>(E)</b> Real time-PCR analyses of versican, VEGF, and GLUT-1 mRNA fold induction in HMDM treated with cobalt chloride (COB). N: normoxia 20.9% O₂, H: hypoxia 0.2% O₂. All incubations, with or without hypoxia, were for 18hrs. Data were normalized to 2MG mRNA levels. Data from 3 (A, B, and C) or 5 (D and E) or 8 (F) independent experiments are expressed as means ± SEM. Data were further analyzed using paired two-tailed t-tests. *** = p <0.001 ** p < 0.01,* = p <0.05</p

Oleoyl coenzyme A regulates interaction of transcriptional regulator RaaS (Rv1219c) with DNA in mycobacteria

We have recently shown that RaaS (regulator of antimicrobial-assisted survival), encoded by Rv1219c in Mycobacterium tuberculosis and by bcg_1279c in Mycobacterium bovis bacillus Calmette-Guérin, plays an important role in mycobacterial survival in prolonged stationary phase and during murine infection. Here, we demonstrate that long chain acyl-CoA derivatives (oleoyl-CoA and, to lesser extent, palmitoyl-CoA) modulate RaaS binding to DNA and expression of the downstream genes that encode ATP-dependent efflux pumps. Moreover, exogenously added oleic acid influences RaaS-mediated mycobacterial improvement of survival and expression of the RaaS regulon. Our data suggest that long chain acyl-CoA derivatives serve as biological indicators of the bacterial metabolic state. Dysregulation of efflux pumps can be used to eliminate non-growing mycobacteria