Hypoxia-inducible factors and innate immunity in liver cancer.

The liver has strong innate immunity to counteract pathogens from the gastrointestinal tract. During the development of liver cancer, which is typically driven by chronic inflammation, the composition and biological roles of the innate immune cells are extensively altered. Hypoxia is a common finding in all stages of liver cancer development. Hypoxia drives the stabilization of hypoxia-inducible factors (HIFs), which act as central regulators to dampen the innate immunity of liver cancer. HIF signaling in innate immune cells and liver cancer cells together favors the recruitment and maintenance of pro-tumorigenic immune cells and the inhibition of anti-tumorigenic immune cells, promoting immune evasion. HIFs represent attractive therapeutic targets to inhibit the formation of an immunosuppressive microenvironment and growth of liver cancer.

Molecular characterization and gene expression analysis of hypoxia-inducible factor and its inhibitory factors in kuruma shrimp Marsupenaeus japonicus.

In shrimp aquaculture, overcrowded farming causes fluctuations in dissolved oxygen concentrations. Low-oxygen conditions (hypoxia) affect shrimp growth. Hypoxia-inducible factor (HIF) is a transcriptional factor in the basic helix-loop-helix/PAS family and is activated in response to hypoxic stress. However, little is known about HIF and other inhibitors of the HIF pathway in crustaceans. In this study, we cloned MjHIF-1α, an inhibitory factor, MjFIH-1 (factor inhibiting HIF-1α), and MjVHL (Von Hippel-Lindau tumor suppressor) from kuruma shrimp (Marsupenaeus japonicus). MjVHL is the first crustacean VHL ortholog to be cloned. MjHIF-1α, MjFIH-1, and MjVHL exhibit significant sequence similarity and share key functional domains with previously described vertebrate and invertebrate genes. As a result of gene expression analysis in various tissues, MjHIF-1α and MjVHL were more highly expressed in the intestine than in any other organ tissues. In hypoxia experiments, HIF-induced expression levels of MjHIF-1α in the hypoxic group increased significantly for 24 h after initiating hypoxia stimulation and expression of MjVHL decreased significantly for 6 h after hypoxia stimulation (P < 0.05).

Hypoxia and hypoxia-inducible factor (HIF) downregulate antigen-presenting MHC class I molecules limiting tumor cell recognition by T cells.

Human cancers are known to downregulate Major Histocompatibility Complex (MHC) class I expression thereby escaping recognition and rejection by anti-tumor T cells. Here we report that oxygen tension in the tumor microenvironment (TME) serves as an extrinsic cue that regulates antigen presentation by MHC class I molecules. In support of this view, hypoxia is shown to negatively regulate MHC expression in a HIF-dependent manner as evidenced by (i) lower MHC expression in the hypoxic TME in vivo and in hypoxic 3-dimensional (3D) but not 2-dimensional (2D) tumor cell cultures in vitro; (ii) decreased MHC in human renal cell carcinomas with constitutive expression of HIF due to genetic loss of von Hippel-Lindau (VHL) function as compared with isogenically paired cells with restored VHL function, and iii) increased MHC in tumor cells with siRNA-mediated knockdown of HIF. In addition, hypoxia downregulated antigen presenting proteins like TAP 1/2 and LMP7 that are known to have a dominant role in surface display of peptide-MHC complexes. Corroborating oxygen-dependent regulation of MHC antigen presentation, hyperoxia (60% oxygen) transcriptionally upregulated MHC expression and increased levels of TAP2, LMP2 and 7. In conclusion, this study reveals a novel mechanism by which intra-tumoral hypoxia and HIF can potentiate immune escape. It also suggests the use of hyperoxia to improve tumor cell-based cancer vaccines and for mining novel immune epitopes. Furthermore, this study highlights the advantage of 3D cell cultures in reproducing hypoxia-dependent changes observed in the TME.

[Role of hypoxia-inducible factor in the pathogenesis of rheumatoid arthritis].

Rheumatoid arthritis (RA) is a destructive chronic autoimmune disease characterized by synovium inflammation, cartilage destruction, bone erosion and the presence of autoantibodies. Hypoxia is a prominent micro-environmental feature in a range of disorders including RA. A combination of increased oxygen consumptionby inflamed resident cells and infiltrating immune cells along with a disrupted blood supply due to vascular dysfunction contribute to tissue hypoxia in RA. Hypoxia in turn regulates a number of key signaling pathways that help adaptation. The primary signaling pathway activated by hypoxia is the hypoxia-inducible factor (HIF) pathway. It has been shown that HIFs are highly expressed in the synovium of RA. HIFs mediate the pathogenesis of RA through inducing inflammation, angiogenesis, cell migration, and cartilage destruction, and inhibiting the apoptosis of synovial cells and inflammatory cells. HIF expressed in RA can be regulated in both oxygen-dependent and independent fashions, like inflammatory cytokines, leading to the aggravation of this disease. Considering the vital role of HIF in the pathogenesis of RA, we reviewed the new advances about hypoxia and RA. In this review, we firstly discussed the hypoxia-inducible factor and its regulation, and then, the pathologic role of hypoxia in RA, mainly elucidating the role of hypoxia in synovitis and cartilage destruction and immune cells. Finally, we provided evidence about the potential therapeutic target for treating RA.

Hypoxia-inducible factor 1 in autoimmune diseases.

Autoimmune disorders are a complicated and varied group of diseases arising from inappropriate immune responses. Recent studies have demonstrated that ongoing inflammatory and immune responses are associated with increased oxygen consumption, a process resulting in localized tissue hypoxia within inflammatory lesions ("inflammatory hypoxia"), in which hypoxia-inducible factor 1 (HIF-1), an oxygen-sensitive transcription factor that allows adaptation to hypoxia environments, has been shown to play an important function. HIF-1 is a regulator of angiogenesis and immune system. Besides, HIF-1-mediated metabolic shift and fibrosis may also play crucial roles in some autoimmune disorders. Firstly, we briefly summarize the role of HIF-1 in angiogenesis, immune responses and fibrosis. Secondly, we will show the major recent findings demonstrating a role for HIF-1 signaling in autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, psoriasis, systemic sclerosis and multiple sclerosis. The growing evidences may prompt HIF-1 to be a new target for treatment of autoimmune diseases.

Targeting the HIF pathway in inflammation and immunity.

Oxygen deprivation (hypoxia) is a frequently encountered condition in both health and disease. Metazoans have evolved an elegant and direct cellular mechanism by which to sense local oxygen levels and mount an adaptive transcriptional response to hypoxia which is mediated by a transcription factor termed the hypoxia-inducible factor (HIF). In normoxia, HIF is repressed primarily through the action of a family of hydroxylases, which target HIFα subunits for degradation in an oxygen-dependent manner. In hypoxia, HIF is rapidly stabilized in cells thus allowing it to regulate the expression of hundreds of genes which promote an adaptive response including genes expressing regulators of angiogenesis, metabolism, growth and survival. Initial studies into the HIF pathway focused mainly on its role in supporting tumor adaptation through enhancing processes such as angiogenesis, glycolytic metabolism and cell survival. More recently however, it has become clear that the HIF pathway also plays a key role in the regulation of immunity and inflammation. In fact, conditional knockout of the HIF-1α subunit has identified key immune roles in T-cells, dendritic cells, macrophages, neutrophils and epithelial cells. In this review, we will consider the role for HIF in the regulation of the immune response and its possible contribution to inflammation. Furthermore, we will consider potential therapeutic strategies, which target the HIF pathway in chronic inflammatory and infectious disease.

[Hypoxia, inflammation and phenotypic plasticity of macrophages: the central role of HIF-1 and NFkappaB].

Decrease of oxygen concentration, i.e. hypoxia, in organism tissues and cells is an important pathogenetic component in a large number of diseases. In these cases hypoxia is not only an important component of diseases pathogenesis, but can also influence immune reactions determining the outcome of diseases. Thus, concentration of macrophages in hypoxic areas and their reaction to hypoxia are the key moments in understanding the mechanisms of hypoxia influence on immunity. Macrophages are of the utmost importance in the congenital immune startup and define the vector of development of the adaptive response. In this review we present updated data on influence of hypoxia on macrophages phenotype and their plasticity, and we also analyze genetic trait of macrophages reaction to hypoxia. Molecular mechanisms of immune cells reaction on hypoxia and the role of transcription factors, HIF-1 and NF-kappaB, are analyzed. As a whole, it allowed to describe an important biological phenomenon - hypoxia-regulated control of macrophages phenotypic plasticity, and to define ways of search of new effective approaches to the management of diseases with hypoxic disturbances.

NFκB and HIF display synergistic behaviour during hypoxic inflammation.

The oxygen-sensitive transcription factor hypoxia inducible factor (HIF) is a key regulator of gene expression during adaptation to hypoxia. Crucially, inflamed tissue often displays regions of prominent hypoxia. Recent studies have shown HIF signalling is intricately linked to that of the pro-inflammatory transcription factor nuclear factor kappa B (NFκB) during hypoxic inflammation. We describe the relative temporal contributions of each to hypoxia-induced inflammatory gene expression and investigate the level of crosstalk between the two pathways using a novel Gaussia princeps luciferase (Gluc) reporter system. Under the control of an active promoter, Gluc is expressed and secreted into the cell culture media, where it can be sampled and measured over time. Thus, Gluc constructs under the control of either HIF or NFκB were used to resolve their temporal transcriptional dynamics in response to hypoxia and to cytokine stimuli, respectively. We also investigated the interactions between HIF and NFκB activities using a construct containing the sequence from the promoter of the inflammatory gene cyclooxygenase 2 (COX-2), which includes functionally active binding sites for both HIF and NFκB. Finally, based on our experimental data, we constructed a mathematical model of the binding affinities of HIF and NFκB to their respective response elements to analyse transcriptional crosstalk. Taken together, these data reveal distinct temporal HIF and NFκB transcriptional activities in response to hypoxic inflammation. Furthermore, we demonstrate synergistic activity between these two transcription factors on the regulation of the COX-2 promoter, implicating a co-ordinated role for both HIF and NFκB in the expression of COX-2 in hypoxic inflammation.

Interactions between nitric oxide and hypoxia-inducible factor signaling pathways in inflammatory disease.

Induction and activation of nitric oxide (NO) synthases (NOS) and excessive production of NO are common features of almost all diseases associated with infection and acute or chronic inflammation, although the contribution of NO to the pathophysiology of these diseases is highly multifactorial and often still a matter of controversy. Because of its direct impact on tissue oxygenation and cellular oxygen (O(2)) consumption and re-distribution, the ability of NO to regulate various aspects of hypoxia-induced signaling has received widespread attention. Conditions of tissue hypoxia and the activation of hypoxia-inducible factors (HIF) have been implicated in hypoxia or in cancer biology, but are also being increasingly recognized as important features of acute and chronic inflammation. Thus, the activation of HIF transcription factors has been increasingly implicated in inflammatory diseases, and recent studies have indicated its critical importance in regulating phagocyte function, inflammatory mediator production, and regulation of epithelial integrity and repair processes. Finally, HIF also appears to contribute to important features of tissue fibrosis and epithelial-to-mesenchymal transition, processes that are associated with tissue remodeling in various non-malignant chronic inflammatory disorders. In this review, we briefly summarize the current state of knowledge with respect to the general mechanisms involved in HIF regulation and the impact of NO on HIF activation. Secondly, we will summarize the major recent findings demonstrating a role for HIF signaling in infection, inflammation, and tissue repair and remodeling, and will address the involvement of NO. The growing interest in hypoxia-induced signaling and its relation with NO biology is expected to lead to further insights into the complex roles of NO in acute or chronic inflammatory diseases and may point to the importance of HIF signaling as key feature of NO-mediated events during these disorders.

A robust automated measure of average antibody staining in immunohistochemistry images.

Identifying and scoring cancer markers plays a key role in oncology, helping to characterize the tumor and predict the clinical course of the disease. The current method for scoring immunohistochemistry (IHC) slides is labor intensive and has inherent issues of quantitation. Although multiple attempts have been made to automate IHC scoring in the past decade, a major limitation in these efforts has been the setting of the threshold for positive staining. In this report, we propose the use of an averaged threshold measure (ATM) score that allows for automatic threshold setting. The ATM is a single multiplicative measure that includes both the proportion and intensity scores. It can be readily automated to allow for large-scale processing, and it is applicable in situations in which individual cells are hard to distinguish. The ATM scoring method was validated by applying it to simulated images, to a sequence of images from the same tumor, and to tumors from different patient biopsies that showed a broad range of staining patterns. Comparison between the ATM score and manual scoring by an expert pathologist showed that both methods resulted in essentially identical scores when applied to these patient biopsies. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Apurinic/apyrimidinic endonuclease 1 is a key modulator of keratinocyte inflammatory responses.

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1) functions in both DNA repair and redox signaling, making it an attractive emerging therapeutic target. However, the role of APE1 in cutaneous inflammatory responses is largely unknown. In this study, we report that APE1 is a key upstream regulator in TLR2-dependent keratinocyte inflammatory responses. We found that nuclear expression of APE1 in epidermal layers was markedly up-regulated in psoriatic skin. APE1 was essential for the transcriptional activation and nuclear translocation of hypoxia-inducible factor-1alpha and NF-kappaB, both of which are crucial for inflammatory signaling in keratinocytes. Moreover, APE1 played a crucial role in the expression of TLR2-mediated inflammatory mediators, including TNF-alpha, CXCL8, and LL-37, in HaCaT cells and human primary keratinocytes. Silencing of APE1 attenuated cyclin D1/cyclin-dependent kinase 4 expression and phosphorylation of ERK1/2 and Akt, thereby affecting keratinocyte proliferation. Importantly, TLR2-induced generation of reactive oxygen species contributed to the nuclear translocation and expression of APE1, suggesting an autoregulatory circuit in which the subcellular localization of APE1 is associated with the production of APE1 per se through reactive oxygen species-dependent signaling. Taken together, these findings establish a role for APE1 as a master regulator of TLR2-dependent inflammatory responses in human keratinocytes.

HIF-1 in the inflammatory microenvironment.

Hypoxia-inducible factor (HIF) is a transcriptional activator that coordinates adaptive responses to hypoxia. An increased activity is recognized in the majority of clinical relevant hypoxic/ischemic episodes and human cancers. However, studies with HIF-1alpha knockout mice revealed an important role of HIF-1 for physiology such as embryogenesis or glycolytic energy production. The discovery that HIF-1 activity is not only restricted to pathological conditions of reduced oxygen availability but also is needed for the normal O2-homeostasis by regulating O2-delivery and consumption opens a diverse spectrum of so far unappreciated HIF-1 functions in several organs, including the immune system. Innate immune responses are orchestrated by macrophages. These cells respond to environmental input signals and in turn generate appropriate answers to initiate resolution of inflammation. It appears that multiple pathways in the inflammatory microenvironment are used to adjust HIF-1alpha levels to affect macrophage biology. This review summarizes mechanisms of HIF activation in mammalian immune cells, especially in macrophages and neutrophils, and outlines how HIF moderates inflammation.

Activation of transcription factors and gene expression by oxidized low-density lipoprotein.

It is well recognized that oxidized LDL (OxLDL) plays a crucial role in the initiation and progression of atherosclerosis. Many biological effects of OxLDL are mediated through signaling pathways, especially via the activation of transcription factors, which in turn stimulate the expression of genes involved in the inflammatory and oxidative stress response or in cell cycle regulation. In this review, we will discuss the various transcription factors activated by OxLDL, the studied cell types, the active compounds of the OxLDL particle, and the downstream genes when identified. Identification of the transcription factors and some of the downstream genes regulated by OxLDL has helped us understand the molecular mechanism involved in generation of the atherosclerotic plaque.

Basic science for the clinician 37: Protecting against autoimmunity-tolerance: mechanisms of negative selection in the thymus.

As noted in previous articles in this series, tolerance, the ability of the immune system to differentiate self from nonself and leave the former alone, is a vital characteristic of a successful (and safe) immune system. With the detection of the molecule called aire (autoimmune regulator), the mechanism whereby autoreactive thymocytes encounter extrathymic proteins within the thymus and therefore are deleted, is now far better understood; aire was the subject of a prior article in this series. The absence of aire leads to autoimmune polyendocrinopathy, proof that aire is the center of an amazing "filtering" system. However, there are other mechanisms at work. Irregularities in expression of other proteins such as hypoxia-induced factor-1 (HIF-1) and CTLA4, have been implicated in autoimmune disease, the former in rheumatoid arthritis, the latter in autoimmune thyroid disease and lupus. Defects in intracellular factors involved in transcription of key apoptotic proteins have also been implicated in the escape of autoreactive thymocytes from the thymus, leading to autoimmune and lymphoproliferative syndromes as well. Changes in the proteins that oversee acetylation of histone lead to differential patterns of gene expression. At least 2 proteins involved in this process, HDAC and nur77, have been implicated in changes in survival of thymocytes. Yet again, there are multiple layers at work in the immune system; I have no idea how many more will be brought to light, which are phylogenetically most ancient or which will prove the most clinically relevant. For now, it is enough to bask in our new-found knowledge and know that the time from laboratory oddity, to animal model development, to therapeutic and/or diagnostic applications grows shorter each year since the molecular biologic revolution.