Antigen-specific downregulation of miR-150 in CD4 T cells promotes cell survival.

MicroRNA-150 (miR-150) has been shown to play a general role in the immune system, but very little is known about its role on CD4+ T cell responses. During T cell responses against superantigen Staphylococcal Enterotoxin A, miR-150 expression was down-regulated in antigen-specific CD4+ T cells but up-regulated in CD8+ T cells. CD4+ and CD8+ T cell clonal expansion was greater in miR-150-KO mice than in WT mice, but miR-150 selectively repressed IL-2 production in CD4+ T cells. Transcriptome analysis of CD4+ T cells demonstrated that apoptosis and mTOR pathways were highly enriched in the absence of miR-150. Mechanistic studies confirmed that miR-150 promoted apoptosis specifically in antigen-specific CD4+ T cells, but not in bystander CD4+ nor in CD8+ T cells. Furthermore, inhibition of mTOR-linked mitochondrial superoxidedismutase-2 increased apoptosis in miR-150-/- antigen-specific CD4+ T. Thus, miR-150 impacts CD4+ T cell helper activity by attenuating IL-2 production along with clonal expansion, and suppresses superoxidedismutase to promote apoptosis.

Evaluating the glycolytic potential of mouse costimulated effector CD8+ T cells ex vivo.

Studying the metabolic fitness of T cells is fundamental to understand how immune responses are regulated. Here, we describe a step-by-step protocol optimized to efficiently generate and isolate effector antigen-specific CD8+ T cells ex vivo using costimulation. We also detail steps to evaluate their metabolic activity using Seahorse technology. This protocol can be used to measure the glycolytic potential of effector murine T cells in response to different manipulations, such as infections, adjuvant studies, gene editing, or metabolite supplementation. For complete details on the use and execution of this protocol, please refer to Agliano et al. (2022).

Nicotinamide breaks effector CD8 T cell responses by targeting mTOR signaling.

Nicotinamide (NAM) shapes T cell responses but its precise molecular mechanism of action remains elusive. Here, we show that NAM impairs naive T cell effector transition but also effector T cells themselves. Although aerobic glycolysis is a hallmark of activated T cells, CD8+ T cells exposed to NAM displayed enhanced glycolysis, yet producing significantly less IFNγ. Mechanistically, NAM reduced mTORC1 activity independently of NAD+ metabolism, decreasing IFNγ translation and regulating T cell transcriptional factors critical to effector/memory fate. Finally, the role of NAM in a biomedically relevant model of lung injury was tested. Specifically, a NAM-supplemented diet reduced systemic IL-2, antigen-specific T cell clonal expansion, and effector function after inhalation of Staphylococcus aureus enterotoxin A. These findings identify NAM as a potential therapeutic supplement that uncouples glycolysis from effector cytokine production and may be a powerful treatment for diseases associated with T cell hyperactivation.

A benzimidazole inhibitor attenuates sterile inflammation induced in a model of systemic autoinflammation in female mice.

Sterile stimuli can trigger inflammatory responses, and in some cases can lead to a variety of acute or chronic diseases. In this study, we hypothesize that a benzimidazole inhibitor may be used as a therapeutic in the treatment of sterile inflammation. In vitro, this inhibitor blocks TLR signalling and inflammatory responses. The benzimidazole inhibitor does not prevent mouse macrophage activation after stimulation with 2,6,10,14-tetramethylpentadecane (TMPD, also known as pristane), a hydrocarbon oil that mimics features of sterile inflammation when injected in vivo. However, C57BL/6J female mice treated with the benzimidazole inhibitor exhibited a significant reduction of pristane-dependent induction of splenocyte number and weight. Conversely, no significant difference was observed in males. Using mass spectrometry, we found that the urine of pristane-injected mice contained increased levels of putative markers for several inflammatory diseases, which were reduced by the benzimidazole inhibitor. To study the mechanism, we showed that pristane-injected mice had increased cell free DNA in serum, which was not impacted by inhibitor treatment. However, chemokine release (e.g. MCP-1, RANTES and TARC) was significantly reduced in inhibitor-treated mice. Thus, the benzimidazole inhibitor might be used as a new drug to block the recruitment of immune cells during sterile inflammatory diseases in humans.

GRK2 enforces androgen receptor dependence in the prostate and prostate tumors.

Metastatic tumors that have become resistant to androgen deprivation therapy represent the major challenge in treating prostate cancer. Although these recurrent tumors typically remain dependent on the androgen receptor (AR), non-AR-driven tumors that also emerge are particularly deadly and becoming more prevalent. Here, we present a new genetically engineered mouse model for non-AR-driven prostate cancer that centers on a negative regulator of G protein-coupled receptors that is downregulated in aggressive human prostate tumors. Thus, prostate-specific expression of a dominant-negative G protein-coupled receptor kinase 2 (GRK2-DN) transgene diminishes AR and AR target gene expression in the prostate, and confers resistance to castration-induced involution. Further, the GRK2-DN transgene dramatically accelerates oncogene-initiated prostate tumorigenesis by increasing primary tumor size, potentiating visceral organ metastasis, suppressing AR, and inducing neuroendocrine marker mRNAs. In summary, GRK2 enforces AR-dependence in the prostate, and the loss of GRK2 function in prostate tumors accelerates disease progression toward the deadliest stage.

Long Noncoding RNAs in Host-Pathogen Interactions.

Long noncoding RNAs (lncRNAs) are key molecules that regulate gene expression in a variety of organisms. LncRNAs can drive different transcriptional and post-transcriptional events that impact cellular functions. Recent studies have identified many lncRNAs associated with immune cell development and activation; however, an understanding of their functional role in host immunity to infection is just emerging. Here, we provide a detailed and updated review of the functional roles of lncRNAs in regulating mammalian immune responses during host-pathogen interactions, because these functions may be either beneficial or detrimental to the host. With increased mechanistic insight into the roles of lncRNAs, it may be possible to design and/or improve lncRNA-based therapies to treat a variety of infectious and inflammatory diseases.

The TLR4 adaptor TRAM controls the phagocytosis of Gram-negative bacteria by interacting with the Rab11-family interacting protein 2.

Phagocytosis is a complex process that eliminates microbes and is performed by specialised cells such as macrophages. Toll-like receptor 4 (TLR4) is expressed on the surface of macrophages and recognizes Gram-negative bacteria. Moreover, TLR4 has been suggested to play a role in the phagocytosis of Gram-negative bacteria, but the mechanisms remain unclear. Here we have used primary human macrophages and engineered THP-1 monocytes to show that the TLR4 sorting adapter, TRAM, is instrumental for phagocytosis of Escherichia coli as well as Staphylococcus aureus. We find that TRAM forms a complex with Rab11 family interacting protein 2 (FIP2) that is recruited to the phagocytic cups of E. coli. This promotes activation of the actin-regulatory GTPases Rac1 and Cdc42. Our results show that FIP2 guided TRAM recruitment orchestrates actin remodelling and IRF3 activation, two events that are both required for phagocytosis of Gram-negative bacteria.

Long Non-coding RNA LincRNA-EPS Inhibits Host Defense Against Listeria monocytogenes Infection.

Long non-coding RNAs (lncRNAs) have emerged as key regulators of gene expression in several biological systems. The long intergenic RNA-erythroid pro-survival (lincRNA-EPS) has been shown to play a critical role in restraining inflammatory gene expression. However, the function of lincRNA-EPS during bacterial infections remains unknown. Here, we demonstrate that following infection with the intracellular bacterium Listeria monocytogenes, both mouse macrophages and dendritic cells lacking lincRNA-EPS exhibit an enhanced expression of proinflammatory cytokine genes, as well as an increased expression of the inducible nitric oxide synthase (iNos) and nitric oxide (NO) production. Importantly, we found that lincRNA-EPS-/- mice intraperitoneally infected with L. monocytogenes exhibit lower bacterial burdens in spleen and liver and produce more NO than control mice. Furthermore, lincRNA-EPS-/- mice are less susceptible to a lethal dose of L. monocytogenes than wild type (WT) mice. Collectively these findings show that lincRNA-EPS suppresses host protective NO expression and impairs the host defense against L. monocytogenes infection.

IRAK4 activity controls immune responses to intracellular bacteria Listeria monocytogenes and Mycobacterium smegmatis.

IL-1 receptor-associated kinase (IRAK) 4 is a central enzyme of the TLR pathways. This study tested the hypothesis that IRAK4 kinase activity is prerequisite for regulating innate immunity during infections with intracellular bacteria. To this end, we analyzed responses of macrophages obtained from mice expressing wild-type (WT) IRAK4 or its kinase-inactive K213M mutant (IRAK4KI ) upon infection with intracellular bacteria Listeria monocytogenes or Mycobacterium smegmatis. In contrast to robust induction of cytokines by macrophages expressing kinase-sufficient IRAK4, IRAK4KI macrophages expressed decreased TNF-α, IL-6, IL-1ß, and C-C motif chemokine ligand 5 upon infection with L. monocytogenes or M. smegmatis. Bacterial infection of IRAK4KI macrophages led to attenuated activation of IRAK1, MAPKs and NF-κB, impaired induction of inducible NO synthase mRNA and secretion of NO, but resulted in elevated microbial burdens. Compared with WT animals, systemic infection of IRAK4KI mice with M. smegmatis or L. monocytogenes resulted in decreased levels of serum IL-6 and CXCL-1 but increased bacterial burdens in the spleen and liver. Thus, a loss of IRAK4 kinase activity underlies deficient cytokine and microbicidal responses during infection with intracellular bacteria L. monocytogenes or M. smegmatis via impaired activation of IRAK1, MAPKs, and NF-κB but increases bacterial burdens, correlating with decreased induction of NO.

The overriding of TRAIL resistance by the histone deacetylase inhibitor MS-275 involves c-myc up-regulation in cutaneous, uveal, and mucosal melanoma.

Malignant melanoma is a highly aggressive tumor which may occur in the skin, eye, and mucous membranes. The prognosis of melanoma remains poor in spite of therapeutic advances, emphasizing the importance of innovative treatment modalities. Currently, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is showing promising clinical responses, however its use is hampered by intrinsic or acquired melanoma resistance to apoptosis. Recently, we showed that the combination of TRAIL with the class I-specific histone deacetylase inhibitor (HDACi) MS-275 was a privileged way to override TRAIL resistance through down-regulation of cellular Fas-associated death domain (FADD)-like interleukin-1beta-converting enzyme-inhibitory protein (c-FLIP). Here, we elucidated the underlying mechanism and provided evidence that a crucial step in the c-FLIP downregulation triggered by MS-275 implies the up-regulation of c-myc, a transcriptional repressor of c-FLIP. Notably, MS-275 caused H3 histone acetylation at the promoter of c-myc and increased its binding to the c-FLIP promoter, that in turn led to reduced c-FLIP gene transcription. Knockdown of c-myc prevented the MS-275-mediated downregulation of c-FLIP and hindered TRAIL-plus MS-275-induced apoptosis. Findings reported here provide additional knowledge tools for a more aware and effective molecular therapy of melanoma.

Epigenetic marks responsible for cadmium-induced melanoma cell overgrowth.

Cadmium (Cd) is a human carcinogen that likely acts via epigenetic mechanisms. However, the precise role of Cd in melanoma remains to be defined. The goals of this study are to: (i) examine the effect of Cd on the proliferation rate of cutaneous and uveal melanoma cells; (ii) identify the genes affected by Cd exposure; (iii) understand whether epigenetic changes are involved in the response to Cd. The cell growth capacity increased at 48 h after Cd treatment at doses ranging from 0.5 to 10 µM. The research on the key genes regulating proliferation has shown that aberrant methylation is responsible for silencing of p16(INK4A) and caspase 8 in uveal and cutaneous melanoma cells, respectively. The methylation and expression patterns of p14(ARF), death receptors 4/5, and E-cadherin remained unmodified after Cd treatment in all the cell lines analyzed. Ectopic expression of p16(INK4A) abolished the overgrowth of uveal melanoma cells in response to Cd and the overexpression of caspase 8 drastically increased the apoptotic rate of Cd-treated cutaneous melanoma cells. In conclusion, our data suggest that hypermethylation of p16(INK4A) and caspase 8 represents the most common event linked to Cd-induced stimulation of cell growth and inhibition of cell death pathway in melanoma.

Epigenetic effects of cadmium in cancer: focus on melanoma.

Cadmium is a highly toxic heavy metal, which has a destroying impact on organs. Exposure to cadmium causes severe health problems to human beings due to its ubiquitous environmental presence and features of the pathologies associated with pro-longed exposure. Cadmium is a well-established carcinogen, although the underlying mechanisms have not been fully under-stood yet. Recently, there has been considerable interest in the impact of this environmental pollutant on the epigenome. Be-cause of the role of epigenetic alterations in regulating gene expression, there is a potential for the integration of cadmium-induced epigenetic alterations as critical elements in the cancer risk assessment process. Here, after a brief review of the ma-jor diseases related to cadmium exposure, we focus our interest on the carcinogenic potential of this heavy metal. Among the several proposed pathogenetic mechanisms, particular attention is given to epigenetic alterations, including changes in DNA methylation, histone modifications and non-coding RNA expression. We review evidence for a link between cadmium-induced epigenetic changes and cell transformation, with special emphasis on melanoma. DNA methylation, with reduced expression of key genes that regulate cell proliferation and apoptosis, has emerged as a possible cadmium-induced epigenetic mechanism in melanoma. A wider comprehension of mechanisms related to this common environmental contaminant would allow a better cancer risk evaluation.