extfieldsofstudy: Biology Medicine

text:
A growth factor-free culture system underscores the coordination between Wnt and BMP signaling in Lgr5+ intestinal stem cell maintenance

Lgr5+ intestinal stem cells (ISCs) drive the fast renewal of intestinal epithelium. Several signaling pathways have been shown to regulate ISC fates. However, it is unclear what are the essential signals to sustain the ISC self-renewal. Here we show that coordination between Wnt and BMP signaling activity is necessary and sufficient to maintain Lgr5+ ISCs self-renewal. The key function of R-spondin1 is to achieve a high activity of Wnt signaling in the organoid culture. Using the GSK3 inhibitor CHIR-99021 and the BMP type I receptor inhibitor LDN-193189, we can maintain Lgr5+ ISCs without growth factors in vitro. Our results define the basic signaling pathways sustaining Lgr5+ ISCs and set up a convenient and economical culture system for their in vitro expansion. This work also set up an example for growth factor-free culture of other adult stem cells.

Introduction
Lgr5 + intestinal stem cells (ISCs) residing at the intestinal crypt bottom are required for the fast renewal of intestinal epithelium. Their fates are determined by environmental cues, including Wnt 1-3 , Notch 4,5 , Hippo 6,7 , bone morphogenetic protein (BMP) [8][9][10] , and epidermal growth factor (EGF) 11 . The establishment of in vitro culture system for Lgr5 + ISCs has greatly facilitated our understanding of how these stem cells are maintained and their fate is determined 12 . Requirement of EGF, Noggin, and R-spondin1 in this culture system emphasizes the importance of those signaling pathways. However, it was reported that EGF is dispensable for Lgr5 + ISC maintenance in vitro 13,14 , and the exact function of R-spondin1 in Lgr5 + ISC maintenance is unclear [15][16][17] . By establishing a novel culture system with chemicals, we not only established a convenient and economic method to expand Lgr5 + ISCs, but also identified the basic signaling pathways for Lgr5 + ISC maintenance.

Results and discussion
Wnt activation and BMP inhibition are necessary and sufficient for Lgr5 + ISCs maintenance in vitro To identify the minimal essential requirement of external signals for Lgr5 + ISCs maintenance, we used various combinations of growth factors to culture small intestinal crypts derived from Lgr5-EGFP-IRES-creERT2 mice 18 , and found that R-spondin1 could sustain organoid survival for 2 weeks, while EGF and Noggin were dispensable (Fig. 1a,  Supplementary Fig. S1a). However, removal of the BMP antagonist Noggin led to a dramatic decrease of Lgr5 + ISCs, whereas EGF had no contribution for Lgr5 + ISCs maintenance (Fig. 1b, c, Supplementary Fig. S1b, c). These data demonstrated the essential role of R-spondin1 and Noggin, but not EGF, in the in vitro maintenance of Lgr5 + ISCs.
R-spondin1 is a Wnt agonist 16,17 , but it cannot be replaced by Wnt ligands in organoid culture. On the contrary, loss-of-function mutations of Adenomatosis Polyposis Coli (APC), the negative regulator of Wnt/βcatenin signaling, could sustain organoid culture without R-spondin1 19,20 . These results prompted us to hypothesize that organoid growth might need a high activity of Wnt/β-catenin signaling, which could not be achieved by Wnt ligand alone. During our study, we found 5 or 10 μM GSK3 inhibitor CHIR-99021 (CHIR) treatment could activate Wnt/β-catenin signaling to the similar level of Wnt plus R-spondin1 with Topflash-luciferase reporter assay in HEK293T cells ( Supplementary Fig. S1d). To test our hypothesis, we used different doses of CHIR to culture organoids and found that low dose CHIR could not sustain organoids survival ( Supplementary Fig. S1e). Consistently, F106A mutation of R-spondin1, which lost its Wnt activation ability 21 (Supplementary Fig. S1d), could not support organoid culture. When CHIR concentration was increased to 10 μM, it alone could maintain the organoid survival and proliferation (Supplementary Fig. S1e, f), although GFP + ISCs were very rare in the organoids ( Supplementary Fig. S1g, h). Although both 5 and 10 μM CHIR could activate Wnt signaling to the similar level in the reporter assay (Supplementary Fig. S1d), only 10 μM CHIR could maintain organoid survival ( Supplementary Fig. S1e). In line with it, only 10 μM CHIR could enhance the Wnt target gene expression to the similar level of R-spondin1 treatment, and the Lgr5 + ISC signature gene expression was significantly higher than treated with 5 μM CHIR in cultured organoids ( Supplementary Fig. S1i). These results suggest that the major function of R-spondin1 in organoid culture is to maintain a high activity of Wnt/β-catenin signaling.
Lgr5 + ISCs can be maintained in a growth factor-free culture system As 10 μM CHIR could substitute R-spondin1 to sustain organoids survival, we tested if CHIR could replace R-spondin1 and cooperate with Noggin to maintain Lgr5 +  Representative bright-field (d), GFP fluorescence images (e), and FACS analysis of GFP expression (f) of small intestinal organoids cultured with ENR and 2ki for 2 months. g Scatter plots comparing the gene expression between ENR and 2ki organoids (spearman correlation is 0.976). The Lgr5 + ISC signature genes are showed in blue dots. h Heat maps showing the RNA expression of Lgr5 + ISC signature genes. i Lgr5-GFP cells sorted from organoids cultured with ENR or 2ki for 2 months were shifted to ENRW culture for 7 days, and then representative images and quantitation of colony numbers were shown. Scale bars, 100 μm. ***P < 0.001, **P < 0.01, and *P < 0.05 analyzed by two-way ANOVA test. Error bars, s.d. n = 4 wells from 2 mice in c and f, n = 9 wells from 3 mice in a, and n = 6 wells from 2 mice in i. Refer to Supplementary Figure 3 for detailed analysis ISCs. As shown in Fig. 1d-f, CHIR and Noggin were able to sustain Lgr5 + ISC self-renewal and proliferation. Then we assessed if Noggin could be replaced by the BMP type I receptor inhibitor LDN-193189 (LDN) to support Lgr5 + ISC self-renewal in the presence of CHIR. As shown in Fig. 1d, e, LDN and CHIR were sufficient to sustain Lgr5 + ISCs without addition of growth factors, and LDN increased Lgr5 + ISCs in a dose dependent manner (Supplementary Fig. S2a).
As 0.5 μM LDN did not further increase the percentage of Lgr5 + ISCs, 10 μM CHIR and 0.2 μM LDN were employed for organoid culture from crypts, and we named this culture system as 2ki culture system. The morphology of the most 2ki-cultured organoids was similar to that of ENR-cultured ones, while some were round shaped, alike the ones cultured with ENR plus Wnt3a (Fig. 1d, Supplementary Fig. S2b). In line with that, 10 μM CHIR induced a high Wnt signaling activity ( Supplementary  Fig. S1d). Further, the colony number was slightly higher in the 2ki culture system ( Supplementary Fig. S2b). Fluorescence-activated cell sorting (FACS) analysis showed that 2ki culture yielded more GFP + cells, but GFP intensity was slightly lower (Fig. 1e). EdU labeling indicated that the cell proliferation ability was similar in the two culture systems (Fig. 1f). Immunoblotting analysis revealed that activation of AKT and p38 was attenuated, and the β-catenin level was slightly increased, while the Smad1/5 phosphorylation and YAP levels remained similar in 2ki-cultured organoids compared to ENRcultured ones ( Supplementary Fig. S2c).
We have previously shown that blebbistatin could promote survival of crypts 22 , and Yin et al. demonstrated that valproic acid (VPA) increased Lgr5 + ISCs in ENRcultured organoids 14 . We then examined their effect in the 2ki culture system and found that blebbistatin markedly increased the organoid number ( Supplementary  Fig. 2d), while VPA had no effect at 0.5 μM, even slightly reduced the number at 1 μM ( Supplementary Fig. S2e).
We further found that the 2ki system could not only culture single Lgr5 + cells to form organoids but also increase the number of formed organoids, even more efficient than ENR plus Wnt3a (ENRW) culture (Fig. 2a). In the single cell-derived organoids, more GFP + cells were detected in the 2ki culture than that in the ENRW culture (Fig. 2b, c).
CHIR and LDN were able to maintain organoids and Lgr5 + ISCs for a long time, and the morphology was similar in 2ki organoids and ENR ones after 10 passages in 2 months (Fig. 2d). The percentage of Lgr5 + ISCs was higher in 2ki organoids, although the GFP intensity was slightly lower (Fig. 2e, f, Supplementary Fig. S2g). Transcriptome analysis also indicated that the gene expression profile, including the Lgr5 + ISC signature genes, was similar in the GFP + ISCs cultured with ENR or 2ki ( Fig. 2g, h). Furthermore, the two culture systems maintained the Lgr5 + ISCs with similar colony formation abilities (Fig. 2i), and the cells cultured with 2ki showed a normal karyotype after 20 passages (2n = 40 chromosomes; Supplementary Fig. S2f). These results together indicate that CHIR and LDN can maintain Lgr5 + ISCs in a long term with normal characteristics.
A high Wnt activity is needed to culture colon crypts 12 . Then we tested whether CHIR and LDN were sufficient to culture colon crypts. As shown in Supplementary Fig. S3a, 2ki culture markedly increased the colony number when compared to ENRW culture. Cell proliferation, GFP + ISC number and GFP intensity were also dramatically increased in 2ki culture ( Supplementary Fig. S3b-d). More Lgr5 + ISCs in 2ki culture could be due to higher Wnt signaling activity mediated by CHIR. Indeed, qPCR showed that in addition to higher mRNA levels of the ISC markers Lgr5 and Ascl2, the mRNA levels of the Wnt targets Axin2 and MMP7 were higher in 2ki organoids than ENRW organoids (Supplementary Fig. S3e). In accordance with the results obtained with small intestine organoids, the mRNA levels of the secretory cell markers Chga and Muc2 were similar in the two culture systems, while the enterocyte marker Alpi was lower in 2ki organoids ( Supplementary Fig. S3e). Immunofluorescence analysis also showed that Muc2 protein levels were similar in the two culture systems (Supplementary Fig. S3f). Single-cell colony formation assay of colon Lgr5 + ISCs showed that 2ki was much better than ENRW to culture colon ISCs (Supplementary Fig. S4a). Colon Lgr5 + ISCs could be maintained in 2ki condition for more than 2 months after 10 passages (Supplementary Fig. S4b-d), and retained the differentiation ability ( Supplementary  Fig. S4e).
Wnt and BMP coordinate with each other to maintain Lgr5 + ISCs The above results indicate that activation of Wnt/βcatenin signaling and inhibition of BMP/Smad signaling are sufficient to maintain the self-renewal of Lgr5 + ISCs. We then explored whether the coordination of the two signaling pathways is necessary to sustain Lgr5 + ISCs. Low-dose CHIR (5 μM) was unable to support the survival of organoids (Fig. 4a). Although adding LDN or rising CHIR to 10 μM could successfully maintain the organoid survival, the Lgr5 + ISCs were well maintained only in the condition with high Wnt/β-catenin signaling activity and abolishment of BMP/Smad signaling (Fig. 4b, c). This is supported by the data that genetic ablation of BMP signaling through disrupting the Alk3 or Smad4 gene greatly enhanced the GFP + cells population in CHIR-cultured organoids (Fig. 4d, e). Apcdeficient Lgr5 + cells could be maintained in the condition absent of Noggin 23 . However, if BMP4 was included in the culture medium, both the colony number and size dramatically decreased (Fig. 4f). In line with this, EdU stained proliferating cells decreased a lot after BMP4 treatment (Fig. 4g). In addition, BMP treatment led to the loss of Lgr5 + ISCs in the Apc mutant organoids (Fig. 4h, i). Consistently, BMP4 downregulated most Lgr5 + ISC signature genes, except Ascl2 and Sox9 (Fig. 4j). BMP also altered differentiation marker expression, increasing the enterocyte marker Alpi Although EGFR signaling is critical for intestinal regeneration and tumor formation 6 , EGF is dispensable as the endogenous EGF activation may be enough to maintain cell proliferation in cultured organoids 13,14 . R-spondin1 can be replaced by the GSK3 inhibitor CHIR, suggesting that the unequal role of Wnt ligands and Rspondin 15 could be the result of their differential activation ability for Wnt/β-catenin signaling. The successful long-term maintenance of Lgr5 + ISCs with the two small molecules CHIR and LDN provides a convenient and cheap culture system for the study and expansion of intestinal stem cells in vitro. This system may also be used for the culture and large quantity expansion of human intestinal stem cells to treat intestinal degeneration diseases.

Mice
Lgr5-EGFP-IRES-creERT2 18 and Apc fl/fl mice were obtained from Jackson Laboratory. Alk3 fl/fl mice 24 were kindly provided by Yuji Mishina and Vil-creERT2 mice 25 were a gift from Sylvie Robine. Smad4 fl/fl mice 26 were from Xiao Yang. All mice were back-crossed into the C57BL/6 genetic background for at least 10 generations. The 8-to 16-week-old mice were used for crypts isolation. For gene-ablation experiments, 6-to 8-week-old mice were injected intraperitoneally with 100 μl tamoxifen in sunflower oil at 20 mg/ml for 5 consecutive days. One month later, Alk3 −/− ;Lgr5-EGFP-IRES-creERT2;Vil-creERT2 and Smad4 −/− ;Lgr5-EGFP-IRES-creERT2;Vil-creERT2 mice were sacrificed for crypts isolation. Apc −/− ; Lgr5-EGFP-IRES-creERT2;Vil-creERT2 mice were used 2-3 months after tamoxifen injection. All animal studies were performed in accordance with the relevant guidelines and under the approval of the Institutional Animal Care and Use Committee of Tsinghua University.

Isolation of intestinal crypts and organoid culture
Intestinal crypts were isolated and cultured as previously described 22 . Briefly, mouse intestine was cut longitudinally and washed three times with cold PBS. Villi were carefully scraped away and small pieces (5 mm) of intestine were incubated in 2 mM EDTA in PBS for 40 min on ice. These pieces were then vigorously suspended in cold PBS and the mixture was passed through 70 μm cell strainer (BD Biosciences). The crypt fraction was enriched through centrifugation (3 min at 300-400 g). Then the crypts were embedded in growth factor-reduced Matrigel (BD Biosciences, 356231) and seeded on 48-well plate or 24-well plate. After polymerization, crypt culture medium (Advanced DMEM/F12 supplemented with Penicillin/Streptomycin, GlutaMAX-I, N2, B27, and Nacetylcysteine (Invitrogen)) containing EGF (50 ng/ml, Invitrogen), Noggin (100 ng/ml, R&D) and R-spondin1 (500 ng/ml, R&D) (ENR) or CHIR-99021 (Selleck, S1263) and LDN-193189 (Selleck, S2618) were added as indicated in the figure legends and refreshed every 2-3 days. For colon culture, 100 ng/ml recombinant Wnt3a protein (R&D, 1324-WN-500/CF) was added in the ENRW group.
For passaging, the organoids embedded in Matrigel in each well were directly suspended in 1 ml cold PBS after removal of medium and were pelleted by centrifugation (3 min at 300-400 g). The pelleted organoids were embedded in fresh Matrigel and seeded on plate followed by addition of culture medium as indicated in the figure legends. For single-cell culture, GFP + cells sorted from FACS were embedded in the matrigel and were cultured in the crypt culture medium as indicated in the figure legends. For organoid colony quantification, about 500 crypts were seeded each well and the survived organoids were counted 3 days later. For passaging, the organoids embedded in Matrigel were suspended in 1 ml cold PBS and pelleted by centrifugation (3 min at 300-400 × g). The pelleted organoids were embedded in fresh Matrigel and seeded on a plate followed by addition of indicated culture medium. For single-cell culture, 5000 GFP + cells sorted from FACS were cultured with the indicated medium together with 10 μM blebbistatin. In single cell culture, LDN was reduced to 100 nM.

Flow cytometry
Fresh intestinal crypts isolated from Lgr5-EGFP-IRES-creERT2 mice were incubated in TrypLE (Invitrogen) for 20 min at 37°C to obtain single-cell suspension. The organoids embedded in Matrigel were first suspended in cold PBS after medium discarding, pelleted by centrifugation (3 min at 300-400 × g) and then incubated in TrypLE for 20 min at 37°C to obtain single-cell suspension. The dissociated cells were stained with propidium iodide (PI) and passed through 40 μm cell strainer (BD), and then single GFP-high cells were analyzed or sorted by flow cytometry (MoFlo XDP, Beckman). PI-negative cells were gated as GFP-positive and GFP-negative populations and analyzed with Summit 5.3 or Flowjo software.

Chromosome analysis
For karyotyping, organoids were pre-treated with colchicine for 6 h before harvested for single cell dissociation using TrypLE. After centrifugation, cell pellets were resuspended with 75 mM KCl and incubated for 15 min at room temperature. Cells were centrifuged, fixed in methanol:acetic acid (3:1) on ice and dropped onto glass slides. Air-dried cells were stained with Giemsa staining, and metaphase spreads were imaged and analyzed. A total of ten metaphases were examined and all had the normal karyotype.

RNA extraction and qRT-PCR
Total RNA was extracted with RNeasy Mini Kit (Qiagen) according to the manufacturer's instruction. cDNA was prepared using Revertra Ace (Toyobo). qRT-PCR was performed with TransStart Green qPCR SuperMix (Transgen Biotech) in triplicates on a LightCycler 480 (Roche) with Gapdh as the reference gene. Data were analyzed according to the ΔCT method.

RNA-seq
Organoids derived from Lgr5-EGFP-IRES-creERT2 mice were cultured in the medium containing ENR or CL for 2 months. The medium was refreshed every 2-3 days, and the organoids were passaged normally. The organoids were dissociated into single cell suspension and single GFP high cells were then sorted by flow cytometry. RNA was purified from 200,000 single GFP high cells using the RNeasy Mini Kit (Qiagen) and converted into cDNA libraries using the Ovation® RNA-Seq System V2 kit (NuGEN). High-throughput sequencing was performed using the Illumina HiSeq X Ten. The RNA-seq was carried out with two biological replicates. The RNA-seq data were uniquely mapped to mm9 genome by Hisat2 (version 2.0.5). The gene expression was calculated by StringTie (version 1.3.1c) using the refFlat database from the UCSC genome browser.

Immunoblotting
Protein lysates were prepared from organoids, and immunoblotting was performed as previously described 22 .

Reporter assay
Cells were transfected with various plasmids as indicated, and at 12 h post-transfection, the cells were treated with small molecules or growth factors. Twenty-four hours later, the cells were harvested for luciferase determination with aluminometer (Berthold Technologies). Reporter activity was normalized to the co-transfected Renilla. For the Topflash reporter, the activity was normalized to Fopflash. Experiments were independently repeated at least three times.

Statistics
Data shown in column graphs represent the mean ± s.d. When normality could be assumed, Student's t-test or two-way ANOVA analysis was used to compare difference between two groups as indicated in the figure legends. *P < 0.05, **P < 0.01, ***P < 0.001. Statistical analysis was performed with GraphPad Prism6 software. The quantification of colony size was performed with ImageJ. Each experiment was independently repeated at least three times.


extfieldsofstudy: Medicine Biology

text:
DNA methylation and repressive histones in the promoters of PD-1, CTLA-4, TIM-3, LAG-3, TIGIT, PD-L1, and galectin-9 genes in human colorectal cancer

Background Colorectal cancer (CRC) is the third most commonly diagnosed human malignancy worldwide. Upregulation of inhibitory immune checkpoints by tumor-infiltrating immune cells (TIICs) or their ligands by tumor cells leads to tumor evasion from host immunosurveillance. Changes in DNA methylation pattern and enrichment of methylated histone marks in the promoter regions could be major contributors to the upregulation of immune checkpoints (ICs) in the tumor microenvironment (TME). Methods Relative expressions of various immune checkpoints and ligands in colon normal tissues (NT) and colorectal tumor tissues (TT) were assessed by qRT-PCR. The epigenetic modifications behind this upregulation were determined by investigating the CpG methylation status of their promoter regions using bisulfite sequencing. Distributions of histone 3 lysine 9 trimethylation (H3K9me3) and histone 3 lysine 27 trimethylation (H3K27me3) in promoter regions of these genes were assessed by chromatin immunoprecipitation (ChIP) assay. Results We found that the expression levels of PD-1, CTLA-4, TIM-3, TIGIT, PD-L1, and galectin-9 were significantly higher in colorectal tumor tissues, compared with colon normal tissues. To study the role of DNA methylation, we checked the promoter CpG methylation of ICs and ligands and found that only CTLA-4 and TIGIT, among other genes, were significantly hypomethylated in TT compared with NT. Next, we checked the abundance of repressive histones (H3K9me3 and H3K27me3) in the promoter regions of ICs/ligands. We found that bindings of H3K9me3 in PD-1 and TIGIT promoters and H3K27me3 in CTLA-4 promotor were significantly lower in TT compared with NT. Additionally, bindings of both H3K9me3 and H3K27me3 in the TIM-3 promoter were significantly lower in TT compared with NT. Conclusion This study shows that both DNA hypomethylation and H3K9me3 and H3K27me3 repressive histones are involved in upregulation of CTLA-4 and TIGIT genes. However, repressive histones, but not DNA hypomethylation, are involved in upregulation of PD-1 and TIM-3 genes in CRC tumor tissue. These epigenetic modifications could be utilized as diagnostic biomarkers for CRC. Electronic supplementary material The online version of this article (10.1186/s13148-018-0539-3) contains supplementary material, which is available to authorized users.

Background
Colorectal cancer (CRC) is the third most common cancer worldwide [1]. Approximately 20% of CRC patients show distinct metastases at diagnosis, and the death rate is estimated to be 26% in both genders [1,2]. The relationship between immune cells and cancer cells within the tumor microenvironment (TME) attains a great interest among researchers. Immune cell-mediated tumor evasion is one of the key mechanisms for the progression and survival of malignant cells [3]. T cells are the chief cytotoxic effector cells that recognize and eliminate tumor cells. Immune response against tumor is initiated by recognition of tumor-antigenic peptides by T cell receptors (TCR) along with co-stimulatory signals, which are required for an effective and prolonged immune response against tumor antigens for successful elimination of malignant cell. In addition to co-stimulatory signals, co-inhibitory signals (immune checkpoints; ICs) are indispensable for maintaining peripheral tolerance and in preventing autoimmunity. The balance between co-stimulatory and co-inhibitory signals determines the amplitude of T cell response [4,5]. The expression of these ICs is utilized by tumor cells to escape from host immunosurveillance [6,7].
It has been reported that epigenetic regulation is one of the key mechanisms behind ICs expression in the TME [8]. Three important epigenetic modifications are reported in the colorectal TME; DNA methylation, post-translational modifications in chromatin-protein interactions, and expression of non-coding RNAs [9,10]. In particular, hypermethylation of the CpG islands (CGIs) enriched in the promoter regions of tumor suppressor genes, induce silencing of these genes [11]. Active demethylation of DNA occurs by the oxidation of 5-methyl cytosine (5-mc) to 5-hydroxymethyl cytosine (5-hmc) and finally to 5-cytosine (5-c) by enzymes belonging to the ten-eleven translocation (TET) family [12]. Mammalian TET family consists of three members; TET1, TET2, and TET3 [12]. It has been reported that promoter demethylation and distribution of repressive histones work together for the upregulation of many genes in cancers [13]. A report showed that the enrichment of repressive histones, histone 3 lysine 9 trimethylation (H3K9me3) and histone 3 lysine 27 trimethylation (H3K27me3) in the promoter regions along with CpG hypermethylation, were the common epigenetic modifications in the colorectal TME [14]. The epigenetic modifications of ICs in colorectal tumor are still not elucidated.
In this study, we investigated expression levels of different immune checkpoints/their ligands, and the epigenetic modifications that could be involved in their upregulation in the colorectal TME. PD-1, CTLA-4, TIM-3, LAG-3, TIGIT immune checkpoints and PD-L1, and galactin-9 ligands were selected due to their important role in tumor immune evasion and their potential as therapeutic targets for immune-mediated therapies. Interestingly, we found that ICs including PD-1, CTLA-4, TIM-3 and TIGIT, and IC ligands including PD-L1 and galactin-9 were significantly upregulated in colorectal tumor tissues (TT), compared with colon normal tissues (NT). Additionally, we found that both DNA hypomethylation and repressive histone binding in the promoter regions are involved in the transcriptional upregulation of CTLA-4 and TIGIT. However, distribution of repressive histones, but not DNA hypomethylation, seems to be involved in the upregulation of PD-1 and TIM-3 in colorectal tumor tissue.

Multiple immune checkpoints/ligands are upregulated in colorectal tumor tissue
Reports showed that tumors attain various mechanisms to circumvent host immunosurveillance [15,16]. One such mechanism is the upregulation of ICs by TIICs and their ligands by tumor cells in the TME. To investigate the transcriptional expression of ICs/ligands in the colorectal TME, we performed real time PCR to determine mRNA levels of ICs/ligands in NT and TT. We found that ICs including PD-1, CTLA-4, TIM-3 and TIGIT, (Fig. 1a) and IC ligands including PD-L1 and galectin-9 ( Fig. 1b) were significantly upregulated in TT compared with NT. However, there was no significant change in LAG-3 expression in TT compared to NT (Fig. 1a). These data show that in the colorectal TME, multiple ICs and ligands are upregulated, which may assist tumor cells to evade host immunosurveillance.
DNA demethylation enzymes are overexpressed in colorectal tumor microenvironment DNA methylation has a predominant role in the silencing of tumor suppressor genes in the TME, and any imbalance in DNA methylation/demethylation genes could result in disease onset and progression [17]. It has been reported that TET1, TET2, and TET3 exhibit both overlapping and discrete functions [18]. In CRC, somatic mutations have been reported in all three TET proteins [19]. These reports prompted us to check the expression of TET1, TET2, and TET3 and methylation enzymes including DNMT3a and DNMT3b in NT and TT. Interestingly, we found all three TETs were significantly increased and DNMTs were significantly decreased in TT compared with NT (Fig. 1c). Out of all TETs, TET2 was more significantly upregulated in TT compared with NT, indicating that TET2 might play a pivotal role in demethylation than TET1 and TET3 in the colorectal TME (Fig. 1c). The reciprocal expressions of TETs and DNMTs are in line with previous findings that the methylation status of the gene is dynamically regulated by TETs and DNMTs [20,21].

Analyses of DNA methylation in the promoter regions of immune checkpoints/ligands in the colorectal tumor microenvironment
Hypermethylation of CpG islands (CpGIs) located in the promoter regions have a major role in gene inactivation in the TME and has been defined in almost all malignancies [22]. In order to check the promoter methylation profile of ICs/ligands, we selected CpGIs in the promotors of PD-1, CTLA-4, TIM-3, LAG-3, TIGIT, and PD-L1 as described previously [23]. In addition to this, we also selected 12 CpGIs in the promoter region of galectin-9. We found that the average demethylation percentages of CTLA-4 and TIGIT in TT were significantly higher compared with NT (Figs. 2b, e and 3a). Additionally, the average demethylation percentages of PD-1 and TIM-3 were higher in TT compared to NT, but not significant (Figs. 2a, c and 3a). In contrast, the demethylation of LAG-3 was reduced in TT compared to NT (Figs. 2d and 3a). These results are in accordance with real-time data that LAG-3 was the only gene, which has lower expression in TT compared to NT (Fig. 1a). Additionally, there were no differences in the demethylation percentages for IC ligands, PD-L1 and galectin-9, in TT compared to NT (Figs. 2f, g and 3a). Interestingly, PD-L1 was completely demethylated in both NT and TT (Fig. 2f ). This is similar to our findings in breast tumor tissues [23]. These data show that all genes do not follow similar mechanisms for their transcriptional upregulation in the TME. The transcriptional upregulation of CTLA-4 and TIGIT might be under the control of DNA hypomethylation. We also checked the corrected demethylation percentage by subtracting the demethylation percentage of NT from corresponding TT and found that the percentages of CTLA-4 and TIGIT were higher than other genes, and there were no significant differences between them (Fig. 3b). In addition, we checked A C B Fig. 1 Expression of immune checkpoints/ligands and methylation/demethylation genes in colorectal tumor and normal colon tissues. RNA isolated from tissues from 14 patients was reverse transcribed to cDNA. Quantitative RT-PCR was performed to assess the expression level of immune checkpoints PD-1, CTLA-4, TIM-3, LAG-3, and TIGIT (a); immune checkpoint ligands PD-L1 and galectin-9 (b); demethylation/methylation enzymes TET1, TET2, TET3, DNMT3a, and DNMT3b (c) from both NT and TT. The relative expression of each gene was normalized to β-actin the corrected demethylation percentages of all genes in individual patients and found that the percentages of CTLA-4 and TIGIT were higher in most of patients compared with other genes (Fig. 3 c, d). These data show that demethylation in promotors might play an important role in the expression of ICs in the TME.

Analyses of the abundance of repressive histones in the promoter regions of immune checkpoints/ligands in the colorectal tumor microenvironment
Our DNA methylation data show that the transcriptional upregulation of ICs/ligands are not completely dependent on the hypomethylation of promoter regions. These results prompted us to check the presence of repressive H3K9me3 and H3K27me3 in the promoter regions of PD-1, CTLA-4, TIM-3, LAG-3, TIGIT, PD-L1, and galectin-9 in the colorectal TME by chromatin immunoprecipitation assays. As controls, we precipitated chromatin from both NT and TT with anti-H3 antibody and confirmed that there is no difference in the distribution of H3 in the promoter regions of all ICs/ligands between NT and TT (Fig. 4). We also used rabbit-IgG as an isotype negative control to confirm that there were no non-specific enrichments (Fig. 4). Interestingly, the abundance of H3K9me3 was significantly lower in TT compared with NT in the promoter regions of PD-1 (Fig. 4a) and TIGIT (Fig. 4e), while H3K27me3 was lower in CTLA-4 promotor (Fig. 4b). Moreover, both H3K9me3 and H3K27me3 were significantly lower in TT in TIM-3 promoter (Fig. 4c). Of note, there was no difference in the distribution of either H3K9me3 or H3K27m3e in the promoter regions of LAG-3, PD-L1, and galectin-9 (Fig. 4d, f,  g). These data show that in the colorectal TME, abundance of repressive histones in TT was significantly lower in the promoter regions of PD-1, CTLA-4, TIM-3, and TIGIT, which may in turn lead to their transcriptional upregulation in TT compared with NT.

Discussion
Evidence shows that immune system actively participates in tumor development by promoting the uncontrolled growth of tumor cells [24]. Cancer cells bind to co-inhibitory molecules on T cell surface such as CTLA-4, PD-1, TIM-3, and LAG-3 which in turn secrete immune-suppressive mediators such as IDO (indoleamine 2,3-dioxygenase) to create an immune subversive environment in the TME [25,26]. We have recently reported that in the breast TME, ICs including PD-1, CTLA-4, TIM-3, and LAG-3 were transcriptionally upregulated in TT compared with NT and both DNA and histone modifications in the TME might be actively involved in this upregulation [23]. Additionally, it has been reported by us and other groups that ICs show elevated expression in the colorectal tumor tissues compared with colon normal tissues [5,27,28]. However, the epigenetic modifications behind this upregulation are still not disclosed.
In this study, we found that expression of ICs including PD-1, CTLA-4, TIM-3, TIGIT, and IC ligands including PD-L1 and galectin-9 was significantly higher in colorectal tumor tissues compared with normal tissues (Fig. 1a, b). These findings are in line with our pervious report that the expression of multiple ICs was elevated in the breast TME [23]. In contrast to CRC TME, we did not find IC ligands, PD-L1 and galectin-9 upregulation in the breast TME [23]. These data show that the expressions of ICs/ligands are different in each cancer type, and precise characterization of the ICs and ligands in each cancer type could have prognostic significance. Moreover, we have previously shown that there were more T cell infiltrates in the colorectal TT compared with NT [28]. In this study, we used tissue samples from the same patients that we had used in our previous study [28].
In order to check DNA epigenetic modifications behind the upregulation of ICs/ligands, we checked the expression of demethylation enzymes (TETs) and methylation enzymes (DNMTs) in the tumor and normal tissues and found that the expressions of demethylation enzymes were significantly higher and methylation enzymes were lower in TT (Fig. 1c). It has been reported that the TET protein level was upregulated in solid tumors [29]. These data prompted us to check the CpG methylation profile of the promoter regions of ICs/ligands. We found that the promoter regions of CTLA-4 and TIGIT were significantly hypomethylated in TT compared with NT. These data suggest that not all ICs are following similar epigenetic modifications to upregulate their expression in the TME. Additionally, there was no significant difference in the demethylation percentage in LAG-3 promoter between NT and TT (Fig. 3a). These results are similar to our previous findings in the breast TME that the promoter regions of PD-1, CTLA-4, and TIM-3 were significantly hypomethylated in TT compared with NT and no change in LAG-3 [23]. Compared to our previous study [23], we found that in both NT and TT of colorectal and breast tumors, the CpGs in the promoter region of PD-L1 have been totally demethylated (Fig. 2f), but the relative expression of PD-L1 was significantly higher only in the colorectal TT compared with NT (Fig. 1b). Taken together, our data suggest that the transcriptional upregulation of ICs/ligands does not solely depend on promoter CpG hypomethylation but also on malignant type.
In addition to CpG methylation, we also investigated whether the histone modifications also participate in the upregulation of ICs/ligands in the colorectal TME. It has been reported that promoter region hypermethylation is often associated with H3K9me3 and H3K27me3 for transcriptional silencing [30]. Herein, we checked H3K9me3 and H3K27me3 markings in the promoter regions of PD-1, CTLA-4, TIM-3, LAG-3, TIGIT, PD-L1, and galectin-9 (Fig. 4). In accordance with our previous findings in breast tumors [23], the distribution of H3K9me3 was lower in colorectal TT of PD-1 (Fig. 4a) and H3K27me3 was lower in TT of the CTLA-4 (Fig. 4b) and TIM-3 (Fig. 4c) promoter regions compared with NT. Moreover, there was no change in distribution of either H3K9me3 or H3K27me3 in the promoter regions of LAG-3, PD-L1 and galectin-9 in colorectal TT compared to NT (Fig. 4d, f, g). We have reported that in the breast TME, the relative expression of LAG-3 was higher in TT compared with NT, and also the distribution of both H3K9me3 and H3K27me3 was lower in TT compared with NT [23]. Of note, in CRC tumor tissue, there was no upregulation in the expression of LAG-3 and also no difference in the distribution of either H3K9me3 or H3K27me3 in TT compared to NT (Figs. 1a and 4d). Taken together, these data show that the expression of ICs and the epigenetic modifications in the TME differ in different malignancy types.

Conclusions
This study advances our knowledge in both molecular and epigenetic modifications behind the upregulation of ICs/ligands in the colorectal TME. We showed that multiple ICs/ligands including PD-1, CTLA-4, TIM-3, TIGIT, PD-L1, and galectin-9 are upregulated in the colorectal TME. The epigenetic modifications, including DNA hypomethylation and less abundance of H3K9me3/H3K27me3 in the promoter regions, could be responsible for their upregulation. Moreover, the transcriptional upregulation of CTLA-4 in tumor tissue might be under the control of both DNA hypomethylation and lower H3K27me3 enrichment, while DNA hypomethylation and lower H3K9me3 enrichment regulate TIGIT expression. Additionally, lower enrichment of H3K9me3 or both H3K9me3 and H3K27me3 markings could be behind the upregulation of PD-1 and TIM-3 expressions in the CRC TME, respectively. These examinations of promoter DNA methylation and distribution of repressive histones in different ICs/ligands could be further utilized as a diagnostic tool for colorectal cancer.

Sample collection
Tumor tissues (TT) and adjacent non-cancerous normal tissues (NT) were obtained from 14 colorectal cancer patients who underwent surgery. All patients provided written informed consent prior to sample collection and none of the patients included in this study received any treatment prior to surgery. Table 1 shows the clinical and pathological characteristics of all patients. The study was executed under ethical approval by the Qatar Biomedical Research Institute, Doha, Qatar (Protocol no. 2017-006).
All experiments were performed in accordance with relevant guidelines and regulations.

RNA and DNA isolation
RNA and DNA were isolated using RNA/DNA/Protein Purification Plus Kit (Norgen Biotek Corp, Ontario, Canada) as per manufacturer's instructions, from 14 fresh-frozen TT and their corresponding NT. Briefly, frozen tissues were grinded thoroughly using mortar and pestle with adequate amount of liquid nitrogen. Tissue fragments were then resuspended with lysis buffer and incubated at 55°C for 10 min. DNA extraction was then performed using the DNA extraction column. The flow-through from DNA extraction was used for RNA purification using RNA extraction column. The flow-through from RNA extraction was then used for protein extraction using the same column. RNA and DNA concentrations were measured using Nanodrop 2000c (Thermo Scientific, MA, USA), and aliquots were stored at − 80°C.

Quantitative real-time PCR (RT-qPCR)
One microgram of RNA from each sample was reverse transcribed into cDNA using QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). RT-qPCR was performed on QuantStudio 7 Flex qPCR (Applied Biosystems, CA, USA) using PowerUP SYBER Green Master Mix, and all data were normalized to β-actin. Non-specific amplifications were checked by using melting curve and agarose gel electrophoresis. The relative changes in target gene expression were determined using comparative threshold cycle (CT) method 2 -ΔΔCT between NT and TT. The primers were designed using Primer3 (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) and Harvard Primer Bank (http://pga.mgh.harvard.edu /primerbank/). Primer sequences are provided in Additional file 1: Table S1a.

CpG methylation analysis by bisulfite sequencing
CpG methylation analyses were performed through bisulfite sequencing as previously described [23]. Briefly, genomic DNA was extracted from NT and TT, and bisulfite treatment was performed using the EZ DNA Methylation Kit (Zymo Research, Irvine, CA, USA). PCR was then performed on the bisulfite-treated DNA for amplification of the promoter regions of PD-1, CTLA-4, TIM-3, LAG-3, TIGIT, PD-L1, and galectin-9 using hot start TaKaRa Taq DNA polymerase (TaKaRa Bio, Shiga, Japan). PCR primers were designed using MethPrimer software (http://www.urogene.org/methprimer/index1.html). Primer details are provided in Additional file 1: Table S1b. PCR products were cloned into the pGemT-easy vector (Promega, Madison, USA) using DNA Ligation Kit, Mighty Mix (TaKaRa Bio). Ten individual clones from  Table S1c). The promoter regions amplified for CpG methylation profile in this study were as previously described [23].

Enzyme disaggregation of tumor and normal tissues for cell isolation
Cell suspensions for ChIP experiments were obtained from frozen NT and TT of five CRC patients by enzyme disaggregation (ED), as previously described [23]. Briefly, thawed tissues were first washed with phosphate-buffered saline (PBS) and mechanically cut into small fragments (2-4 mm) using a surgical scalpel. Tissues were then suspended into RPMI-1640 with 1% penicillin/streptomycin and enzyme cocktail consisting of 1 mg/ml collagenase and 100 μg/ ml hyluronidase type V (all from Sigma-Aldrich, UK) and incubated at 37°C under slow rotation for 60 min. The resulting cell suspension was then passed through a 100 μm BD Falcon cell strainer (BD Biosciences, Oxford, UK), washed with serum free RPMI-1640, and resuspended in RPMI-1640 enriched with 10% FCS and 1% penicillin/streptomycin for further analyses.

Chromatin immunoprecipitation assay (ChIP)
ChIP analysis was performed using Magna ChIP A/G chromatin immunoprecipitation kit (Merck Millipore, MA, USA) as per manufacturer's protocol on cells isolated from NT and TT by ED. Briefly, nuclear lysate was prepared as per manufacturer's protocol and sonicated using Covaris S2 system (Covaris, MA, USA) to make small DNA fragments (100-200 base pairs) and then incubated with ChIP grade anti-Histone H3 rabbit mAb (Active Motif, CA, USA), anti-Histone H3 (tri methyl K9) rabbit mAb (Abcam Cambridge, UK), and anti-Histone H3 (tri methyl K27) rabbit mAb (Abcam). Isotypematched control antibodies were used as negative controls. Immune complexes containing DNA fragments were precipitated using Magna A/G beads (supplied with the kit). Relative enrichment of target regions in the precipitated DNA fragments was analyzed by qPCR using PowerUP SYBER Green Master Mix (Applied Biosystems) on QuantStudio 7 Flex platform (Applied Biosystems). Sequences of primers are listed in Additional file 1: Table S1d. All data were normalized to input controls. Non-specific amplification was checked by using melting curve and agarose gel electrophoresis.

Sanger sequencing
Purified plasmid DNA samples were subjected to sequencing using 3130X Genetic Analyzer (Applied Biosystems). Cycle sequencing reactions of samples were performed using M-13 forward/reverse primers and Big-Dye Treminator V3.1 (Applied Biosystems), using thermal conditions: 95°C for 5 min, 35 cycles of 95°C for 30 s, and 60°C for 4 min. DNA was precipitated after PCR reaction using 125 mm EDTA and 95% ethanol and incubated at − 20°C for 30 min. DNA was then washed twice with 70% ethanol followed by denaturation using formaldehyde. Denatured DNA was then loaded into analyzer for sequencing. Sequencing data were analyzed using Bisulfite Sequencing DNA Methylation Analysis (BISMA) software (Jacobs University, Germany).

Statistical analyses
Statistical analyses were performed using GraphPad Prism 5 software (GraphPad Software, USA). Paired t test was carried out on samples within groups that passed the Shapiro-Wilk normality test. Nonparametric/Wilcoxon matchedpairs signed-rank tests were performed on samples that did not pass normality test. A P value of < 0.05 was considered statistically significant. The P values are represented as ***P < 0.001, **P < 0.01, *P < 0.05. The data are presented as mean + standard error of the mean (SEM).

Additional file
Additional file 1: Table S1. Primer sequences used in this study.


s2fieldsofstudy: Biology Medicine

text:
Concentrated Ambient PM2.5-Induced Inflammation and Endothelial Dysfunction in a Murine Model of Neural IKK2 Deficiency

Background: Exposure to ambient fine particulate matter (PM2.5) is associated with cardiovascular mortality, but underlying pathophysiologic mechanisms are not fully understood. Hypothalamic inflammation, characterized by the activation of Inhibitor kappaB kinase 2/Nuclear factor kappaB (IKK2/NF-κB) signaling pathway, may play an important role in the pathogenesis of cardiovascular diseases. We recently demonstrated that hypothalamic inflammation is increased in mice exposed to concentrated ambient PM2.5 (CAP). Objectives: In the present study, we used a neuron-specific IKK2 knockout mouse model to examine the role of neural IKK2 expression and hypothalamic inflammation in the pathophysiologic effects of PM2.5. Methods: We assessed inflammatory and vascular responses in Nestin-creIKK2flox/flox (IKK2Neu-KO) and littermate Nestin-creIKK2flox/+ (control) mice after 4 mo of exposure to filtered air (FA) or CAP. Results: CAP exposure was associated with significantly higher tumor necrosis factor-α (TNFα) and interleukin (IL)-6 mRNA in the hypothalamus of control mice, but not IKK2Neu-KO mice. In addition, CAP exposure–induced increases in bronchoalveolar lavage fluid (BALF) leukocytes, pulmonary macrophage infiltration and IL-6 expression, plasma TNFα and IL-1β levels, adipose macrophage infiltration and IL-1β expression, and endothelial dysfunction were reduced or absent in IKK2Neu-KO mice compared with controls. Conclusions: Our findings support a role of neural IKK2 in CAP exposure–induced local and systemic pro-inflammatory cytokine expression, pulmonary and adipose inflammation, and endothelial dysfunction, thus providing insight into pathophysiologic mechanisms that may mediate effects of PM2.5 exposure. https://doi.org/10.1289/EHP2311

Introduction
Exposure to ambient fine particulate matter (PM 2:5 ) is associated with increased risk of cardiovascular morbidity and mortality (Brook et al. 2010). However, while the majority of inhaled PM 2:5 are known to deposit in the airway and not enter the systemic circulation (U.S. EPA 2009), mechanisms that link PM 2:5 with cardiovascular effects remain elusive. The putative mechanisms for this include a) egress from the lung of PM 2:5 components, b) autonomic nervous system (ANS) dysfunction, and c) inflammation (Brook et al. 2010). Notably, despite that inhaled nanoparticles have been identified in remote organs, the pathophysiological role of these nanoparticles remain controversial, as their concentrations are low and inconsistent (Kreyling et al. 2009;Oberdörster and Utell 2002;Semmler-Behnke et al. 2008). PM 2:5 exposure is associated with decreased heart rate variability (Brook 2005;Chen and Hwang 2005;Godleski et al. 2000;Gong et al. 2008;Magari et al. 2001;Sivagangabalan et al. 2011;Tankersley et al. 2004), indicating sympathetic nervous system (SNS) activation and/or parasympathetic nervous system withdrawal (Brook 2008;Karemaker 1999). However, because the nervous system can adapt to chronic stimulation, ANS dysfunction has been thought to play a trivial role in the mediation of the long-term health effects of PM 2:5 exposure (Brook et al. 2010). Evidence that cardiovascular effects of PM 2:5 are mediated by inflammation is stronger, given the established role of PM 2:5 in the of various cardiovascular diseases and evidence of inflammatory responses to PM 2:5 exposure in humans (Peters et al. 2001;Pope 2000;Utell et al. 2002) and various animal models (Kennedy et al. 1998;Quay et al. 1998;van Eeden et al. 2001;Vogel et al. 2005).
Putative effect mechanisms that involve ANS dysfunction or inflammation are not mutually exclusive, but may affect the same or different stages of pathogenesis, and may have synergistic effects (Brook et al. 2010). For example, ANS dysfunction may impact various inflammatory responses (Lambert et al. 2015;Pavlov and Tracey 2017), and increases in circulating proinflammatory cytokines/mediators resulting from PM 2:5 -related pulmonary or systemic inflammation may cause ANS dysfunction (Dunn 2000;Turnbull and Rivier 1999;Zhang et al. 2003). More importantly, there is emerging evidence that ANS dysfunction may also play a critical role in the long-term regulation of cardiovascular function (DiBona 2013;Froeschl et al. 2013;Simonds and Cowley 2013), which appears to be dependent on an inflammatory response in the hypothalamus. For example, rats and mice fed a high-fat diet (HFD) showed rapid increases in hypothalamic inflammation, which may contribute to HFD feeding-induced long-term effects, including obesity and insulin resistance (Thaler et al. 2012). Inhibition or deletion of IKK2 in some hypothalamic neurons was sufficient to abolish HFD feeding-induced ANS dysfunction, and subsequently, normalized blood pressure in a mouse model (Purkayastha et al. 2011).
Collectively, these data have highlighted a crucial role of an IKK2-dependent hypothalamic inflammatory response in the pathophysiology of various cardiovascular diseases.
The IKK2=NF-jB signaling pathway is pivotal in both acute and chronic inflammatory responses (Pahl 1999). Previous mechanistic studies by our laboratory (Kampfrath et al. 2011;Wang et al. 2017) and others (Feng et al. 2017;Jeong et al. 2017;Song et al. 2017;Zhang et al. 2017) have demonstrated that exposure to ambient PM 2:5 activates the IKK2=NF-jB pathway, which is believed to play a key role the pathophysiologic effects of PM 2:5 (Brook et al. 2010) in various tissues. Notably, we recently reported that chronic exposure of C57Bl/6J mice to ambient PM 2:5 resulted in activation of IKK2=NF-jB pathway in the hypothalamus, abnormal SNS activation, and hypertension (Ying et al. 2014), while in the KKay mouse model of type 2 diabetes, intracerebroventricular injection of an IKK2 inhibitor reduced PM 2:5 exposure-induced abnormalities in glucose homeostasis (Liu et al. 2014). These findings suggest that an IKK2-dependent hypothalamic inflammatory response may be crucial in the development of adverse health effects in response to ambient PM 2:5 exposure. In the present study, to verify the role of hypothalamic inflammation in the pathophysiologic effects of ambient PM 2:5 , we exposed Nestin-creIKK2 flox=flox (IKK2 Neu-KO ) mice and littermate controls (Nestin-creIKK2 flox= + ) to filtered air (FA) or concentrated ambient PM 2:5 (CAP), and assessed their inflammatory and vascular responses.

Animals
University of Maryland, Baltimore (UMB) is an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-accredited institution. All procedures of this study were approved by the Institutional Animal Care and Use Committee at UMB, and all the animals were treated humanely and with regard for alleviation of suffering. Nestin-cre transgenic mice in the C57Bl/6J background were obtained from Jackson Laboratories (Stock No.: 003,771). IKK2 flox=flox mice were generated as previously described (Li et al. 2003) and back-crossed with C57Bl/6J for three generations. Nestin-creIKK2 flox=flox (IKK2 Neu-KO ) and littermate Nestin-creIKK2 flox= + (control) mice were generated through in-house crossing of Nestin-creKK2 flox= + and IKK2 flox=flox . The pups were genotyped at the age of 1 wk by polymerase chain reaction (PCR), and the genotyping results were verified before exposure to FA/CAP. Concentrated Ambient PM 2.5 Exposure IKK2 Neu-KO and control mice (male, 8-12 wk old) were grouped into four age-matched groups and randomly designated to be subjected to exposure to FA (six IKK2 Neu-KO and seven controls) or CAP (seven IKK2 Neu-KO and seven controls) from May 2016 to September 2016 for a total duration of 4 mo in a mobile trailer with a 12-h light/12-h dark cycle, temperatures of 18-25°C, and relative humidity of 40-60%. Because we failed to observe CAP exposure-induced impairment of glucose tolerance in female mice, male mice only were used in the present study. The mobile trailer was located on the campus of the UMB. Animal exposure and the monitoring of exposure atmosphere and ambient aerosol were performed as previously described using a versatile aerosol concentration enrichment system that was modified for long-term exposures (Geller et al. 2005;Maciejczyk et al. 2005). The exposure protocol comprised exposures for 6 h/d, 5 d/wk (no exposure took place during weekends). Ambient aerosol and exposure atmosphere were monitored as previously described (Maciejczyk et al. 2005). The elemental composition of CAP was determined by inductively coupled plasma mass spectroscopy (for trace element analysis as previously described (Mirowsky et al. 2013;Wang et al. 2017).

Bronchoalveolar Lavage and Lung Histopathology
After euthanasia by overdose of isoflurane, the mouse trachea was cannulated, and the right primary bronchus was closed off with a ligature. Through the tracheal cannula, 0:5 mL sterile phosphatebuffered saline with 0:1 mM ethylenediaminetetraacetic acid (EDTA) was instilled and withdrawn to recover bronchoalveolar lavage fluid (BALF). This was repeated twice. The total number of cells in the collected BALF (around 1:5 mL) was estimated using a hemocytometer. Cytospin slides were prepared using Shandon Cytospin 3™ (Shandon) and stained with Diff-Quik solution (EMS). Differential cell counts for neutrophils, eosinophils, macrophages/ monocytes, and lymphocytes were assessed by a pathologist who was blinded to the grouping.
Following BALF collection, the right lung was harvested. About one-third were cut and fixed with 4% paraformaldehyde, and the rest were snap frozen in liquid nitrogen and then kept at −80 C. To assess the inflammation in the lung, tissue blocks were embedded in paraffin, 5-lm-thick sections were cut, and the sections were subjected to hematoxylin and eosin staining. Three consecutive sections per sample were used for histopathology. Images covering all the tissue area were taken by a laboratory technician who was blinded to the grouping, and all images were then sent to and quantitated by the pathologist (blinded to the grouping, too).

Plasma Cytokine Analysis
To rapidly harvest blood samples for cytokine analysis, mouse retroorbital blood samples were collected in EDTA-treated tubes and subjected to centrifugation for 15 min at 2,000 × g using a refrigerated centrifuge. The supernatants (plasma) were then transferred to new tubes and stored at −80 C until assessments of cytokine levels. Plasma cytokine levels were assessed using BD™ Cytometric Bead Array Kit (BD Biosciences) per manufacturer's instructions. Briefly, 25 lL=mouse plasma were incubated with the beads, and signaling was quantified by flow cytometry.

Real-Time Polymerase Chain Reactions
Mouse heads were removed before bronchoalveolar lavage. Hypothalamus, hippocampus, cortex, and olfactory bulb were immediately isolated. Epididymal adipose tissues were harvested after bronchoalveolar lavage. All these samples were snap frozen in liquid nitrogen and kept at −80 C. Total RNA was extracted and purified using the Trizol reagent (Invitrogen) from epididymal adipose tissue, lung, hypothalamus, hippocampus, cortex, and olfactory bulb. The quality of RNA was assessed by determination of the ratio of absorbance at 260 nm to absorbance at 280 nm by nanodrop. Two micrograms of total DNase-treated RNA were reverse transcribed into cDNA using High Capacity cDNA Reverse Transcription Kits (Applied Biosystems™) per manufacturer's instruction. Real-time PCR was performed using LightCycler ® 480 SYBR Green I Master in the LightCycler ® (Roche). Reactions were performed in a total volume of 10 lL containing 1 lL cDNA, 0:2 lM of each primer, and 5 lL of the SYBR ® Green reaction mix. The amplification protocol was as follows: 95 C=5 min (95 C=10 s, 60 C=20 s, and 72 C=30 sÞ × 45. Following amplification, a dissociation curve analysis was performed to ensure purity of PCR product. The specific sense and antisense primers for tumor necrosis factor-a ðTNFaÞ, interleukin (IL)-6, IL-1b, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (the reference housekeeping gene) were previously described ). In each sample, 2 DDCt was calculated and used to represent its gene expression level.

Western Blotting
Standard techniques were performed with primary antibodies: mouse Anti-IKK2 (Millipore), mouse Anti-b-Actin (Sigma-Aldrich), and mouse Anti-GAPDH (Cell Signaling Technology). Signals were detected by chemiluminescence and analyzed by densitometry.

Vascular Function Assay
After euthanasia, mouse thoracic aorta was quickly removed and cleaned in physiological salt solution (PSS) containing (mM): NaCl, 130; NaHCO 3 , 14.9; KCl, 4.7; KH 2 PO 4 , 1.18; MgSO 4 -7H 2 O, 1.18; CaCl 2 -2H 2 O, 1.56; EDTA, 0.026; and glucose, 5.5. The aorta was cut into 2-mm rings. The aortic rings were then mounted in a muscle bath containing PSS at 37°C and bubbled with 95% O 2 -5% CO 2 . Isometric force generation was recorded with a Multi Myograph System (Danish Myo Technology). A resting tension of 4 mN was imposed on each ring, and the rings were allowed to equilibrate for 1 h. To test vascular function, the contractile responses of aortic rings to phenylephrine (PE) in the absence or presence of an NOS inhibitor x-nitro-L-arginine methyl ester (L-NAME; 100 lM) were assessed in an accumulative manner. To analyze the endothelial function, aortic rings were precontracted with PE (1 lM), and acetylcholine (ACh) or sodium nitroprusside (SNP) was added in an accumulative manner.

Statistics
All data are expressed as means ± standard errors of the mean ðSEMsÞ unless noted otherwise. Statistical tests were performed using one-way or two-way analysis of variance with Bonferroni post tests or unpaired t-tests using GraphPad Prism (version 4.1.2; GraphPad Software). The significance level was set at p < 0:05.

Exposure Characterization
The average PM 2:5 concentrations in the FA and CAP chambers were 3:6 ± 1:9 and 71:6 ± 33:2 lg=m 3 , respectively. Table 1 shows the elemental composition of CAP and ambient PM 2:5 . Although identifying PM 2:5 emission sources is generally beyond the scope of this paper, the relatively high ratio of Na/Al reflects more contribution by the marine source than the crustal source, which is consistent with the geographic proximity of the study site to the ocean (Huang et al. 1999), whereas the relatively high sulfur suggests that the study site was most strongly affected by secondary aerosols, which are likely to include emissions from coal-fired utility boilers located regionally (Huang et al. 1999).
Characterization of IKK 2 Neu-KO and Evaluation of Pro-Inflammatory Cytokines after Exposure to Filtered Air or Concentrated Ambient PM 2:5 In order to examine the role of neural inflammation in the PM 2:5 exposure-related pathophysiology, we generated Nestin-creIKK2 flox=flox (IKK2 Neu-KO ) and littermate Nestin-creIKK2 flox= + (control). Except for lower body weight at 12 wk of age (mean 22:3 ± 1:1 vs. 20:3 ± 0:6 g for controls and IKK2 Neu-KO , respectively, n = 8=group, student's t-test p < 0:05), adult IKK2 Neu-KO mice were similar in appearance to adult control mice. Figure 1A shows the deletion of IKK2 in the brain of IKK2 Neu-KO . To confirm the tissue-specific deletion of IKK2 in IKK2 Neu-KO , we assessed IKK2 protein levels in different tissues. Figures 1B and C reveal that compared to control, IKK2 Neu-KO had significantly decreased expression of IKK2 in the brain but not lung, heart, and epididymal adipose tissue, corroborating the neuronspecific deletion of IKK2 in these mice. Consistent with our previous study (Ying et al. 2014), Figures 1D and E show that CAP exposure significantly increased the hypothalamic expression of TNFa and IL-6 mRNAs. Neural IKK2 knockout did not significantly alter the hypothalamic expression levels of TNFa and IL-6 mRNAs in FA-exposed mice, but significantly reduced them in CAP-exposed mice. Given that recent studies have suggested that some components of ambient PM 2:5 may enter the brain through the olfactory system (Maher et al. 2016), we isolated olfactory bulb, cortex, and hippocampus, and assessed their expression of TNFa and IL-6 mRNAs. Except for a marked trend of increase in the expression of IL-6 mRNA in the hippocampus of CAP-exposed control mice vs. that of FAexposed control mice, no any other remarkable difference was observed ( Figures 1F-K).
Evaluation of Pulmonary Inflammation after Exposure to Filtered Air or Concentrated Ambient PM 2:5 Central inflammation has been implicated in the regulation of peripheral inflammatory responses (Liu et al. 2014), and pulmonary inflammation is widely believed to be essential in the pathophysiology due to exposure to ambient PM 2:5 (Brook et al. 2010). To determine whether central inflammation plays a role in the inflammatory response of the lung to PM 2:5 inhalation, we performed BALF cell differentiation. Figures 2A-D reveal that exposure to CAP significantly increased BALF total cells and macrophages in control mice. Consistent with many previous studies (Filep et al. 2016;Yoshizaki et al. 2017), although neutrophil infiltration is the hallmark of inflammation, we did not observe significant increase in BALF neutrophils in CAP-exposed mice ( Figure 2D). This may be a reflection of the crucial role of neutrophils in acute but not chronic inflammatory response. BALF cell numbers were not significantly different between CAP-vs. FAexposed neural IKK2-deficient mice, which suggests that CAP exposure-induced pulmonary inflammation may be regulated by a central mechanism. To confirm this, we performed lung histological analysis. The percentage of alveolar macrophages was significantly higher in CAP-exposed vs. FA-exposed IKK2 Neu-KO mice and control mice, but was significantly lower in CAPexposed IKK2 Neu-KO mice vs. CAP-exposed controls ( Figure  2E-F). Pulmonary TNFa and IL-6 expression was higher in CAP-exposed vs. FA mice, though the difference was significant only for controls ( Figure 2G-I). In addition, pulmonary IL-6 mRNA expression was significantly lower in CAP-exposed IKK2 Neu-KO mice than in CAP-exposed controls ( Figure 2H).

Evaluation of Systemic and Adipose Inflammation after Exposure to Filtered Air or Concentrated Ambient PM 2:5
Systemic inflammation is one of the putative mechanisms that mediate the development of extrapulmonary abnormalities due to Representative Western blot (B) and mean ± standard deviation ðSDÞ IKK2 protein expression in brain, fat, lung, and heart tissues from male control (n = 8) and IKK2 Neu-KO (n = 7) mice at 10-16 wk of age. *p < 0:05 vs. control, one-way analysis of variance (ANOVA). (D-K) TNFa and IL-6 mRNA expression in hypothalamus, hippocampus, cortex, and olfactory bulb tissues harvested from control and IKK2 Neu-KO mice (n = 6 − 7=group) at 8-12 wk of age following 4 mo of exposure to concentrated ambient particulate matter ðPMÞ 2:5 (CAP) or filtered air (FA). *p < 0:05 vs. FA, # p < 0:05 vs. control, ANOVA. exposure to ambient PM 2:5 (Brook et al. 2010). In control mice, CAP vs. FA exposure was associated with higher plasma TNFa, IL-1b, and IL-6 levels (significant for IL-1b and IL-6 only) ( Figure 3A-C). In CAP-exposed IKK2 Neu-KO mice, plasma TNFa, IL-1b, and IL-6 were lower than in CAP-exposed controls (significant for TNFa and IL-1b only). In addition, IL-6 was significantly higher in CAP-vs. FA-exposed IKK2 Neu-KO mice.
( Figure 3C). In addition, exposure to ambient PM 2:5 has been shown to induce marked inflammation in adipose tissues, and the latter is believed to be subsequent to systemic inflammation and play a role in the PM 2:5 exposure-related abnormalities in energy and glucose metabolism (Brook et al. 2010). CAP vs. FA exposure was associated with significantly higher IL-1b mRNA expression in epididymal adipose tissue from both IKK2 Neu-KO and control mice, but was not associated with significant differences in TNFa or IL-6 expression ( Figure 4A-C). IL-1b mRNA expression in adipose tissue was significantly lower in FAexposed IKK2 Neu-KO mice vs. FA controls and in CAP-exposed IKK2 Neu-KO mice vs. CAP controls ( Figure 4C). Macrophage infiltration in epididymal adipose tissue was significantly higher in CAP-exposed IKK2 Neu-KO and control mice relative to FAexposed IKK2 Neu-KO and control mice, respectively, but was significantly lower in CAP-exposed IKK2 Neu-KO mice than in CAPexposed controls ( Figure 4D-E). Environmental Health Perspectives 027003-5 Evaluation of Endothelial Dysfunction after Exposure to Filtered Air or Concentrated Ambient PM 2:5 Endothelial dysfunction may contribute to PM 2:5 exposure-related cardiometabolic abnormalities, such as insulin resistance and hypertension (Brook et al. 2010). CAP vs. FA exposure was associated with a significantly higher aortic contractile response to PE in both IKK2 Neu-KO and control mice, though the maximal PE response was significantly lower in CAP-exposed IKK2 Neu-KO mice than in CAP-exposed controls ( Figure 5A and Table 2). As increased contractile response may result from increased contractility of vascular smooth muscle and/or decreased endotheliumdependent relaxation, we next assessed the aortic response to an endothelium-dependent vasodilator, Ach. CAP vs. FA exposure was associated with a significantly reduced Ach-induced aortic relaxation in both IKK2 Neu-KO and control mice, though the maximal effect was significantly lower in CAP-exposed IKK2 Neu-KO mice than in CAP-exposed controls ( Figure 5B and Table 2). In contrast, we did not observe any significant effect of CAP exposure and neural IKK2 knockout on aortic contractile response to PE in the presence of a NOS inhibitor L-NAME ( Figure 5C) and aortic relaxation by endothelium-independent vasodilator SNP ( Figure 5D). Together, these findings support a role of central inflammation in the mediation of ambient PM 2:5 exposureinduced endothelial dysfunction.

Discussion
Findings from experimental studies of mice suggest that exposure to ambient PM 2:5 may induce central inflammation characterized by activation of the IKK2=NF-jB signaling pathway in the hypothalamus (Liu et al. 2014;Ying et al. 2014). However, to our knowledge, the role of central inflammation in pathophysiologic responses to ambient PM 2:5 exposure has not yet been systemically investigated. In the present study, we exploited a mouse model with reduced neuronal IKK2 expression to examine the role of central inflammation in PM 2:5 exposure-induced peripheral inflammation and endothelial dysfunction. The main findings from the present study are that neural IKK2 reduction appears to inhibit CAP exposure-induced increases in a) hypothalamic TNFa and IL-6 mRNA expression; b) pulmonary, systemic, and adipose tissue inflammatory responses; and c) endothelial dysfunction. These findings provide further support for a role of central inflammation in the development of adverse cardiometabolic responses to ambient PM 2:5 exposure.
Central inflammation, particularly in the hypothalamus, has been shown to play an important role in the pathogenesis of various cardiometabolic diseases such as obesity, hypertension, and diabetes (Han et al. 2016). We previously reported that exposure to ambient PM 2:5 was associated with increased expression of pro-inflammatory cytokines and activation of IKK2 in the mouse hypothalamus (Ying et al. 2014). As in our previous study, we demonstrated that CAP exposure increased expression of pro-inflammatory cytokines in the hypothalamus. In addition, we demonstrated that these responses to CAP exposure were significantly inhibited or abolished in mice with reduced neural IKK2 expression. We found that, on average, IKK2 protein expression was only 31 ± 12% lower in the brains of IKK2 Neu-KO mice compared with Nestin-creIKK2 flox= + controls, consistent with neuron-specific knockout by Nestin-cre (Tronche et al. 1999) and the presence of cells other than neurons in brain, including astrocytes and microglia that may play a role in local inflammatory responses (Douglass et al. 2017;Jassam et al. 2017). Nonetheless, the partial reduction in brain IKK2 expression appeared to be sufficient to reduce or eliminate CAP exposure-induced expression of TNFa and IL-6 mRNA in the hypothalamus. A recent study reported hypothalamic inflammation induced by a single-day exposure to an HFD was inhibited in vagotomized mice, suggesting that the vagal afferent nerve may be responsible for transmitting gutderived inflammatory signals to the hypothalamus (Waise et al. 2015). Further studies are needed to determine whether a similar mechanism might be involved in CAP-induced hypothalamic inflammation.
In addition to systemic circulation, foreign materials may also gain access to the brain via the olfactory mucosa, known as olfactory bulb transmission (Tjälve et al. 1996). Given the previous demonstration of ambient particles in the brain (Maher et al. 2016) and increasing evidence that exposure to ambient PM 2:5 correlates to various brain insults (Underwood 2017), olfactory bulb transmission has been suspected to be an important mechanism for the access of ambient PM 2:5 to the brain (Underwood 2017). In the present study, CAP vs. FA exposure was associated with significantly higher expression of the pro-inflammatory cytokines TNFa and IL-6 in the hypothalamus, whereas in the olfactory bulb, TNFa expression was higher in CAP-vs. FA-exposed control mice, but not significantly, and there was a nonsignificant decrease in IL-6 expression. Additional research is needed to clarify the potential for olfactory bulb transmission of PM 2:5 to the brain, and its influence, if any, on central inflammation. Another finding from the present study was that increases in plasma TNFa, IL-1b, and IL-6 in CAP-vs. FA-exposed control mice were reduced or eliminated in IKK2 Neu-KO mice. This suggests a potential role of the central nervous system in inflammatory responses to CAP, consistent with our previous finding that intracerebrovascular administration of an IKK2 inhibitor was associated with reduced systemic inflammatory responses to CAP exposure (Liu et al. 2014). Moreover, as hypothalamic inflammation has been shown to correlate to ANS dysfunction (Han et al. 2016), these present data are also consistent with increasing evidence that ANS regulates systemic inflammatory responses (Lambert et al. 2015;Pavlov and Tracey 2017). Our finding that CAP exposure-induced pulmonary inflammation was reduced in IKK2 Neu-KO mice compared with controls (Figure 2), consistent with involvement of a central neural mechanism in pathophysiologic responses to PM 2:5 exposure, may seem inconsistent with compelling evidence that pulmonary inflammation in response to ambient PM 2:5 is induced through toll-like receptors (TLRs)and/or pattern recognition receptors (PRRs)-dependent mechanisms (Bekki et al. 2016;Fuertes et al. 2013;Shoenfelt et al. 2009;Zhao et al. 2012). However, previous studies have also reported evidence of close cross-talk between the brain and lung (Davison et al. 2012;Engel et al. 2015;Winklewski et al. 2014).
Exposure to ambient PM 2:5 has been associated with endothelial dysfunction in humans (O'Neill et al. 2005;Wauters et   2005; Ying et al. 2013), and effects of PM 2:5 on endothelial function have been implicated in the pathogenesis of cardiometabolic diseases associated with PM 2:5 exposure, such as atherosclerosis, hypertension, and type 2 diabetes mellitus (Corban et al. 2017). As such, endothelial dysfunction is widely believed to be central in the pathophysiology due to exposure to PM 2:5 (Brook et al. 2010). In the present study, although CAP exposure was associated with evidence of endothelial dysfunction in both control and IKK2 Neu-KO mice, the response was significantly less pronounced in the mice with reduced neural IKK2 expression compared with controls, consistent with a role of central inflammation in PM 2:5 exposure-related effects. Many studies have demonstrated that systemic inflammation may lead to endothelial dysfunction (Piccardi et al. 2017).

Conclusion
In summary, our findings support a role of neural IKK2 in CAP exposure-induced local and systemic pro-inflammatory cytokine expression, pulmonary and adipose inflammation, and endothelial dysfunction, and thus advance knowledge our understanding of the contribution of central inflammation to the pathophysiologic effects of ambient PM 2:5 exposure.