Description: PALLD is an actin cross-linker supporting cellular mechanical tension. However, its involvement in the regulation of phagocytosis, a cellular activity essential for innate immunity and physiological tissue turnover, is unclear. We report that PALLD is highly induced along with all- trans -retinoic acid–induced maturation of myeloid leukemia cells, to promote Ig- or complement-opsonized phagocytosis. PALLD mechanistically facilitates phagocytic receptor clustering by regulating actin polymerization and c-Src dynamic activation during particle binding and early phagosome formation. PALLD is also required at the nascent phagosome to recruit phosphatase oculocerebrorenal syndrome of Lowe, which regulates phosphatidylinositol-4,5-bisphosphate hydrolysis and actin depolymerization to complete phagosome closure. Collectively, our results show a new function for PALLD as a crucial regulator of the early phase of phagocytosis by elaborating dynamic actin polymerization and depolymerization.

Description: Long intergenic noncoding RNAs (lincRNAs) are long noncoding transcripts (〉200 nt) from the intergenic regions of annotated protein-coding genes. One of the most highly induced lincRNAs in macrophages upon TLR ligation is lincRNA-Cox2, which was recently shown to mediate the activation and repression of distinct classes of immune genes in innate immune cells. We report that lincRNA-Cox2 , located at chromosome 1 proximal to the PG-endoperoxide synthase 2 ( Ptgs2/Cox2 ) gene, is an early-primary inflammatory gene controlled by NF-B signaling in murine macrophages. Functionally, lincRNA-Cox2 is required for the transcription of NF-B–regulated late-primary inflammatory response genes stimulated by bacterial LPS. Specifically, lincRNA-Cox2 is assembled into the switch/sucrose nonfermentable (SWI/SNF) complex in cells after LPS stimulation. This resulting lincRNA-Cox2/SWI/SNF complex can modulate the assembly of NF-B subunits to the SWI/SNF complex, and ultimately, SWI/SNF-associated chromatin remodeling and transactivation of the late-primary inflammatory-response genes in macrophages in response to microbial challenge. Therefore, our data indicate a new regulatory role for NF-B–induced lincRNA-Cox2 as a coactivator of NF-B for the transcription of late-primary response genes in innate immune cells through modulation of epigenetic chromatin remodeling.

Description: More than 350 million people are chronically infected with hepatitis B virus, and dysfunctional T cell responses contribute to persistent viral infection and immunopathogenesis in chronic hepatitis B (CHB). However, the underlying mechanisms of T cell hyporesponsiveness remain largely undefined. Given the important role of microRNA-146a (miR-146a) in diverse aspects of lymphocyte function, we investigated the potential role and mechanism of miR-146a in regulating T cell immune responses in CHB. We found that miR-146a expression in T cells is significantly upregulated in CHB compared with healthy controls, and miR-146a levels were correlated with serum alanine aminotransaminase levels. Both inflammatory cytokines and viral factors led to miR-146a upregulation in T cells. Stat1 was identified as a miR-146a target that is involved in antiviral cytokine production and the cytotoxicity of CD4 + and CD8 + T cells. In vitro blockage of miR-146a in T cells in CHB greatly enhanced virus-specific T cell activity. Therefore, our work demonstrates that miR-146a upregulation in CHB causes impaired T cell function, which may contribute to immune defects and immunopathogenesis during chronic viral infection.

Description: Critical roles of IL-27 in autoimmune diseases and infections have been reported; however, the contribution of endogenous IL-27 to tumor progression remains elusive. In this study, by using IL-27p28 conditional knockout mice, we demonstrate that IL-27 is critical in protective immune response against methyl-cholanthrene–induced fibrosarcoma and transplanted B16 melanoma, and dendritic cells (DCs) are the primary source. DC-derived IL-27 is required for shaping tumor microenvironment by inducing CXCL-10 expression in myeloid-derived suppressor cells and regulating IL-12 production from DCs, which lead to the recruitment and activation of NK and NKT cells resulting in immunological control of tumors. Indeed, reconstitution of IL-27 or CXCL-10 in tumor site significantly inhibits tumor growth and restores the number and activation of NK and NKT cells. In summary, our study identifies a previous unknown critical role of DC-derived IL-27 in NK and NKT cell–dependent antitumor immunity through shaping tumor microenvironment, and sheds light on developing novel therapeutic approaches based on IL-27.

Description: Anti-dsDNA Ab is reported to be the central pathogenic autoantibody involved in systemic lupus erythematosus (SLE) pathogenesis. However, the mechanisms involved in anti-dsDNA Ab production remain unclear. Recent evidence indicated that DNA-containing immune complexes (ICs) in circulation (termed "circulating DNA-containing ICs"), which are one of the hallmarks of SLE, might be involved in autoantibody production. In this study, we explored their potential role in anti-dsDNA Ab production and the underlying mechanisms in patients with SLE. We demonstrated that circulating DNA-containing ICs were able to induce anti-dsDNA Ab. Of note, HMGB1 in circulating DNA-containing ICs was crucial for anti-dsDNA Ab induction. The HMGB1 content of circulating DNA-containing ICs also correlated positively with anti-dsDNA Ab production in patients with SLE. Further, we revealed that the TLR2/MyD88/microRNA-155 (miR-155) pathway was pivotal for HMGB1 to confer anti-dsDNA Ab induction, and Ets-1 was a functional target of miR-155 in the induction of anti-dsDNA Ab by circulating DNA-containing ICs. Finally, we validated the expression of miR-155 and Ets-1 and their correlation with anti-dsDNA Ab production in patients with SLE. To our knowledge, this is the first report of the crucial role of HMGB1 in autoantibody production mediated by the TLR2/MyD88/miR-155/Ets-1 pathway. These findings identify a novel mechanism to account for the persistent production of anti-dsDNA Ab in SLE and a clue for developing a novel therapeutic strategy against SLE.

Description: 18 F-FPPRGD2, which was approved for clinical study recently, has favorable properties for integrin targeting and showed potential for antiangiogenic therapy and early response monitoring. However, the time-consuming multiple-step synthesis may limit its widespread applications in the clinic. In this study, we developed a simple lyophilized kit for labeling PRGD2 peptide ( 18 F-AlF-NOTA-PRGD2, denoted as 18 F-alfatide) using a fluoride–aluminum complex that significantly simplified the labeling procedure. Methods: Nine patients with a primary diagnosis of lung cancer were examined by both static and dynamic PET imaging with 18 F-alfatide, and 1 tuberculosis patient was investigated using both 18 F-alfatide and 18 F-FDG imaging. Standardized uptake values were measured in tumors and other main organs at 30 min and 1 h after injection. Kinetic parameters were calculated by Logan graphical analysis. Immunohistochemistry and staining intensity quantification were performed to confirm the expression of integrin α v β 3 . Results: Under the optimal conditions, the whole radiosynthesis including purification was accomplished within 20 min with a decay-corrected yield of 42.1% ± 2.0% and radiochemical purity of more than 95%. 18 F-alfatide PET imaging identified all tumors, with mean standardized uptake values of 2.90 ± 0.10. Tumor-to-muscle and tumor-to-blood ratios were 5.87 ± 2.02 and 2.71 ± 0.92, respectively. Conclusion: 18 F-alfatide can be produced with excellent radiochemical yield and purity via a simple, 1-step, lyophilized kit. PET scanning with 18 F-alfatide allows specific imaging of α v β 3 expression with good contrast in lung cancer patients. This technique might be used for the assessment of angiogenesis and for planning and response evaluation of cancer therapies that would affect angiogenesis status and integrin expression levels.

Description: G protein–coupled receptor kinases (GRKs) phosphorylate the activated form of G protein–coupled receptors leading to receptor desensitization and downregulation. We have recently shown that the chemokine receptor, CXCR2, couples to GRK6 to regulate cellular responses including chemotaxis, angiogenesis, and wound healing. In this study, we investigate the role of GRK6 in tumorigenesis using murine models of human lung cancer. Mice deficient in GRK6 (GRK6 –/– ) exhibited a significant increase in Lewis lung cancer growth and metastasis relative to control littermates (GRK6 +/+ ). GRK6 deletion had no effect on the expression of proangiogenic chemokine or vascular endothelial growth factor, but upregulated matrix metalloproteinase (MMP)-2 and MMP-9 release, tumor-infiltrating PMNs, and microvessel density. Because β-arrestin-2–deficient (βarr2 –/– ) mice exhibited increased Lewis lung cancer growth and metastasis similar to that of GRK6 –/– , we developed a double GRK6 –/– /βarr2 –/– mouse model. Surprisingly, GRK6 –/– /βarr2 –/– mice exhibited faster tumor growth relative to GRK6 –/– or βarr2 –/– mice. Treatment of the mice with anti-CXCR2 Ab inhibited tumor growth in both GRK6 –/– and GRK6 –/– /βarr2 –/– animals. Altogether, the results indicate that CXCR2 couples to GRK6 to regulate angiogenesis, tumor progression, and metastasis. Deletion of GRK6 increases the activity of the host CXCR2, resulting in greater PMN infiltration and MMP release in the tumor microenvironment, thereby promoting angiogenesis and metastasis. Because GRK6 –/– /βarr2 –/– showed greater tumor growth relative to GRK6 –/– or βarr2 –/– mice, the data further suggest that CXCR2 couples to different mechanisms to mediate tumor progression and metastasis.

Description: Janus kinase (JAK) 2 plays a pivotal role in the tumorigenesis of signal transducers and activators of transcription (STAT) 3 constitutively activated solid tumors. JAK2 mutations are involved in the pathogenesis of various types of hematopoietic disorders, such as myeloproliferative disorders, polycythemia vera, essential thrombocythemia, and primary myelofibrosis. Thus, small-molecular inhibitors targeting JAK2 are potent for therapy of these diseases. In this study, we screened 1,062,608 drug-like molecules from the ZINC database and 2080 natural product chemicals. We identified a novel JAK family kinase inhibitor, dehydrocrenatidine, that inhibits JAK-STAT3–dependent DU145 and MDA-MB-468 cell survival and induces cell apoptosis. Dehydrocrenatidine represses constitutively activated JAK2 and STAT3, as well as interleukin-6–, interferon- α– , and interferon- –stimulated JAK activity, and STAT phosphorylation, and suppresses STAT3 and STAT1 downstream gene expression. Dehydrocrenatidine inhibits JAKs-JH1 domain overexpression–induced STAT3 and STAT1 phosphorylation. In addition, dehydrocrenatidine inhibits JAK2-JH1 kinase activity in vitro. Importantly, dehydrocrenatidine does not show significant effect on Src overexpression and epidermal growth factor–induced STAT3 activation. Our results indicate that dehydrocrenatidine is a JAK-specific inhibitor.

Description: Tenascin-C is an extracellular matrix glycoprotein that is expressed by injured tissues and by various cancers. Recent publications showed that tenascin-C expression by cancer lesions predicts tumor growth, metastasis, and angiogenesis, suggesting tenascin-C as a potential therapeutic target. Currently there is no noninvasive method to determine tumoral tenascin-C expression in vivo. To address the need for an agent to image and quantify tenascin-C, we report the development of a radioactive PET tracer based on a tenascin-C–specific single-stranded DNA aptamer (tenascin-C aptamer). Methods: Tenascin-C aptamer was radiolabeled with 18 F and 64 Cu. PET imaging studies for the evaluation of tumor uptake and pharmacokinetics of tenascin-C aptamer were performed in comparison to a nonspecific scrambled aptamer (Sc aptamer). Results: The labeled tenascin-C aptamer provided clear visualization of tenascin-C–positive but not tenascin-C–negative tumors. The uptake of tenascin-C aptamer was significantly higher than that of Sc aptamer in tenascin-C–positive tumors. The labeled tenascin-C aptamer had fast clearance from the blood and other nonspecific organs through the kidneys, resulting in high tumor contrast. Conclusion: Our data suggest that suitably labeled tenascin-C aptamer can be used as a PET tracer to image tumor expression of tenascin-C with a high tumor-to-background ratio and might provide insightful and personalized medical data that will help determine appropriate treatment and monitoring.

Description: By sensing viral nucleic acids, host innate receptors elicit signaling pathways converging on TBK1-IFN regulatory factor (IRF)3 axis in mediating IFN-αβ induction and defense mechanisms. In contrast, viruses have evolved with diverse immune evasion/interference mechanisms to undermine innate receptor signaling and IFN response. In this regard, approaches enabling host to overcome such immune evasion/interference mechanisms are urgently needed to combat infections by epidemic/pandemic viruses. In this study, we report that protein kinase CK2 serves as a key component controlling TBK1 and IRF3 activation in IFN-inducing TLR, RIG-I–like receptors, and cGAS/STING signaling pathways. Accordingly, knocking down of CK2 expression or genetic ablation of its kinase activity resulted in elevated IFN-αβ response in response to infection by DNA and RNA viruses. Moreover, PP2A was identified as one of the intermediate phosphatases responsible for CK2-regulated IFN response, suggesting that CK2 may regulate TBK1 and IRF3 activation indirectly. Importantly, blockade of CK2 activity by small molecule inhibitor was able to activate TBK1, whereby eliciting effective host defense mechanisms against hepatitis C virus infection. Taken together, our results identify CK2 as a novel regulator of TBK1 and IRF3 and suggest that targeting CK2 by small molecular inhibitor may be a viable approach to prevent and treat viral infections.