Boswellic acid exerts potent anticancer effects in HCT-116 human colon cancer cells mediated via the induction of apoptosis, cell cycle arrest, cell migration inhibition and inhibition of PI3K/AKT signalling pathway

The Editors of JBUON issue an Expression of Concern to 'Boswellic acid exerts potent anticancer effects in HCT-116 human colon cancer cells mediated via induction of apoptosis, cell cycle arrest, cell migration inhibition and inhibition of PI3K/AKT signalling pathway', by Dan Wang, Shuke Ge, Jichang Bai, Yongwei Song, JBUON 2018;23(2):340-345; PMID:29745074. Following the publication of the above article, readers drew to our attention that part of the data was possibly unreliable. We sent emails to the authors with a request to provide the raw data to prove the originality, but received no reply. Therefore, as we continue to work through the issues raised, we advise readers to interpret the information presented in the article with due caution. We thank the readers for bringing this matter to our attention. We apologize for any inconvenience it may cause.

BATF2 prevents glioblastoma multiforme progression by inhibiting recruitment of myeloid-derived suppressor cells.

The basic leucine zipper ATF-like transcription factor 2 (BATF2) has been implicated in inflammatory responses and anti-tumour effects. Little, however, is known regarding its extracellular role in maintaining a non-supportive cancer microenvironment. Here, we show that BATF2 inhibits glioma growth and myeloid-derived suppressor cells (MDSCs) recruitment. Interestingly, extracellular vesicles (EVs) from BATF2-overexpressing glioma cell lines (BATF2-EVs) inhibited MDSCs chemotaxis in vitro. Moreover, BATF2 inhibited intracellular SDF-1α and contributes to decreased SDF-1α in EVs. In addition, BATF2 downregulation-induced MDSCs recruitment were reversed by blocking SDF-1α/CXCR4 signalling upon AMD3100 treatment. Specifically, detection of EVs in 24 pairs of gliomas and healthy donors at different stages revealed that the abundance of BATF2-positive EVs in plasma (BATF2+ plEVs) can distinguish stage III-IV glioma from stage I-II glioma and healthy donors. Taken together, our study identified novel regulatory functions of BATF2 in regulating MDSCs recruitment, providing a prognostic value in terms of the number of BATF2+ plEVs in glioma stage.

LncRNA miR143HG suppresses miR-21 through methylation to inhibit cell invasion and migration.

OBJECTIVE: This study aimed to investigate the role of lncRNA miR143HG in laryngeal squamous cell carcinoma (LSCC). METHODS: Quantitative polymerase chain reaction (PCR) and paired t test were used to measure and compare expression levels of miR143HG and miR-21 in LSCC and nontumor tissues. To analyze the interactions between miR143HG and miR-21, UM-SCC-17A cells were transfected miR143HG expression vector or miR-21 mimic. The effects of miR143HG and miR-21 overexpression on UM-SCC-17A cell invasion and migration were analyzed by transwell assays. RESULTS: We found that miR143HG was downregulated in LSCC and inversely correlated with miR-21. In LSCC cells, miR143HG overexpression led to the downregulated expression of miR-21, whereas miR-21 overexpression failed to affect miR143HG. Methylation-specific PCR results showed that miR143HG overexpression led to increased methylation of miR-21. Low expression levels of miR143HG were correlated with poor survival. Overexpression of miR143HG led to decreased, whereas miR-21 overexpression resulted in increased rate of LSCC cell migration and invasion. In addition, miR-21 overexpression led to reduced effects of miR143HG on cell invasion and migration. CONCLUSION: Therefore, miR143HG suppresses miR-21 via methylation to regulate cell behaviors in LSCC. LEVEL OF EVIDENCE: NA Laryngoscope, 130:E640-E645, 2020.

Lung inflammation stalls Th17-cell migration en route to the central nervous system during the development of experimental autoimmune encephalomyelitis.

Recruiting pathogenic T cells to the central nervous system (CNS) is a critical step during the development of experimental autoimmune encephalomyelitis (EAE). Here, we report that the absence of autophagy and microtubule-associated protein 1A/1B-light chain 3-associated phagocytosis significantly delayed the onset of EAE in Atg7 conditional knockout (Atg7 CKO) mice in myeloid cells. T-helper cell-cell priming appeared to be normal in the Atg7 CKO mice, but the mice showed significant accumulation of Th17 cells in the lung. The data suggested that the stalling of Th17 cells in the lung en route to the CNS caused the delay. The lung of Atg7 CKO mice, in which we previously demonstrated spontaneous mild inflammation, showed high expression of CCL20, a chemokine that attracts Th17 cells. We have also shown that LPS intranasal instillation delayed EAE onset, suggesting that pulmonary inflammation has an impact on EAE development. Based on our data, therapeutic immunomodulation targeted to the lung, rather than systemically, might be a possible future option to treat multiple sclerosis.

Inhibition of CXCL12-mediated chemotaxis of Jurkat cells by direct immunotoxicants.

Directional migration of cells to specific locations is required in tissue development, wound healing, and immune responses. Immune cell migration plays a crucial role in both innate and adaptive immunity. Chemokines are small pro-inflammatory chemoattractants that control the migration of leukocytes. In addition, they are also involved in other immune processes such as lymphocyte development and immune pathology. In a previous toxicogenomics study using the Jurkat T cell line, we have shown that the model immunotoxicant TBTO inhibited chemotaxis toward the chemokine CXCL12. In the present work, we aimed at assessing a novel approach to detecting chemicals that affect the process of cell migration. For this, we first evaluated the effects of 31 chemicals on mRNA expression of genes that are known to be related to cell migration. With this analysis, seven immunotoxicants were identified as potential chemotaxis modulators, of which five (CoCl2 80 µM, MeHg 1 µM, ochratoxin A 10 µM, S9-treated ochratoxin A 10 µM, and TBTO 100 nM) were confirmed as chemotaxis inhibitor in an in vitro trans-well chemotaxis assay using the chemokine CXCL12. The transcriptome data of the five compounds together with previously obtained protein phosphorylation profiles for two out of five compounds (i.e., ochratoxin A and TBTO) revealed that the mechanisms behind the chemotaxis inhibition are different for these immunotoxicants. Moreover, the mTOR inhibitor rapamycin had no effect on the chemotaxis of Jurkat cells, indicating that the mTOR pathway is not involved in CXCL12-mediated chemotaxis of Jurkat cells, which is opposite to the findings on human primary T cells (Munk et al. in PLoS One 6(9):e24667, 2011). Thus, the results obtained from the chemotaxis assay conducted with Jurkat cells might not fully represent the results obtained with human primary T cells. Despite this difference, the present study indicated that some compounds may exert their immunotoxic effects through inhibition of CXCL12-mediated chemotaxis.

SMAD4 loss triggers the phenotypic changes of pancreatic ductal adenocarcinoma cells.

BACKGROUND: SMAD4 is a gastrointestinal malignancy-specific tumor suppressor gene found mutated in one third of colorectal cancer specimens and half of pancreatic tumors. SMAD4 inactivation by allelic deletion or intragenic mutation mainly occurs in the late stage of human pancreatic ductal adenocarcinoma (PDAC). Various studies have proposed potential SMAD4-mediated anti-tumor effects in human malignancy; however, the relevance of SMAD4 in the PDAC molecular phenotype has not yet been fully characterized. METHODS: The AsPC-1, CFPAC-1 and PANC-1 human PDAC cell lines were used. The restoration or knockdown of SMAD4 expression in PDAC cells were confirmed by western blotting, luciferase reporter and immunofluorescence assays. In vitro cell proliferation, xenograft, wound healing, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), Western blotting, and immunohistochemistry analysis were conducted using PDAC cells in which SMAD4 was either overexpressed or knocked down. RESULTS: Here, we report that re-expression of SMAD4 in SMAD4-null PDAC cells does not affect tumor cell growth in vitro or in vivo, but significantly enhances cells migration in vitro. SMAD4 restoration transcriptionally activates the TGF-ß1/Nestin pathway and induces expression of several transcriptional factors. In contrast, SMAD4 loss in PDAC leads to increased expression of E-cadherin, vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR) and CD133. Furthermore, SMAD4 loss causes alterations to multiple kinase pathways (particularly the phosphorylated ERK/p38/Akt pathways), and increases chemoresistance in vitro. Finally, PDAC cells with intact SMAD4 are more sensitive to TGF-ß1 inhibitor treatment to reduced cell migration; PDAC cells lacking SMAD4 showed decreased cell motility in response to EGFR inhibitor treatment. CONCLUSIONS: This study revealed the molecular basis for SMAD4-dependent differences in PDAC with the aim of identifying the subset of patients likely to respond to therapies targeting the TGF-ß or EGFR signaling pathways and of identifying potential therapeutic interventions for PDAC patients with SMAD4 defects.

MLK3 regulates fMLP-stimulated neutrophil motility.

INTRODUCTION: Mixed lineage kinase 3 (MLK3) is part of the intracellular regulatory system that connects extracellular cytokine or mitogen signals received through G-protein coupled receptors to changes in gene expression. MLK3 activation stimulates motility of epithelial cells and epithelial-derived tumor cells, but its role in mediating the migration of other cell types remains unknown. Since neutrophils play a crucial role in innate immunity and contribute to the pathogenesis of several diseases, we therefore examined whether MLK3 might regulate the motility of mouse neutrophils responding to a chemotactic stimulus, the model bacterial chemoattractant fMLP. METHODS: The expression of Mlk3 in mouse neutrophils was determined by immunocytochemistry and by RT-PCR. In vitro chemotaxis in a gradient of fMLP, fMLP-stimulated random motility, fMLP-stimulated F-actin formation were measured by direct microscopic observation using neutrophils pre-treated with a novel small molecule inhibitor of MLK3 (URMC099) or neutrophils obtained from Mlk3-/- mice. In vivo effects of MLK3 inhibition were measured by counting the fMLP-induced accumulation of neutrophils in the peritoneum following pre-treatment with URMC099 in wild-type C57Bl/6 or mutant Mlk3-/- mice. RESULTS: The expression of Mlk3 mRNA and protein was observed in neutrophils purified from wild-type C57Bl/6 mice but not in neutrophils from mutant Mlk3-/- mice. Chemotaxis by wild-type neutrophils induced by a gradient of fMLP was reduced by pre-treatment with URMC099. Neutrophils from C57Bl/6 mice pretreated with URMC099 and neutrophils from Mlk3-/- mice moved far less upon fMLP-stimulation and did not form F-actin as readily as untreated neutrophils from C57Bl/6 controls. In vivo recruitment of neutrophils into the peritoneum by fMLP was significantly reduced in wild-type mice treated with URMC099, as well as in untreated Mlk3-/- mice-thereby confirming the role of MLK3 in neutrophil migration. CONCLUSIONS: Mlk3 mRNA is expressed in murine neutrophils. Genetic or pharmacologic inhibition of MLK3 blocks fMLP-mediated motility of neutrophils both in vitro and in vivo, suggesting that MLK3 may be a therapeutic target in human diseases characterized by exuberant neutrophil migration.

Cyclooxygenase-2 deficiency in macrophages leads to defective p110γ PI3K signaling and impairs cell adhesion and migration.

Cyclooxygenase (Cox)-2 dependent PGs modulate several functions in many pathophysiological processes, including migration of immune cells. In this study, we addressed the role of Cox-2 in macrophage migration by using in vivo and in vitro models. Upon thioglycolate challenge, CD11b(+) F4/80(+) macrophages showed a diminished ability to migrate to the peritoneal cavity in cox-2(-/-) mice. In vivo migration of cox-2(-/-) macrophages from the peritoneal cavity to lymph nodes, as well as cell adhesion to the mesothelium, was reduced in response to LPS. In vitro migration of cox-2(-/-) macrophages toward MCP-1, RANTES, MIP-1α, or MIP-1ß, as well as cell adhesion to ICAM-1 or fibronectin, was impaired. Defects in cell migration were not due to changes in chemokine receptor expression. Remarkably, cox-2(-/-) macrophages showed a deficiency in focal adhesion formation, with reduced phosphorylation of paxillin (Tyr(188)). Interestingly, expression of the p110γ catalytic subunit of PI3K was severely reduced in the absence of Cox-2, leading to defective Akt phosphorylation, as well as cdc42 and Rac-1 activation. Our results indicate that the paxillin/p110γ-PI3K/Cdc42/Rac1 axis is defective in cox-2(-/-) macrophages, which results in impaired cell adhesion and migration.

GILZ overexpression inhibits endothelial cell adhesive function through regulation of NF-κB and MAPK activity.

Glucocorticoid-induced leucine zipper (GILZ) is an anti-inflammatory protein first identified in T lymphocytes. We recently observed that GILZ is highly expressed in synovial endothelial cells in rheumatoid arthritis. However, the function of GILZ in endothelial cells is unknown. To investigate the actions of GILZ in this cell type, we induced GILZ expression in HUVECs via transient transfection. GILZ overexpression significantly reduced the capacity of TNF-stimulated HUVECs to support leukocyte rolling, adhesion, and transmigration. These effects were associated with decreased expression of E-selectin, ICAM-1, CCL2, CXCL8, and IL-6. Experiments in a human microvascular endothelial cell line demonstrated that TNF-inducible NF-κB activity was significantly inhibited by overexpression of GILZ. Exogenous GILZ inhibited TNF-induced NF-κB p65 DNA binding, although this occurred in the absence of an effect on p65 nuclear translocation, indicating that the mechanism of action of exogenous GILZ in endothelial cells differs from that reported in other cell types. GILZ overexpression also inhibited TNF-induced activation of p38, ERK, and JNK MAPKs, as well as increased expression of the MAPK inhibitory phosphatase, MKP-1. In contrast, silencing endogenous GILZ in glucocorticoid-treated HUVECs did not alter their capacity to support leukocyte interactions. These data demonstrate that exogenous GILZ exerts inhibitory effects on endothelial cell adhesive function via a novel pathway involving modulation of NF-κB p65 DNA binding and MAPK activity. Induction of GILZ expression in endothelial cells may represent a novel therapeutic modality with the potential to inhibit inflammatory leukocyte recruitment.

α1ß1 integrin-mediated adhesion inhibits macrophage exit from a peripheral inflammatory lesion.

Integrins are adhesion molecules critical for the recruitment of leukocytes from blood into peripheral tissues. However, whether integrins are also involved in leukocyte exit from peripheral tissues via afferent lymphatics to the draining lymph node remains poorly understood. In this article, we show that adhesion by the collagen IV-binding integrin α1ß1 unexpectedly inhibited macrophage exit from inflamed skin. We monitored macrophages exiting mouse footpads using a newly developed in situ pulse labeling technique. Blockade of α1ß1 integrin or genetic deletion (Itga1(-/-)) increased macrophage exit efficiency. Chemotaxis assays through collagen IV showed more efficient migration of Itga1(-/-) macrophages relative to wild type. Given that macrophages are key orchestrators of inflammation, α1ß1 integrin adhesion may represent a mechanism for regulating inflammatory responses by controlling macrophage exit or persistence in inflamed tissues.

miR-22 is down-regulated in gastric cancer, and its overexpression inhibits cell migration and invasion via targeting transcription factor Sp1.

Accumulating evidence has shown that microRNAs are involved in multiple processes in cancer development and progression. Recently, miR-22 has been identified as a tumor-suppressing microRNA in many human cancers. However, the specific function of miR-22 in gastric cancer is unclear at this point. In this study, we first measured miR-22 expression level in 30 pairs of gastric cancer and matched normal tissues, two normal and six gastric cancer cell lines by real-time quantitative RT-PCR. We found that the expression of miR-22 in gastric cancer tissues and cell lines was much lower than that in normal control, respectively. Transfection of miR-22 expression plasmid could significantly inhibit the cell migration and invasion in SGC-7901 and NCL-N87 gastric cancer cell lines. Moreover, we also showed that Sp1 was negatively regulated by miR-22 at the posttranscriptional level, via a specific target site within the 3'UTR by luciferase reporter assay. The expression of Sp1 was inversely correlated with miR-22 expression in gastric cancer tissues, and knockdown of Sp1 by siRNA inhibited cell malignant behaviors. Thus, our findings suggest that miR-22 acts as tumor suppressor by targeting the Sp1 gene and inhibiting gastric cancer cell migration and invasion. The findings of this study contribute to current understanding of the functions of miR-22 in gastric cancer.

Clearance of activated stellate cells for hepatic fibrosis regression: molecular basis and translational potential.

Hepatic fibrosis, characterized by abnormal accumulation of extracellular matrix (ECM), is a common pathological process of many chronic liver diseases. A growing number of studies have shown that the activation of hepatic stellate cells (HSCs) plays an important role in the pathogenesis of hepatic fibrosis. Inhibiting the activation of HSCs and accelerating the clearance of activated HSCs may be effective strategies for resolution of hepatic fibrosis. Therefore, understanding the underlying mechanisms of clearance of activated HSCs and the therapeutic implications is an active subject of research. Studies have shown that apoptosis, immune clearance, phenotype reversion and senescence are involved in clearance of activated HSCs. In this review, we will discuss the mechanisms of clearance of activated HSCs and their potential in resolution of hepatic fibrosis.

Rho-family GTPase Cdc42 controls migration of Langerhans cells in vivo.

Epidermal Langerhans cells (LCs) of the skin represent the prototype migratory dendritic cell (DC) subtype. In the skin, they take up Ag, migrate to the draining lymph nodes, and contribute to Ag transport and immunity. Different depletion models for LCs have revealed contrasting roles and contributions of this cell type. To target the migratory properties of DCs, we generated mice lacking the Rho-family GTPase Cdc42 specifically in DCs. In these animals, the initial seeding of the epidermis with LCs is functional, resulting in slightly reduced Langerhans cell numbers. However, Cdc42-deficient LCs fail to leave the skin in steady state as well as upon stimulation, as they do not enter the skin-draining afferent lymph vessels. Similarly, also other Cdc42-deficient migratory DC subsets fail to home properly to the corresponding draining lymph nodes. We used this novel mouse model, in which LCs are locked out, to demonstrate that these cells contribute substantially to priming of Ag-specific CD4 and CD8 T cell responses upon epicutaneous immunization, but could not detect a role in the induction of contact hypersensitivity to various doses of hapten.

CD47 deficiency does not impede polymorphonuclear neutrophil transmigration but attenuates granulopoiesis at the postacute stage of colitis.

Previous studies have suggested that CD47, an essential cell-surface protein, plays an important role in polymorphonuclear neutrophil (PMN) transmigration across tissue cells and extracellular matrix. In the current study, the role of CD47 in PMN transmigration and infiltration into tissues was further evaluated by investigating the function of CD47(-/-) PMN and inflammatory conditions induced in CD47(-/-) mice. Using in vitro time-course assays, we found that CD47(-/-) PMN exhibited no impediment, but slightly enhanced response to and transmigration toward, the chemoattractant fMLF. In vivo analysis in CD47(-/-) mice by inducing acute peritonitis and aggressive colitis observed consistent results, indicating that both PMN and monocytes effectively infiltrated inflammatory sites despite the absence of CD47 on these leukocytes or the surrounding tissue cells. Although PMN transmigration was not delayed in CD47(-/-) mice, fewer PMN were found in the intestine at the postacute/chronic stage of chronic colitis induced with sustained low-dose dextran sulfate sodium. Further analysis suggested that the paucity of PMN accumulation was attributable to attenuated granulopoiesis secondary to assessed lower levels of IL-17. Administration of exogenous IL-17A markedly increased PMN availability and rapidly rendered severe colitis in CD47(-/-) mice under dextran sulfate sodium treatment.

Suppression of vaccine immunity by inflammatory monocytes.

Vaccine adjuvant-induced inflammation augments vaccine immunity in part by recruiting APCs to vaccine draining lymph nodes (LNs). However, the role of one APC subtype, inflammatory monocytes, in regulating vaccine immunity in healthy animals has not been fully examined in detail. Therefore, vaccine-mediated monocyte recruitment and subsequent immune responses were investigated using murine vaccination models and in vitro assays. Recruitment of inflammatory monocytes to vaccine draining LNs was rapid and mediated primarily by local production of MCP-1, as revealed by studies in MCP-1(-/-) mice. Interrupting monocyte recruitment to LNs by either transient monocyte depletion or monocyte migration blockade led to marked amplification of both cellular and humoral immune responses to vaccination. These results were most consistent with the idea that rapidly mobilized inflammatory monocytes were actually suppressing vaccine responses. The suppressive nature of vaccine-elicited monocytes was confirmed using in vitro cocultures of murine monocytes and T cells. Furthermore, it was determined that inflammatory monocytes suppressed T cell responses by sequestering cysteine, as cysteine supplementation in vitro and in vivo appreciably augmented vaccine responses. These findings indicated, therefore, that vaccination-elicited inflammation, although necessary for effective immunity, also generated potent counter-regulatory immune responses that were mediated primarily by inflammatory monocytes. Therefore, interrupting monocyte-mediated vaccine counterregulatory responses may serve as an effective new strategy for broadly amplifying vaccine immunity.

An antibody to the sixth Ig-like domain of VCAM-1 inhibits leukocyte transendothelial migration without affecting adhesion.

VCAM-1 plays a key role in leukocyte trafficking during inflammatory responses. However, molecular mechanisms underlying this function have not been clearly elucidated. In this study, using phage display technology, we developed a rabbit/human chimeric VCAM-1 Ab, termed VCAM-1 domain 6 (VCAM-1-D6), which specifically recognizes aa 511-599 within the sixth Ig-like domain. We report that the VCAM-1-D6 Ab blocked U937 cell transmigration across activated HUVECs but did not alter adhesion of U937 cells to the HUVECs. We also demonstrate that VCAM-1-D6 does not alter TNF-α-stimulated endothelial cell chemokine or cytokine production. Furthermore, through in vivo efficacy testing using a mouse islet allograft model, we demonstrate that VCAM-1-D6 significantly alleviates allograft rejection by blocking leukocyte infiltration to the grafted islets. Taken together, our results suggest that the VCAM-1-D6 Ab may block VCAM-1-mediated inflammation and could be a useful tool in treating inflammatory diseases.

Impaired humoral immunity and tolerance in K14-VEGFR-3-Ig mice that lack dermal lymphatic drainage.

Lymphatic vessels transport interstitial fluid, soluble Ag, and immune cells from peripheral tissues to lymph nodes (LNs), yet the contribution of peripheral lymphatic drainage to adaptive immunity remains poorly understood. We examined immune responses to dermal vaccination and contact hypersensitivity (CHS) challenge in K14-VEGFR-3-Ig mice, which lack dermal lymphatic capillaries and experience markedly depressed transport of solutes and dendritic cells from the skin to draining LNs. In response to dermal immunization, K14-VEGFR-3-Ig mice produced lower Ab titers. In contrast, although delayed, T cell responses were robust after 21 d, including high levels of Ag-specific CD8+ T cells and production of IFN-γ, IL-4, and IL-10 upon restimulation. T cell-mediated CHS responses were strong in K14-VEGFR-3-Ig mice, but importantly, their ability to induce CHS tolerance in the skin was impaired. In addition, 1-y-old mice displayed multiple signs of autoimmunity. These data suggest that lymphatic drainage plays more important roles in regulating humoral immunity and peripheral tolerance than in effector T cell immunity.

Defective macrophage migration in Gαi2- but not Gαi3-deficient mice.

Various heterotrimeric G(i) proteins are considered to be involved in cell migration and effector function of immune cells. The underlying mechanisms, how they control the activation of myeloid effector cells, are not well understood. To elucidate isoform-redundant and -specific roles for Gα(i) proteins in these processes, we analyzed mice genetically deficient in Gα(i2) or Gα(i3). First, we show an altered distribution of tissue macrophages and blood monocytes in the absence of Gα(i2) but not Gα(i3). Gα(i2)-deficient but not wild-type or Gα(i3)-deficient mice exhibited reduced recruitment of macrophages in experimental models of thioglycollate-induced peritonitis and LPS-triggered lung injury. In contrast, genetic ablation of Gα(i2) had no effect on Gα(i)-dependent peritoneal cytokine production in vitro and the phagocytosis-promoting function of the Gα(i)-coupled C5a anaphylatoxin receptor by liver macrophages in vivo. Interestingly, actin rearrangement and CCL2- and C5a anaphylatoxin receptor-induced chemotaxis but not macrophage CCR2 and C5a anaphylatoxin receptor expression were reduced in the specific absence of Gα(i2). Furthermore, knockdown of Gα(i2) caused decreased cell migration and motility of RAW 264.7 cells, which was rescued by transfection of Gα(i2) but not Gα(i3). These results indicate that Gα(i2), albeit redundant to Gα(i3) in some macrophage activation processes, clearly exhibits a Gα(i) isoform-specific role in the regulation of macrophage migration.

Downregulation of integrin receptor-signaling genes by Epstein-Barr virus EBNA 3C via promoter-proximal and -distal binding elements.

Epstein-Barr virus (EBV) establishes a persistent latent infection in B lymphocytes and is associated with the development of numerous human tumors. Epstein-Barr nuclear antigen 3C (EBNA 3C) is essential for B-cell immortalization, has potent cell cycle deregulation capabilities, and functions as a regulator of both viral- and cellular-gene expression. We performed transcription profiling on EBNA 3C-expressing B cells and identified several chemokines and members of integrin receptor-signaling pathways, including CCL3, CCL4, CXCL10, CXCL11, ITGA4, ITGB1, ADAM28, and ADAMDEC1, as cellular target genes that could be repressed by the action of EBNA 3C alone. Chemotaxis assays demonstrated that downregulation of CXCL10 and -11 by EBNA 3C is sufficient to reduce the migration of cells expressing the CXCL10 and -11 receptor CXCR3. Gene repression by EBNA 3C was accompanied by decreased histone H3 lysine 9/14 acetylation and increased histone H3 lysine 27 trimethylation. In an EBV-positive cell line expressing all latent genes, we identified binding sites for EBNA 3C at ITGB1 and ITGA4 and in a distal regulatory region between ADAMDEC1 and ADAM28, providing the first demonstration of EBNA 3C association with cellular-gene control regions. Our data implicate indirect mechanisms in CXCL10 and CXCL11 repression by EBNA 3C. In summary, we have unveiled key cellular pathways repressed by EBNA 3C that are likely to contribute to the ability of EBV-immortalized cells to modulate immune responses, adhesion, and B-lymphocyte migration to facilitate persistence in the host.

Cutting edge: loss of α4 integrin expression differentially affects the homing of Th1 and Th17 cells.

The neutralization of α4 integrin is currently used as treatment in several autoimmune diseases and is thought to prevent the entry of most immune cells in target tissues. In this study, we showed that selective deletion of α4 integrin in T cells did not prevent but delayed the development of experimental autoimmune encephalomyelitis. Whereas both Th1 and Th17 cells infiltrate the CNS of wild-type mice, T cells present in the CNS of mice lacking α4 integrin were mainly enriched in Th17 cells, suggesting that this T cell subset uses other integrins to access the CNS. In contrast, α4 integrin expression is important for Th1 cells to enter the CNS and for the stability of their Th1-associated genetic program. Therefore, our data suggest that anti-α4 integrin Ab treatment may be more efficient in the treatment of Th1- rather than Th17-mediated disease.