Changes in H3K27ac following lipopolysaccharide stimulation of nasopharyngeal epithelial cells

Background

The epithelium is the first line of defense against pathogens. Notably the epithelial cells lining the respiratory track are crucial in sensing airborne microbes and mounting an effective immune response via the expression of target genes such as cytokines and chemokines. Gene expression regulation following microbial recognition is partly regulated by chromatin re-organization and has been described in immune cells but data from epithelial cells is not as detailed. Here, we report genome-wide changes of the H3K27ac mark, characteristic of activated enhancers and promoters, after stimulation of nasopharyngeal epithelial cells with the bacterial endotoxin Lipopolysaccharide (LPS).

Results

In this study, we have identified 626 regions where the H3K27ac mark showed reproducible increase following LPS induction in epithelial cells. This indicated that sensing of LPS led to opening of the chromatin in our system. Moreover, this phenomenon seemed to happen extensively at enhancers regions and we could observe instances of Super-enhancer formation. As expected, LPS-increased H3K27ac regions were found in the vicinity of genes relevant for LPS response and these changes correlated with up-regulation of their expression. In addition, we found the induction of H3K27ac mark to overlap with the binding of one of the NF-kB members and key regulator of the innate immune response, RELA, following LPS sensing. Indeed, inhibiting the NF-kB pathway abolished the deposition of H3K27ac at the TNF locus, a target of RELA, suggesting that these two phenomena are associated.

Conclusions

Enhancers’ selection and activation following microbial or inflammatory stimuli has been described previously and shown to be mediated via the NF-kB pathway. Here, we demonstrate that this is also likely to occur in the case of LPS-sensing by nasopharyngeal epithelial cells as well. In addition to validating previous findings, we generated a valuable data set relevant to the host immune response to epithelial cell colonizing or infecting pathogens.

     

超声处理对PRC2相关蛋白染色质免疫共沉淀测序结果的影响

超声处理是染色质免疫共沉淀(Chromatin immunoprecipitation,Ch IP)实验过程中染色质片段化的重要手段之一。选用多梳抑制复合物2 (Polycomb repressive complex 2,PRC2)相关蛋白组蛋白甲基转移酶EZH2及其催化产物H3K27me3为代表,研究不同分子量大小的蛋白在不同超声处理时间下对染色质免疫共沉淀测序(Chromatin immunoprecipitation sequencing,Ch IP-seq)实验的影响,结果表明在启动子区或非启动子区,不同超声时间下小分子量组蛋白H3K27me3结合位点的注释基因均无明显差异,说明超声时间对组蛋白Ch IP-seq数据影响不大。与组蛋白不同,超声时间从10 min延长至20 min后启动子区EZH2新增结合位点的注释基因能够显著聚类在与肌动蛋白丝组装等相关通路上。超声20min相比10min,非启动子区EZH2新增基因的GO(Gene ontology)聚类通路要远多于丢失基因,且超声30 min相比20 min,非启动子区丢失基因的GO聚类通路要远多于新增基因,这些通路大多与RNA聚合酶Ⅱ(RNApolymeraseⅡ,RNAPII)、器官发育、细胞形态发生相关。这表明超声时间不足或过长均会导致EZH2的基因组定位信息的不全。另外,超声主要影响启动子区中的PRC2非结合区域及二价启动子区域的EZH2结合位点,还影响非启动子区中PRC2结合区域、PRC2不结合区域以及活化态增强子区域的EZH2结合位点。综上,建议对大分子量的染色质修饰相关蛋白优化超声处理时间,使形成的染色质片段聚集在100–500 bp可获得比较全面的基因组信息。对小分子量的组蛋白来说,超声时间对Ch IP-seq结果影响不大。     

Cooperation of erythrocytes with leukocytes in immune response of a teleost <Emphasis Type="Italic">Oplegnathus fasciatus</Emphasis>

The most abundant cell type in the blood of mammals and fish is erythrocyte. Unlike mammalian erythrocyte, fish erythrocyte is nucleated. The functional differentiation of teleost erythrocyte is insufficient compared with that of mammals. Therefore, fish erythrocyte may have different functions from that of mammals. Functional interaction between erythrocyte and leukocyte was confirmed by the cDNA microarray newly constructed in this study to investigate characterization of rock bream erythrocyte. In this study, different immune related genes of erythrocytes were annotated by microarray analysis. Microarray analysis showed that a total of 338 genes were up-regulated in co-cultured erythrocytes with leukocytes by LPS stimulation when comparing to erythrocytes stimulated LPS. Many genes in erythrocyte of rock bream were up-regulated in presence of leukocytes, suggesting that erythrocytes interact with leukocytes to trigger expression of various genes associated with the immune system. Our results provide valuable information that direct and indirect immunological function of fish erythrocyte.     

Microglia initiate central nervous system innate and adaptive immune responses through multiple TLRs

Microglia are the resident macrophage-like population in the CNS. Microglia remain quiescent until injury or infection activates the cells to perform effector inflammatory and APC functions. Our previous studies have shown that microglia infected with a neurotropic strain of Theiler's murine encephalomyelitis virus secreted innate immune cytokines and up-regulated costimulatory molecules and MHC class II, enabling the cells to present viral and myelin Ags to CD4+ T cells. Recently, TLRs have been shown to recognize pathogen-associated molecular patterns and initiate innate immune responses upon interaction with infectious agents. We examined TLR expression on brain microglia and their functional responses upon stimulation with various TLR agonists. We report that mouse microglia express mRNA for all of the recently identified TLRs, TLR1-9, used for recognition of bacterial and viral molecular patterns. Furthermore, stimulation of quiescent microglia with various TLR agonists, including LPS (TLR4), peptidoglycan (TLR2), polyinosinic-polycytidylic acid (TLR3), CpG DNA (TLR9), and infection with viable Theiler's murine encephalomyelitis virus, activated the cells to up-regulate unique patterns of innate and effector immune cytokines and chemokines at the mRNA and protein levels. In addition, TLR stimulation activated up-regulation of MHC class II and costimulatory molecules, enabling the microglia to efficiently present myelin Ags to CD4+ T cells. Thus, microglia appear to be a unique and important component of both the innate and adaptive immune response, providing the CNS with a means to rapidly and efficiently respond to a wide variety of pathogens.     

Identification of noncoding transcripts from within CENP-A chromatin at fission yeast centromeres

The histone H3 variant CENP-A is the most favored candidate for an epigenetic mark that specifies the centromere. In fission yeast, adjacent heterochromatin can direct CENP-A(Cnp1) chromatin establishment, but the underlying features governing where CENP-A(Cnp1) chromatin assembles are unknown. We show that, in addition to centromeric regions, a low level of CENP-A(Cnp1) associates with gene promoters where histone H3 is depleted by the activity of the Hrp1(Chd1) chromatin-remodeling factor. Moreover, we demonstrate that noncoding RNAs are transcribed by RNA polymerase II (RNAPII) from CENP-A(Cnp1) chromatin at centromeres. These analyses reveal a similarity between centromeres and a subset of RNAPII genes and suggest a role for remodeling at RNAPII promoters within centromeres that influences the replacement of histone H3 with CENP-A(Cnp1).     

Expression of the adaptor protein hematopoietic Src homology 2 is up-regulated in response to stimuli that promote survival and differentiation of B cells

Analysis of hematopoietic Src homology 2 (HSH2) protein expression in mouse immune cells demonstrated that it is expressed at low levels in resting B cells but not T cells or macrophages. However, HSH2 expression is up-regulated within 6-12 h in response to multiple stimuli that promote activation, differentiation, and survival of splenic B cells. HSH2 expression is increased in response to anti-CD40 mAb, the TLR ligands LPS and CpG DNA, and B lymphocyte stimulator (BLyS), a key regulator of peripheral B cell survival and homeostasis. Stimulation of B cells with anti-CD40 mAb, LPS, CpG DNA, or BLyS has previously been shown to induce activation of NF-kappaB. In agreement with this finding, up-regulation of HSH2 expression in response to these stimuli is blocked by inhibitors of NF-kappaB activation and is potentiated by stimulation with PMA, suggesting that HSH2 expression is dependent on NF-kappaB activation. In contrast to CD40, BAFF receptor, TLR4, and TLR9 mediated signaling, stimulation of splenic B cells via the BCR was not observed to induce expression of HSH2 unless the cells had been stimulated previously through CD40. Finally, HSH2 expression is down-regulated in splenic B cells in response to stimulation with IL-21, which has been shown to induce apoptosis, even in the presence of anti-CD40 mAb, LPS, or CpG DNA. IL-21 stimulation also results in down-regulation of antiapoptotic proteins such as Bcl-x(L) and up-regulation of proapoptotic proteins like Bim. Therefore, HSH2 expression is coordinately up-regulated with known antiapoptotic molecules and directly correlates with B cell survival.     

In vitro and in vivo evaluation of intrinsic immunogenicity of reporter and insulin gene therapy plasmids

BACKGROUND: Plasmid DNA vectors offer the potential of safe gene therapy avoiding viral vector-mediated toxicity and immunogenicity. As plasmid DNA is bacterial in origin, presence of bacterial lipopolysaccharide (LPS) or unmethylated CpG dinucleotides may stimulate host innate immunity. METHODS: Primary cultures of mouse and rat dendritic cells were established and incubated with bacterial lipopolysaccharide; immunostimulatory CpG oligodeoxynucleotide; control GpC oligodeoxynucleotide; and a range of (pVR1012) plasmids encoding transgenes with increasing CpG content (wild-type and mutant human preproinsulin; non-mammalian eukaryotic eGFP reporter gene; and bacterial beta-galactosidase reporter gene). IL-12 secretion was assayed to determine in vitro plasmid immunogenicity. Local inflammatory response following intramuscular injection of these plasmids, with or without a non-ionic carrier SP1017, was characterised in vivo. RESULTS: Dose-responsive LPS and CpG stimulation of IL-12 secretion from dendritic cells was demonstrated. All plasmids induced significant IL-12 secretion in comparison to control unstimulated cells. The beta-galactosidase plasmid had highest CpG content and induced significantly higher IL-12 secretion than constructs containing a eukaryotic transgene. Injection of rat muscle with the beta-galactosidase construct induced greater inflammatory response than human preproinsulin constructs. This was further enhanced by SP1017. At 2 days post-injection, monocyte/macrophage injection site infiltration predominated with CD8-positive lymphocytes predominating at 7 days. There was no evidence of transgene expression in infiltrating immune cells. CONCLUSIONS: Dendritic cell immunostimulation may be employed as an in vitro bioassay of innate immune response to plasmid DNA vectors during evaluation for clinical gene therapy.