L-Plastin Promotes Gastric Cancer Growth and Metastasis in a Helicobacter pylori cagA-ERK-SP1-Dependent Manner.

Actin cytoskeleton dynamic rearrangement is required for tumor cell metastasis and is a key characteristic of Helicobacter pylori (H. pylori)-infected host cells. Actin cytoskeleton modulation is coordinated by multiple actin-binding proteins (ABP). Through Kyoto encyclopedia of gene and genomes database, GEPIA website, and real-time PCR data, we found that H. pylori infection significantly induced L-plastin, a key ABP, in gastric cancer cells. We further explored the regulation and function of L-plastin in H. pylori-associated gastric cancer and found that, mechanistically, H. pylori infection induced gastric cancer cells to express L-plastin via cagA-activated ERK signaling pathway to mediate SP1 binding to L-plastin promoter. Moreover, this increased L-plastin promoted gastric cancer cell proliferation and migration in vitro and facilitated the growth and metastasis of gastric cancer in vivo. Finally, we detected the expression pattern of L-plastin in gastric cancer tissues, and found that L-plastin was increased in gastric cancer tissues and that this increase of L-plastin positively correlated with cagA + H. pylori infection status. Overall, our results elucidate a novel mechanism of L-plastin expression induced by H. pylori, and a new function of L-plastin-facilitated growth and metastasis of gastric cancer, and thereby implicating L-plastin as a potential therapeutic target against gastric cancer. IMPLICATIONS: Our results elucidate a novel mechanism of L-plastin expression induced by H. pylori in gastric cancer, and a new function of L-plastin-facilitated gastric cancer growth and metastasis, implicating L-plastin as a potential therapeutic target against gastric cancer.

TREML4 receptor regulates inflammation and innate immune cell death during polymicrobial sepsis.

Sepsis is a biphasic disease characterized by an acute inflammatory response, followed by a prolonged immunosuppressive phase. Therapies aimed at controlling inflammation help to reduce the time patients with sepsis spend in intensive care units, but they do not lead to a reduction in overall mortality. Recently, the focus has been on addressing the immunosuppressive phase, often caused by apoptosis of immune cells. However, molecular triggers of these events are not yet known. Using whole-genome CRISPR screening in mice, we identified a triggering receptor expressed on myeloid cells (TREM) family receptor, TREML4, as a key regulator of inflammation and immune cell death in sepsis. Genetic ablation of Treml4 in mice demonstrated that TREML4 regulates calcium homeostasis, the inflammatory cytokine response, myeloperoxidase activation, the endoplasmic reticulum stress response and apoptotic cell death in innate immune cells, leading to an overall increase in survival rate, both during the acute and chronic phases of polymicrobial sepsis.

CXCR4+ cells are increased in lung tissue of patients with idiopathic pulmonary fibrosis.

BACKGROUND: CXCR4, a transmembrane-receptor located on epithelial cells that is activated by CXCL12, may have a role in IPF via migration of CXCR4+ fibrocytes to the lung. However, its expression has not been fully characterised in idiopathic pulmonary fibrosis (IPF) or other fibrotic interstitial lung diseases (ILDs). CXCL12 is constitutively expressed in the bone marrow, and levels of CXCR4 regulate control of this signalling pathway. The aim of this study was to profile the expression of CXCR4 in lung tissue and peripheral circulation of patients with IPF and other fibrotic ILDs. METHODS: Expression of CXCR4 on peripheral blood mononuclear cells (PBMCs) was examined by flow cytometry in 20 patients with IPF and 10 age-matched non-disease control (NDC) donors. Levels of CXCL12 in human plasma were measured by ELISA. Expression of CXCR4, CXCL12, CD45, and e-cadherin was assessed in IPF (n = 10), other fibrotic ILD (n = 8) and NDC (n = 10) lung tissue by multiplex immunohistochemistry (OPAL) and slides were scanned using a Vectra 3 scanner. Cells were quantified with computer automated histological analysis software (HALO). RESULTS: In blood, the number of CXCR4+ cells was lower but the level of CXCL12 was higher in patients with IPF compared to NDC donors. Elevated CXCR4 expression was detected in lung tissue from patients with IPF and other fibrotic ILDs compared to NDC. There were higher levels of CXCR4+/e-cadherin+/CXCL12+ (epithelial) cells in IPF lung tissue compared to NDC, but there was no difference in the numbers of CXCR4+/CD45+/CXCL12+ (myeloid) cells between the two groups. CONCLUSIONS: This report demonstrates that CXCR4 is overexpressed not only in IPF but also in other ILDs and expression is particularly prominent within both honeycomb cysts and distal airway epithelium. This observation supports the hypothesis that CXCR4 may drive tissue fibrosis through binding its specific ligand CXCL12. Although CXCR4 expressing cells could be either of epithelial or myeloid origin it appears that the former is more prominent in IPF lung tissue. Further characterization of the cells of the honeycomb cyst may lead to a better understanding of the fibrogenic processes in IPF and other end-stage fibrotic ILDs.

Monocyte apoptotic bodies are vehicles for influenza A virus propagation.

The disassembly of apoptotic cells into small membrane-bound vesicles termed apoptotic bodies (ApoBDs) is a hallmark of apoptosis; however, the functional significance of this process is not well defined. We recently discovered a new membrane protrusion (termed beaded apoptopodia) generated by apoptotic monocytes which fragments to release an abundance of ApoBDs. To investigate the function of apoptotic monocyte disassembly, we used influenza A virus (IAV) infection as a proof-of-concept model, as IAV commonly infects monocytes in physiological settings. We show that ApoBDs generated from IAV-infected monocytes contained IAV mRNA, protein and virions and consequently, could facilitate viral propagation in vitro and in vivo, and induce a robust antiviral immune response. We also identified an antipsychotic, Haloperidol, as an unexpected inhibitor of monocyte cell disassembly which could impair ApoBD-mediated viral propagation under in vitro conditions. Together, this study reveals a previously unrecognised function of apoptotic monocyte disassembly in the pathogenesis of IAV infections.

Plexin B2 Is a Regulator of Monocyte Apoptotic Cell Disassembly.

Billions of cells undergo apoptosis daily and often fragment into small, membrane-bound extracellular vesicles termed apoptotic bodies (ApoBDs). We demonstrate that apoptotic monocytes undergo a highly coordinated disassembly process and form long, beaded protrusions (coined as beaded apoptopodia), which fragment to release ApoBDs. Here, we find that the protein plexin B2 (PlexB2), a transmembrane receptor that regulates axonal guidance in neurons, is enriched in the ApoBDs of THP1 monocytes and is a caspase 3/7 substrate. To determine whether PlexB2 is involved in the disassembly of apoptotic monocytes, we generate PlexB2-deficient THP1 monocytes and demonstrate that lack of PlexB2 impairs the formation of beaded apoptopodia and ApoBDs. Consequently, the loss of PlexB2 in apoptotic THP1 monocytes impairs their uptake by both professional and non-professional phagocytes. Altogether, these data identify PlexB2 as a positive regulator of apoptotic monocyte disassembly and demonstrate the importance of this process in apoptotic cell clearance.

Semiquantitative Proteomics Enables Mapping of Murine Neutrophil Dynamics following Lethal Influenza Virus Infection.

Neutrophils are rapidly deployed innate immune cells, and excessive recruitment is causally associated with influenza-induced pathologic conditions. Despite this, the complete set of influenza lethality-associated neutrophil effector proteins is currently unknown. Whether the expression of these proteins is predetermined during bone marrow (BM) neutrophil maturation or further modulated by tissue compartment transitions has also not been comprehensively characterized at a proteome-wide scale. In this study, we used high-resolution mass spectrometry to map how the proteomes of murine neutrophils change comparatively across BM, blood, and the alveolar airspaces to deploy an influenza lethality-associated response. Following lethal influenza infection, mature neutrophils undergo two infection-dependent and one context-independent compartmental transitions. Translation of type I IFN-stimulated genes is first elevated in the BM, preceding the context-independent downregulation of ribosomal proteins observed in blood neutrophils. Following alveolar airspace infiltration, the bronchoalveolar lavage (BAL) neutrophil proteome is further characterized by a limited increase in type I IFN-stimulated and metal-sequestering proteins as well as a decrease in degranulation-associated proteins. An influenza-selective and dose-dependent increase in antiviral and lipid metabolism-associated proteins was also observed in BAL neutrophils, indicative of a modest capacity for pathogen response tuning. Altogether, our study provides new and comprehensive evidence that the BAL neutrophil proteome is shaped by BM neutrophil maturation as well as subsequent compartmental transitions following lethal influenza infection.

Influenza A Virus Infection Induces Viral and Cellular Defective Ribosomal Products Encoded by Alternative Reading Frames.

The importance of antiviral CD8+ T cell recognition of alternative reading frame (ARF)-derived peptides is uncertain. In this study, we describe an epitope (NS1-ARF21-8) present in a predicted 14-residue peptide encoded by the +1 register of NS1 mRNA in the influenza A virus (IAV). NS1-ARF21-8 elicits a robust, highly functional CD8+ T cell response in IAV-infected BALB/c mice. NS1-ARF21-8 is presented from unspliced NS mRNA, likely from downstream initiation on a Met residue that comprises the P1 position of NS1-ARF21-8 Derived from a 14-residue peptide with no apparent biological function and negligible impacts on IAV infection, infectivity, and pathogenicity, NS1-ARF21-8 provides a clear demonstration of how immunosurveillance exploits natural errors in protein translation to provide antiviral immunity. We further show that IAV infection enhances a model cellular ARF translation, which potentially has important implications for virus-induced autoimmunity.

Memory regulatory T cells home to the lung and control influenza A virus infection.

Memory regulatory T cells (mTregs) have been demonstrated to persist long-term in hosts after the resolution of primary influenza A virus (IAV) infection. However, whether such IAV infection-experienced (IAV-experienced) mTregs differentiate into a phenotypically and functionally distinct Treg subset and what function they play at the infection site remains poorly defined. In this study, we characterized the phenotype, examined the responsiveness and assessed the suppressive function of IAV-experienced memory Tregs (mTregs). In comparison with inexperienced naïve Tregs (nTregs), mTregs exhibited elevated expression of CD39, CD69, CD103, cytotoxic T lymphocyte-associated antigen-4, leukocyte function-associated antigen-1 and programmed cell death-1 and could be activated in an antigen-specific manner in vitro and in vivo. When mTregs and nTregs were adoptively cotransferred into recipient mice, mTregs had a competitive advantage in migrating to the IAV-infected lungs. mTregs were more capable of controlling in vitro proliferation of CD4+ and CD8+ T cells and suppressed CD40 and CD86 upregulation on bone marrow-derived dendritic cells. Adoptively transferred mTregs, but not adoptively transferred nTregs, significantly attenuated body weight loss, lung pathology and immune cell infiltration into the infected lungs after IAV infection. These results suggest that mTregs generated after IAV infection differentiate into a phenotypically distinct and functionally enhanced Treg subset that can be activated in an antigen-specific manner to exert immunosuppression. We propose vaccination to induce such mTregs as a potential novel strategy to protect against severe IAV infection.

Chenodeoxycholic Acid from Bile Inhibits Influenza A Virus Replication via Blocking Nuclear Export of Viral Ribonucleoprotein Complexes.

Influenza A virus (IAV) infection is still a major global threat for humans, especially for the risk groups: young children and the elderly. The currently licensed antiviral drugs target viral factors and are prone to viral resistance. In recent years, a few endogenous small molecules from host, such as estradiol and omega-3 polyunsaturated fatty acid (PUFA)-derived lipid mediator protection D1 (PD1), were demonstrated to be capable of inhibiting IAV infection. Chenodeoxycholic acid (CDCA), one of the main primary bile acids, is synthesized from cholesterol in the liver and classically functions in emulsification and absorption of dietary fats. Clinically, CDCA has been used in the treatment of patients with cholesterol gallstones for more than five decades. In this study, we showed that CDCA attenuated the replication of three subtypes of influenza A virus, including a highly pathogenic H5N1 strain, in A549 and MDCK cell cultures with IC50 ranging from 5.5 to 11.5 µM. Mechanistically, CDCA effectively restrained the nuclear export of viral ribonucleoprotein (vRNP) complexes. In conclusion, as an endogenous physiological small molecule, CDCA can inhibit IAV replication in vitro, at least in part, by blocking vRNP nuclear export, and affords further studies for development as a potential antiviral agent against IAV infections.

Platycodin D Suppresses Type 2 Porcine Reproductive and Respiratory Syndrome Virus In Primary and Established Cell Lines.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a continuous threat to the pork industry as it continues to cause significant economic loss worldwide. Currently, vaccination strategies provide very limited protection against PRRSV transmission. Consequently, there is an urgent need to develop new antiviral strategies. Platycodin D (PD) is one of the major bioactive triterpenoid saponins derived from Platycodon grandiflorum, a traditional Chinese medicine used as an expectorant for pulmonary diseases and a remedy for respiratory disorders. Here, we demonstrate that PD exhibits potent activity against PRRSV infection in Marc-145 cells and primary porcine alveolar macrophages. PD exhibited broad-spectrum inhibitory activities in vitro against high pathogenic type 2 PRRSV GD-HD strain and GD-XH strain as well as classical CH-1a and VR2332 strains. PD at concentrations ranging 1⁻4 µM significantly inhibited PRRSV RNA synthesis, viral protein expression and progeny virus production in a dose-dependent manner. EC50 values of PD against four tested PRRSV strains infection in Marc-145 cells ranged from 0.74 to 1.76 µM. Mechanistically, PD inhibited PRRSV replication by directly interacting with virions therefore affecting multiple stages of the virus life cycle, including viral entry and progeny virus release. In addition, PD decreased PRRSV- and LPS-induced cytokine (IFN-α, IFN-ß, IL-1α, IL-6, IL-8 and TNF-α) production in PAMs. Altogether, our findings suggested that PD is a potent inhibitor of PPRSV infection in vitro. However, further in vivo studies are necessary to confirm PD as a potential novel and effective PPRSV inhibitor in swine.

Altered NKp30, NKp46, NKG2D, and DNAM-1 Expression on Circulating NK Cells Is Associated with Tumor Progression in Human Gastric Cancer.

Natural killer (NK) cell activity is influenced by a complex integration of signaling pathways activated downstream of both activating and inhibitory surface receptors. The tumor microenvironment can suppress NK cell activity, and there is a great clinical interest in understanding whether modulating tumor-mediated NK cell suppression and/or boosting preexisting NK cell numbers in cancer patients is therapeutically viable. To this light, we characterized the surface receptor phenotypes of peripheral blood NK cells and examined their clinical relevance to human gastric cancer (GC). We found that the proportion of peripheral blood NK cells which expressed the activating receptors NKp30, NKp46, NKG2D, and DNAM-1 was significantly decreased in GC patients compared to healthy donors, and that this decrease was positively associated with tumor progression. At the same time, plasma TGF-ß1 concentrations were significantly increased in GC patients and negatively correlated with the proportion of NKp30, NKp46, NKG2D, and DNAM-1 expressing NK cells. Furthermore, TGF-ß1 significantly downregulated the expression of NKp30, NKp46, NKG2D, and DNAM-1 on NK cells in vitro, and the addition of galunisertib, an inhibitor of the TGF-ß receptor subunit I, reversed this downregulation. Altogether, our data suggest that the decreased expression of activating receptors NKp30, NKp46, NKG2D, and DNAM-1 on peripheral blood NK cells is positively associated with GC progression, and that TGF-ß1-mediated NK cell suppression may be a therapeutically targetable characteristic of GC.

The induction and consequences of Influenza A virus-induced cell death.

Infection with Influenza A virus (IAV) causes significant cell death within the upper and lower respiratory tract and lung parenchyma. In severe infections, high levels of cell death can exacerbate inflammation and comprise the integrity of the epithelial cell barrier leading to respiratory failure. IAV infection of airway and alveolar epithelial cells promotes immune cell infiltration into the lung and therefore, immune cell types such as macrophages, monocytes and neutrophils are readily exposed to IAV and infection-induced death. Although the induction of cell death through apoptosis and necrosis following IAV infection is a well-known phenomenon, the molecular determinants responsible for inducing cell death is not fully understood. Here, we review the current understanding of IAV-induced cell death and critically evaluate the consequences of cell death in aiding either the restoration of lung homoeostasis or the progression of IAV-induced lung pathologies.

CD45+CD33lowCD11bdim myeloid-derived suppressor cells suppress CD8+ T cell activity via the IL-6/IL-8-arginase I axis in human gastric cancer.

Myeloid-derived suppressor cells (MDSCs) are a prominent component of the pro-tumoral response. The phenotype of and mechanisms used by MDSCs is heterogeneous and requires more precise characterization in gastric cancer (GC) patients. Here, we have identified a novel subset of CD45+CD33lowCD11bdim MDSCs in the peripheral blood of GC patients compared to healthy individuals. CD45+CD33lowCD11bdim MDSCs morphologically resembled neutrophils and expressed high levels of the neutrophil marker CD66b. Circulating CD45+CD33lowCD11bdim MDSCs effectively suppressed CD8+ T cells activity through the inhibition of CD8+ T cell proliferation and interferon-γ (IFN-γ) and granzyme B (GrB) production. The proportion of CD45+CD33lowCD11bdim MDSCs also negatively correlated with the proportion of IFN-γ+CD8+ T cell in the peripheral blood of GC patients. GC patient serum-derived IL-6 and IL-8 activated and induced CD45+CD33lowCD11bdim MDSCs to express arginase I via the PI3K-AKT signaling pathway. This pathway contributed to CD8+ T cell suppression as it was partially rescued by the blockade of the IL-6/IL-8-arginase I axis. Peripheral blood CD45+CD33lowCD11bdim MDSCs, as well as IL-6, IL-8, and arginase I serum levels, positively correlated with GC progression and negatively correlated with overall patient survival. Altogether, our results highlight that a subset of neutrophilic CD45+CD33lowCD11bdim MDSCs is functionally immunosuppressive and activated via the IL-6/IL-8-arginase I axis in GC patients.

Inhibition of proanthocyanidin A2 on porcine reproductive and respiratory syndrome virus replication in vitro.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a widely prevalent and endemic swine pathogen that causes significant economic losses for the global pig industry annually. Currently, the most prevalent strategy for PRRSV control remains the prevention of virus transmission, with highly effective therapeutic agents and vaccines still lacking. Proanthocyanidin A2 (PA2) belongs to the family of tea polyphenols, which have been reported to exhibit a range of biological activities including anti-oxidative, cardio-protective, anti-tumoural, anti-bacterial, anti-viral, and anti-inflammatory effects in vitro as well as in vivo. Here, we demonstrate that PA2 exhibits potent anti-viral activity against PRRSV infection in Marc-145 cells. Similar inhibitory effects were also found in porcine alveolar macrophages, the primary target cell type of PRRSV infection in pigs in vivo. For traditional type II PRRSV CH-1a strain and high pathogenic GD-XH strain and GD-HD strain, PA2 exhibited broad-spectrum and comparable inhibitory activities in vitro with EC50 ranging from 2.2 to 3.2 µg/ml. Treatment of PRRSV-infected Marc-145 cells with PA2 significantly inhibited viral RNA synthesis, viral protein expression and progeny virus production in a dose-dependent manner. In addition, PA2 treatment reduced gene expressions of cytokines (TNF-α, IFN-α, IL-1ß and IL-6) induced by PRRSV infection in PAMs. Mechanistically, PA2 inhibited PRRSV replication by targeting multiple pathways including blockade of viral entry and progeny virus release. Altogether, our findings suggest that PA2 has the potential to serve as a novel prophylactic and therapeutic strategies against PRRSV infection.

The spleen in liver cirrhosis: revisiting an old enemy with novel targets.

The spleen is a secondary lymphoid organ which can influence the progression of multiple diseases, notably liver cirrhosis. In chronic liver diseases, splenomegaly and hypersplenism can manifest following the development of portal hypertension. These splenic abnormalities correlate with and have been postulated to facilitate the progression of liver fibrosis to cirrhosis, although precise mechanisms remain poorly understood. In this review, we summarize the literature to highlight the mechanistic contributions of splenomegaly and hypersplenism to the development of liver cirrhosis, focusing on three key aspects: hepatic fibrogenesis, hepatic immune microenvironment dysregulation and liver regeneration. We conclude with a discussion of the possible therapeutic strategies for modulating splenic abnormalities, including the novel potential usage of nanomedicine in non-surgically targetting splenic disorders for the treatment of liver cirrhosis.

Tumor-Associated Monocytes/Macrophages Impair NK-Cell Function via TGFß1 in Human Gastric Cancer.

Natural killer (NK) cells are a major component of the host antitumor immune response in human cancer. However, the nature, functional regulation, and clinical relevance of NK cells in gastric cancer remain largely unknown. In this study, we showed that the percentages of NK cells in tumors were significantly decreased, and low percentages of tumor-infiltrating NK cells were positively correlated with poor survival and disease progression. Although the expression of activating and inhibitory receptors on NK cells was shown to be not different between tumor and nontumor tissues, NK cells in tumors had impaired effector functions, characterized by decreased IFNγ, TNFα, and Ki-67 expression. We found that tumor-infiltrating monocytes/macrophages were physically close to NK cells, and their percentages negatively correlated with IFNγ+ and TNFα+ NK-cell percentages. Ex vivo study showed that isolated tumor-associated monocytes/macrophages could impair NK-cell expression of IFNγ, TNFα, and Ki-67. Blockade of TGFß1 attenuated such monocytes/macrophages-mediated impairment of NK-cell function. Our data suggest that human NK-cell function was impaired by tumor-associated monocytes/macrophages, and that restoring NK-cell function may be an important therapeutic strategy to prevent tumor immune escape in gastric cancer. Cancer Immunol Res; 5(3); 248-56. ©2017 AACR.

Isolation of cell type-specific apoptotic bodies by fluorescence-activated cell sorting.

Apoptotic bodies (ApoBDs) are membrane-bound extracellular vesicles that can mediate intercellular communication in physiological and pathological settings. By combining recently developed analytical strategies with fluorescence-activated cell sorting (FACS), we have developed a method that enables the isolation of ApoBDs from cultured cells to 99% purity. In addition, this approach also enables the identification and isolation of cell type-specific ApoBDs from tissue, bodily fluid and blood-derived samples.

The contributions of lung macrophage and monocyte heterogeneity to influenza pathogenesis.

The lung myeloid cell microenvironment comprises airway, alveolar and interstitial macrophages, peripheral blood recruited lung monocytes as well as residential and migratory dendritic cell subsets. Findings from fate mapping, parabiosis, transcriptome and epigenome profiling studies now indicate that tissue macrophage and monocyte subsets possess specialized functions which differentially impact homoeostatic tolerance, pathogen detection and pathogen killing. In the lungs, residential alveolar macrophages are catabolic and immunosuppressive in contrast to the classically pro-inflammatory repertoire of lung monocytes and monocyte-derived dendritic cells recruited during acute inflammation. Here, we review the identity and functions of all lung macrophage and monocyte subsets during homoeostasis and acute lung inflammation, with a special focus on their contributions to influenza virus detection, clearance and the development of influenza-induced lung pathologies. Subsequent implications for the development of new therapeutic targets against influenza-induced lung pathologies will also be discussed.

Saikosaponin A inhibits influenza A virus replication and lung immunopathology.

Fatal influenza outcomes result from a combination of rapid virus replication and collateral lung tissue damage caused by exaggerated pro-inflammatory host immune cell responses. There are few therapeutic agents that target both biological processes for the attenuation of influenza-induced lung pathology. We show that Saikosaponin A, a bioactive triterpene saponin with previouslyestablished anti-inflammatory effects, demonstrates both in vitro and in vivo anti-viral activity against influenza A virus infections. Saikosaponin A attenuated the replication of three different influenza A virus strains, including a highly pathogenic H5N1 strain, in human alveolar epithelial A549 cells. This anti-viral activity occurred through both downregulation of NF-κB signaling and caspase 3-dependent virus ribonucleoprotein nuclear export as demonstrated by NF-κB subunit p65 and influenza virus nucleoprotein nuclear translocation studies in influenza virus infected A549 cells. Critically, Saikosaponin A also attenuated viral replication, aberrant pro-inflammatory cytokine production and lung histopathology in the widely established H1N1 PR8 model of influenza A virus lethality in C57BL/6 mice. Flow cytometry studies of mouse bronchoalveolar lavage cells revealed that SSa exerted immunomodulatory effects through a selective attenuation of lung neutrophil and monocyte recruitment during the early peak of the innate immune response to PR8 infection. Altogether, our results indicate that Saikosaponin A possesses novel therapeutic potential for the treatment of pathological influenza virus infections.