Description: Long intergenic noncoding RNAs (lincRNAs) are long noncoding transcripts (〉200 nt) from the intergenic regions of annotated protein-coding genes. We report here that the lincRNA gene lincRNA-Tnfaip3 , located at mouse chromosome 10 proximal to the tumor necrosis factor α-induced protein 3 ( Tnfaip3 ) gene, is an early-primary response gene controlled by nuclear factor-B (NF-B) signaling in murine macrophages. Functionally, lincRNA- Tnfaip3 appears to mediate both the activation and repression of distinct classes of inflammatory genes in macrophages. Specifically, induction of lincRNA-Tnfaip3 is required for the transactivation of NF-B-regulated inflammatory genes in response to bacterial LPSs stimulation. LincRNA-Tnfaip3 physically interacts with the high-mobility group box 1 (Hmgb1), assembling a NF-B/Hmgb1/lincRNA-Tnfaip3 complex in macrophages after LPS stimulation. This resultant NF-B/Hmgb1/lincRNA-Tnfaip3 complex can modulate Hmgb1-associated histone modifications and, ultimately, transactivation of inflammatory genes in mouse macrophages in response to microbial challenge. Therefore, our data indicate a new regulatory role of NF-B-induced lincRNA-Tnfaip3 to act as a coactivator of NF-B for the transcription of inflammatory genes in innate immune cells through modulation of epigenetic chromatin remodeling.—Ma, S., Ming, Z., Gong, A.-Y., Wang, Y., Chen, X., Hu, G., Zhou, R., Shibata, A., Swanson, P. C., Chen, X.-M. A long noncoding RNA, LincRNA-Tnfaip3, acts as a coregulator of NF-B to modulate inflammatory gene transcription in mouse macrophages.

Description: Siglecs are sialic acid-binding Ig-like lectins that recognize sialoglycans via amino-terminal V-set domains. CD33-related Siglecs (CD33rSiglecs) on innate immune cells recognize endogenous sialoglycans as "self-associated molecular patterns" (SAMPs), dampening immune responses via cytosolic immunoreceptor tyrosine-based inhibition motifs that recruit tyrosine phosphatases. However, sialic acid-expressing pathogens subvert this mechanism through molecular mimicry. Meanwhile, endogenous host SAMPs must continually evolve to evade other pathogens that exploit sialic acids as invasion targets. We hypothesized that these opposing selection forces have accelerated CD33rSiglec evolution. We address this by comparative analysis of major CD33rSiglec (Siglec-3, Siglec-5, and Siglec-9) orthologs in humans, chimpanzees, and baboons. Recombinant soluble molecules displaying ligand-binding domains show marked quantitative and qualitative interspecies differences in interactions with strains of the sialylated pathogen, group B Streptococcus , and with sialoglycans presented as gangliosides or in the form of sialoglycan microarrays, including variations such as N -glycolyl and O -acetyl groups. Primate Siglecs also show quantitative and qualitative intra- and interspecies variations in expression patterns on leukocytes, both in circulation and in tissues. Taken together our data explain why the CD33rSiglec-encoding gene cluster is undergoing rapid evolution via multiple mechanisms, driven by the need to maintain self-recognition by innate immune cells, while escaping 2 distinct mechanisms of pathogen subversion.—Padler-Karavani, V., Hurtado-Ziola, N., Chang, Y.-C., Sonnenburg, J. L., Ronaghy, A., Yu, H., Verhagen, A., Nizet, V., Chen, X., Varki, N., Varki, A., Angata, T. Rapid evolution of binding specificities and expression patterns of inhibitory CD33-related Siglecs in primates.

Description: This study was designed to evaluate and characterize the molecular basis of antitumor activity of naturally occurring resveratrol (RES; 3,5,4'-trihydroxy- trans -stilbene) derivatives. The compounds were isolated from plants in previous studies and characterized spectroscopically. The antitumor activities of 31 RES derivatives, including dimers, trimers, and tetramers of RES, were evaluated using cell-based assays and validated on a murine model. Several trimeric and a tetrameric stilbenoids induced tumor cell apoptosis or growth arrest of several tumor cell lines with IC 50 values (2.8–19.7 μM), significantly lower than that of RES (IC 50 〉70 μM). Using pauciflorol B (PauB) as an example, we showed that the compound induced apoptosis p53 dependently, inducing p53 accumulation and p53-modulated gene expression in cells with wild-type p53, but not in those with nonfunctional p53. Reexpression of p53 in p53-null cells rescued cell death response. In parallel, the MAPK/p38 was activated and critical for PauB-induced killing. Interestingly, activation of p38 in p53 deficient cells was sufficient to drive cells into senescence via the p16–pRb pathway. Finally, PauB dose-dependently inhibited tumor growth on nude mice. Naturally occurring trimeric and tetrameric stilbenoids are potent antitumor agents. Those compounds exert antitumor effect through p53-dependent induction of apoptosis or senescence.—Qiao, H., Chen, X., Xu, L., Wang, J., Zhao, G., Hou, Y., Ge, H. M., Tan, R-X., Li, E. Antitumor effects of naturally occurring oligomeric resveratrol derivatives.

Description: Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 or PKD2 , and it affects over 10 million people worldwide. It is characterized by cyst formation in the kidney, liver and pancreas. Dosage changes in PKD1/PKD2 are important in ADPKD pathogenesis; therefore, their expression and function has to be strictly regulated. However, how they are regulated remain poorly understood. Recent studies have linked PKD2 regulation to endoplasmic reticulum (ER) stress that is implicated in neuronal, cardiac, and renal diseases. One major ER stress downstream is phosphorylation of eukaryotic initiation factor eIF2α by kinase PERK, which attenuates global protein translation and enhances translation of selected proteins. Here, we showed in several mammalian cell lines that PKD2 protein expression is up-regulated by different stresses that all increase phosphorylated eIF2α (P-eIF2α). Increasing P-eIF2α by overexpression or inhibiting the phosphatase activity resulted in increased PKD2. PCR and polysome-binding assays showed that ER stress does not affect the PKD2 mRNA level but increase its binding with ribosomes, indicating that P-eIF2α translationally up-regulates PKD2. By mutation analysis, we found that the upstream open reading frame (uORF) in the 5'-untranslated region of PKD2 mRNA represses PKD2 translation. Thus, ER stress and P-eIF2α translationally up-regulates PKD2 through bypassing the inhibitory uORF.—Yang, J., Zheng, W., Wang, Q., Lara, C., Hussein, S., and Chen, X.-Z. Translational up-regulation of polycystic kidney disease protein PKD2 by endoplasmic reticulum stress.

Description: Long intergenic noncoding RNAs (lincRNAs) can regulate the transcription of inflammatory genes and thus may represent a new group of inflammatory mediators with a potential pathogenic role in inflammatory diseases. Here, our genome-wide transcriptomic data show that TNF-α stimulation caused up-regulation of 171 lincRNAs and down-regulation of 196 lincRNAs in murine intestinal epithelial cells in culture. One of the up-regulated lincRNAs, lincRNA-Cox2, is an early-responsive lincRNA induced by TNF-α through activation of the NF-ĸB signaling pathway. Knockdown of lincRNA-Cox2 resulted in reprogramming of the gene expression profile in intestinal epithelial cells in response to TNF-α stimulation. Specifically, lincRNA-Cox2 silencing significantly ( P 〈 0.05) enhanced the transcription of Il12b , a secondary late-responsive gene induced by TNF-α. Mechanistically, lincRNA-Cox2 promoted the recruitment of the Mi-2/nucleosome remodeling and deacetylase (Mi-2/NuRD) repressor complex to the Il12b promoter region. Recruitment of the Mi-2/NuRD complex was associated with decreased H3K27 acetylation and increased H3K27 dimethylation at the Il12b promoter region, which might contribute to Il12b trans-suppression by lincRNA-Cox2. Thus, our data demonstrate a novel mechanism of epigenetic modulation by lincRNA-Cox2 on Il12b transcription, supporting an important role for lincRNAs in the regulation of intestinal epithelial inflammatory responses.—Tong, Q., Gong, A.-Y., Zhang, X.-T., Lin, C., Ma, S., Chen, J., Hu, G., Chen, X.-M. LincRNA-Cox2 modulates TNF-α–induced transcription of Il12b gene in intestinal epithelial cells through regulation of Mi-2/NuRD-mediated epigenetic histone modifications.

Description: Isoflurane and propofol are known to depress cardiac contraction, but the molecular mechanisms involved are not known. In this study, we determined whether decreasing myofilament Ca 2+ responsiveness underlies anesthesia-induced depression of contraction and uncovered the molecular targets of isoflurane and propofol. Force and intracellular Ca 2+ ([Ca 2+ ] i ) were measured in rat trabeculae superfused with Krebs-Henseleit solution, with or without propofol or isoflurane. Photoaffinity labeling of myofilament proteins with meta-Azi-propofol (AziP m ) and Azi-isoflurane (Azi-iso) and molecular docking were also used. Both propofol and isoflurane dose dependently depressed force from low doses (propofol, 27 ± 6 μM; isoflurane, 1.0 ± 0.1%) to moderate doses (propofol, 87 ± 4 μM; isoflurane, 3.0 ± 0.25%), without significant alteration [Ca 2+ ] i . During steady-state activations in both intact and skinned preparations, propofol and isoflurane depressed maximum Ca 2+ -activated force and increased the [Ca 2+ ] i required for 50% of activation. Myofibrils photolabeled with AziP m and Azi-iso identified myosin, actin, and myosin light chain as targets of the anesthetics. Several adducted residues in those proteins were located in conformationally sensitive regions that underlie contractile function. Thus, propofol and isoflurane decrease force development by directly depressing myofilament Ca 2+ responsiveness and have binding sites in key regions for contraction in both actin and myosin.—Meng, T., Bu, W., Ren, X., Chen, X., Yu, J., Eckenhoff, R. G., Gao, W. D. Molecular mechanism of anesthetic-induced depression of myocardial contraction.

Description: It was reported that TNF receptor type II signaling, which has the capacity to stimulate CD4 + forkhead box P3 + (Foxp3 + ) regulatory T cells (Tregs), activated the noncanonical NF- B pathway in an IKK α -dependent manner. Therefore, we studied the role of IKK α in the homeostasis of Treg population. To this end, we generated a mouse strain with conditional knockout of IKK α in CD4 cells ( Ikkα f/f :CD4.Cre) that showed a 〉60% reduction in the number of Tregs in the thymus and peripheral lymphoid tissues, whereas the number of Foxp3 – effector T cells (Teffs) remained at a normal level. The function of Tregs deficient in IKK α was examined using Rag1 –/– mice cotransferred with naive CD4 cells (nCD4s). Although wild-type (WT) Tregs inhibited colitis induced by transfer of WT nCD4s, IKK α -deficient Tregs failed to do so, which was associated with their inability to reconstitute Rag1 –/– mice. Furthermore, nCD4s deficient in IKK α also failed to reconstitute Rag1 –/– mice and were defective in proliferative responses in vitro and in vivo . Thus, our study reveals a novel role of IKK α in the maintenance of a normal Treg population and in the control of expansion of CD4 T cells. These properties of IKK α may be exploited as therapeutic strategies in the treatment of major human diseases.—Chen, X., Willette-Brown, J., Wu, X., Hu, Y., Howard, O. M. Z., Hu, Y., Oppenheim, J. J. IKK α is required for the homeostasis of regulatory T cells and for the expansion of both regulatory and effector CD4 T cells.

Description: Separation of germ cells from somatic cells is a widespread feature of animal sexual reproduction, with a core set of germ cell factors conserved among diverse animals. It is not known what controls their conserved gonad-specific expression. Core components of epigenetic machinery are ancient, but its role in conserved tissue expression regulation remains unexplored. We found that promoters of the reproductive genes BOULE and DAZL exhibit differential DNA methylation, consistent with their gonad-specific expression in humans and mice. Low or little promoter methylation from the testicular tissue is attributed to spermatogenic cells of various stages in the testis. Such differential DNA methylation is present in the orthologous promoters not only of other mammalian species, but also of chickens and fish, supporting a highly conserved epigenetic mechanism. Furthermore, hypermethylation of DAZL and BOULE promoters in human sperm is associated with human infertility. Our data strongly suggest that epigenetic regulation may underlie conserved germ-cell–specific expression, and such a mechanism may play an important role in human fertility.—Zhang, C., Xue, P., Gao, L., Chen, X., Lin, K., Yang, X., Dai, Y., Xu, E. Y. Highly conserved epigenetic regulation of BOULE and DAZL is associated with human fertility.

Description: The immune systems can be altered by spaceflight in many aspects, but microgravity-related mucosal immune changes and its clinical significance have not been well studied. The purpose of this study was to investigate whether simulated microgravity influences the intestinal homeostasis and increases the susceptibility to colon inflammation. The hindlimb unloading (HU) mouse model was used to simulate the microgravity condition. Three percent dextran sulfate sodium (DSS) was given to mice to induce colitis. Compared to ground control (Ctrl) mice, the HU ones revealed an impaired intestinal homeostasis and increased susceptibility to DSS-induced colitis. This includes an early-onset, 4-fold expansion of segmented filamentous bacteria (SFB), more than 2-fold decrease in regulatory T (T reg ) cell numbers and IL-10 production, ~2-fold increase in colonic IL-1β expression, 2-fold increase in circulating neutrophils, and colonic neutrophil infiltration. The application of antibiotics ameliorated the T reg and IL-10 reductions but did not significantly dampen neutrophilia and elevated expression of colonic IL-1β. These results indicate that the intestinal microflora and innate immune system both respond to simulated microgravity and together, contribute to the proinflammatory shift in the gut microenvironment. The data also emphasize the necessity for evaluating the susceptibility to inflammatory bowel diseases (IBDs) in distant space travels.—Li, P., Shi, J., Zhang, P., Wang, K., Li, J., Liu, H., Zhou, Y., Xu, X., Hao, J., Sun, X., Pang, X., Li, Y., Wu, H., Chen, X., Ge, Q. Simulated microgravity disrupts intestinal homeostasis and increases colitis susceptibility.

Description: The mechanism by which IFN-α regulates the host response to Mycobacterium tuberculosis ( M.tb ) infection in humans is poorly understood. In the present study, we found that freshly isolated pleural fluid mononuclear cells (PFMCs) from tuberculous pleural effusion but not peripheral blood mononuclear cells (PBMCs) spontaneously expressed IFN-α and IL-1β in vivo . In addition, exogenous IFN-α significantly inhibited production of IL-1β in PFMCs after stimulation with Bacillus Calmette-Guérin (BCG). To further evaluate the effect of endogenous IFN-α on BCG-induced IL-1β production, a neutralizing antibody to IFN-α was added to the cultures of BCG-stimulated PFMCs. As expected, neutralization of IFN-α by antibody significantly enhanced the production of IL-1β. Notably, we showed that IFN-α inhibited production of IL-1β through 2 distinct mechanisms: IFN-α signaling, via the STAT1 transcription factor, suppressed caspase-1-dependent IL-1β maturation, and IFN-α induced the production of IL-10 in a STAT1-dependent manner in which IL-10 reduced the abundance of IL-1β. In contrast, we found that IFN-α enhanced the production of IFN-, and IFN- also suppressed IL-1β production in the PFMCs during BCG stimulation. Our findings demonstrate that IFN-α employs distinct pathways for regulating IL-1β production and reveal that in the case of M.tb infection, the induction of IFN-α and IFN- might be associated with M.tb immune escape and disease progression in infected humans.—Ma, J., Yang, B., Yu, S., Zhang, Y., Zhang, X., Lao, S., Chen, X., Li, B., Wu, C. Tuberculosis antigen-induced expression of IFN-α in tuberculosis patients inhibits production of IL-1β.