Th1 immune maturation effects of Nocardia rubra cell-wall skeleton via PI3K/Akt/PAX8 regulatory axis

Abstract

Nocardia rubra cell-wall skeleton (Nr-CWS) is reported as an external immunotherapeutic enhancer with the advantage of antitumor effect on human cancers. However, the immune regulatory role of Nr-CWS is not fully illustrated. We studied mouse CD4⁺ T lymphocytes isolated from mice spleen were induced by Nr-CWS and observed that the differentiation of Th1 CD4⁺ T cells and the cytokines of IL-2, TNF-α, IFN-γ were all enhanced by Nr-CWS. Furthermore, RNA sequencing was conducted to investigate the different mRNA profiling induced by Nr-CWS. We observed that paired box 8 (PAX8) was significantly up-regulated in Nr-CWS-treated Th1 cells compared to control. As a transcription factor, chromatin immunoprecipitation sequencing was carried out to study the genome-wide distribution of PAX8. Interestingly, we found that the binding domain of PAX8 was elevated by Nr-CWS, and the target genes associated with these binding sites showed a positive correlation between their transcription and PAX8 binding strength. Finally, we determined that Nr-CWS could enhance the activity of the PI3 K/Akt signaling pathway. Akt agonist could mimic the effect of Nr-CWS for PAX8 up-regulation, while Akt inhibitor compromised the expression of PAX8. Taken together, we determined a novel role of Nr-CWS in boosting the activity of Th1 maturation via the PI3 K/Akt/PAX8 axis.

Introduction

From a variety of sources, lung carcinoma has become the most deadly type of solid cancer in the world. There are approximately 95% of lung malignancies that are classified as non-small cell lung cancer (NSCLC) or smaller cell lung cancer (SCLC). ¹ The survival of stage IV disease decreases gradually to four months as the stage progresses, hence early intervention is crucial. Systemic chemotherapy has traditionally been undertaken for advanced-stage NSCLC is not compatible enough within the current system. ² Although targeting the genetic variants or mutations such as epidermal growth factor receptor (EGFR) or an anaplastic lymphoma kinase (ALK) fusion oncogene (EGFR) has been achieved certain therapeutic effects on NSCLC, these available targets are still too scarce for clinic application. ³ Immunotherapy is the most effective new treatment for lung cancer.^4,5

During the process that immune cells wipe out and eliminate tumor cells, immunoediting generally experiences three stages between the interaction of cancer and immune system.^6–9 The first phase is the direct elimination by the response of activated immunosurveillance. Immunosurveillance can determine and kill cells that have become malignant or potentially malignant owing to the naturally occurring genetic DNA damage. Dendritic cells introduce the tumor antigens and stimulate the development of tumor-specific CD4⁺ and CD8⁺ T cells that enables the destruction of cancer cells. Immunosurveillance is widely acknowledged as the stage of detectable and early tumor development. ¹⁰ The second stage is equilibrium, whereas the immune system continues to destroy tumor cells that are not destroyed in the first stage of elimination. Tumor cells are still in contact with the immune system. The third phase of immunoediting is evasion or escape. Cancer cells can develop and metastasize to escape from the immune system. ¹¹ The immune system usually becomes overwhelmed when malignant cells rapidly thrive. Multiple approaches allow malignant cells to evade elimination by the immune system, including the suppression of the immune system by the tumor itself or by genetic acquisitions that allow immune suppression.

Nocardia rubra cell wall skeleton (Nr-CWS) is a component derived from a gram-positive bacterium called N.rubra. It has been found that Nr-CWS exerts antitumor effects against lung cancer and interferes with the complicated immune network created by immunocompetent cells’ interactions in vitro and in vivo several decades ago.^12,13 Several recent discoveries suggest that Nr-CWS can stimulate the activity of macrophages, dendritic cells, natural killer (NK) cells, CD4⁺ and CD8⁺ T lymphocytes, and serve as a promising immunotherapy strategy.^14–16 However, the molecular machinery of Nr-CWS driving the immune response is still not fully elucidated. The current study aimed to determine the epigenetic role of Nr-CWS in the capability of differentiation of T lymphocytes. We mainly focused on the activities of T cells and attempted to investigate the underlying mechanism of immune activation by Nr-CWS against human cancer.

Materials and methods

Cell culture

Spleen was isolated from ten female C57BL/6 mice aged six-week-old and put into the pre-placed cell strainer, and added into the dish of 5 mL sterile PBS. The spleen was fully ground with the handle of a sterile syringe, and the cells were gently shaken until fully suspended. The cells were collected and centrifuged at 400 ×  g for 5 min at 4 ^oC. 5 mL modified ACK lysis buffer (10 mM KHCO₃, 0.15 M NH₄Cl, 0.1 M EDTA) ⁷ was gently blown and mixed, which was lysed on ice for 5 min. 10 mL PBS was added to terminate the reaction, followed by 400 ×  g centrifugation for 5 min. After discarding the supernatant, 5 mL RPMI 1640 medium (HyClone) with 10% FBS, 2 mM L-glutamine, 50 μM β-mercaptoethanol and 1x antibiotics was added for cell re-suspension and counting. Cells were cultured at 37 ^oC, 5% CO₂ for 48 h with 15 μg/mL Nr-CWS (Sinopharm Chemical Reagent), 10 nM Palomid 529 (Cat. No. A8618, APExBIO) or 20 μM 740 Y-P (Cat. No. B5246, APExBIO).

Flow cytometric assay for cell isolation

The antibodies used for cytometric assay were listed as FITC anti-mouse CD3 antibody (AM003E01, LiankeBio), BV450 anti-mouse CD45 (368523, Biolegend), PerCP-Cy5.5 anti-mouse CD4 antibody (AM00407, LiankeBio), PE-Cy7 anit-mouse CD8 (AM048010, LiankeBio), APC anti-mouse IL-4 (AM0I405, LiankeBio), PE anti-mouse IFN-γ antibody (AM0IF04, LiankeBio), PE anti-mouse IL-17A antibody (AM0I1704, LiankeBio), APC mouse IgG1 isotype (CMG105, LiankeBio), APC anti-mouse CD25 antibody (AM02505, LiankeBio), PE anti-mouse FOXP3 antibody (AM0F04, LiankeBio).

Enzyme-linked immunosorbent assay (ELISA)

The production of cytokines within culture supernatant including IL-2, TNF-α and IFN-γ from Th1 CD4⁺ T cells treated with or without Nr-CWS for 48 h were analyzed using Multiskan FC microplate reader (ThermoFisher Scientific) according to the manufacturer's instruction.

RNA sequencing (RNA-seq)

Total 1 × 10⁸ CD4⁺ cells were processed for cytometric assay. Total RNA of the harvested cells were extracted using TRIZOL (Thermo Fisher Scientific). ¹⁷ Nanodrop 2000 (Thermo Fisher Scientific) and Agilent Bioanalyzer 2100 (Agilent) were used to evaluate RNA concentration and quality. Four μg of RNA from different experimental groups was prepared cDNA library using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB) following the manufacturer's instructions, and sequenced on an Illumina HiSeq platform.

Adaptors and low-quality reads were removed from the raw data using Trimmomatic. The quality of the rest of the clean reads was checked using FastQC, and aligned to the latest human genome (assembly GRCh38) using Hisat2. FPKM values of assembled transcripts were estimated as their expression levels using the StringTie algorithm with default parameters. Differentially expressed genes (DEGs) among the groups were assessed using the R package Ballgown, and p-value was adjusted for the significance of differences using the Benjamini & Hochberg (BH). Ensembl Genome Browser database was used for transcript and gene annotation (http://www.ensembl.org/index.html). The R package ClusterProfiler was used to annotate the DEGs using Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.^18,19

Chromatin immunoprecipitation sequencing (chIP-seq)

Briefly, cells were incubated with 1 mL lysis buffer (20 mM HEPES [pH 7.9], 350 mM NaCl, 0.1% NP-40, 1 mM DTT, 0.2 mM PMSF, 2 mg/mL leupeptin and 2 mg/mL aprotinin) on ice for 30 min, then harvested nucleus by highest speed centrifugation. Genomic DNA in the supernatant was fragmented into 200–500 bp debris, and incubated with 1 μg IP-grade antibodies of PAX8 (Novus, NBP1-32440) at 4 °C overnight after saving 10% lysates as input. 30 μL Protein A beads were incubated for 2 h with at 4 °C to pull down the bound DNA fragments. For high-throughput sequencing,^20,21 we added 3′-dA overhangs to the PAX8 enriched or input DNA and ligated them with the adapter to construct a DNA library. The fragments with the appropriate size of DNA libraries were picked up and processed on an Illumina Hiseq2000 platform for sequencing. The Data preprocessing was similar with RNA-seq including removal of adaptors and low-quality reads, quality control of the clean reads, data alignment and mapping, peak calling (p < 0.01), and annotation.

Western blot assay

The protein lysate of Th1 cells was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to vinylidene fluoride (PVDF) membranes (Bio-Rad Laboratories). The non-specific antigens were blocked within 5% skim milk in PBST for 2 h, and then incubated with appropriately diluted primary antibodies against PAX8 (1:2000), p-Akt (1:1000, Novus, AF887), Akt1 (1:2000, Novus, NBP2-01724) or Tublin (1:5000, Novus, NB100-690) at 4 C overnight. PVDF membrane was washed by TBST for three times and then incubated with HRP-conjugated secondary antibodies (1:10000, Proteintech Group). The blotting bands were developed with ECL plus immunoblotting detection reagents (Thermo Fisher Scientific), and pictured and analyzed using Image lab.

Statistical analysis

Data is shown as the mean ± standard deviation for at least three independent duplications. The variables among different groups were analyzed using one-way ANOVA. The p-value less than 0.05 was considered as statistical significance.

Results

Activation of Cd4⁺ T cells and Th1 immune response induced by Nr-CWS in vitro

Primary CD4⁺ T cells were isolated from adolescent female mice spleen by CD3 and CD4. The condition of 15 μg/mL Nr-CWS treatment per million cells for 48 h as previously described^16,22 was carried out. Each subset of CD4⁺ T population including Th0, Th1, Th2, Th17 and Treg cells was further harvested and cultured in vitro (Figure 1A). We observed that only the count of Th1 cells was significantly elevated, indicating that the Th1 immune response was activated remarkably by Nr-CWS (Figure 1B). Consistently, we also verified the increased cytokines including IL-2, TNF-α, and IFN-γ secreted by Th1 cells in the supernatant (Figure 1C). Taken together, we determined that the Th1 lymphocyte subset was substantially stimulated by Nr-CWS.

Figure 1.

Maturation of Th1 subset of CD4⁺ population enhanced by Nr-CWS. (A) Cytometric assay shows the different subsets of CD4⁺ population from mouse splenocytes. Red arrows indicate the obvious increased Th1 cell count in Nr-CWS compared to control. (B) Cell count statistical analysis shows the changes of the proportion of each subset of CD4⁺ population with and without Nr-CWS treatment. (C) ELISA shows the changes of cytokines of IL-2, TNF-α and IFN-γ of Th1 subset with and without Nr-CWS treatment. The data from triplicate experiments were processed as mean ± standard error and compared by one-way ANOVA test “*” means significance with a p-value less than 0.05 vs. the control group.

Unique up-regulation of PAX8 in Th1 subset of Cd4⁺ population induced by Nr-CWS

To further investigate the molecular role of Nr-CWS in Th1 differentiation and activation, Th1 cells with and without Nr-CWS treatment were conducted RNA-seq to search the potential genes responding to Nr-CWS. Total 980 differential expressed genes (DEGs) were obtained including 562 up-regulated and 418 down-regulated genes in Nr-CWS treatment compared to control (log₂FC > 1 or < −1, p < 0.05) (Figure 2A). Some genes reported to be associated with immune response such as insulin like growth factor binding protein 2 (IGFBP2) (log₂FC = 4.184, p = 6.25 × 10⁻⁶), interleukin 2 (IL2) (log₂FC = 3.802, p = 5.26 × 10⁻⁵), interleukin 4 (IL4) (log₂FC = 3.269, p = 3.48 × 10⁻⁵), retinoid X receptor alpha (RXRA) (log₂FC = −4.267, p = 2.25 × 10⁻⁷), interferon lambda 1 (IFNL1) (log₂FC = −3.539, p = 4.35 × 10⁻⁵) and interleukin 17A (IL17A) (log₂FC = −3.028, p = 9.64 × 10⁻⁴) were all significantly changed induced by Nr-CWS (Figure 2B, Table S1). Additionally, we also noticed that a novel DEG PAX8 was up-regulated by Nr-CWS (log₂FC = 2.457, p = 0.002). Moreover, GO analysis indicated that multiple functions were connected with chemokine-mediated signaling pathway, cell apoptosis, regulation of response to external stimulus, and positive regulation of immune response. The main signaling pathways including PI3 K/Akt and cytosolic DNA-sensing were closely associated with Nr-CWS-mediated effect (Figure 2C). Collectively, Nr-CWS impacted with the expression of a large number of target genes in Th1 CD4⁺ subset through numerous crucial signaling pathways.

Figure 2.

RNA profiling of Th1 subset of CD4⁺ population enhanced by Nr-CWS. (A) Heatmap shows the differential expressed genes (log₂FC > 1 or < −1, p < 0.05) between two biological repetitions with and without Nr-CWS treatment. (B) Volcano plot shows 562 up-regulated (red) and 418 down-regulated (blue) genes compared between Nr-CWS group and control. PAX8 is highlighted (log₂FC = 2.457, p = 0.002). (C) Bubble chart shows GO and KEGG analysis of the differential expressed genes.

PAX8 binds to target genes’ promoter for Th1 differentiation

To investigate the role of PAX8 in Nr-CWS-mediated Th1 cells differentiation and activation, ChIP-seq was conducted to study the genome-wide binding regions of PAX8. With the increase of PAX8 expression, its binding peaks on the genome also showed a significant elevation (Figure 3A). 412 new binding sites were obtained and only an extremely few sites were lost induced by Nr-CWS compared to control (Figure 3B). The target genes associated with these additional binding sites showed a positive correlation between the elevated levels of transcript and PAX8 binding strength (r = 0.462, p = 0.002) (Figure 3C). From the results above, the molecular mechanism of Nr-CWS on enhancing Th1 CD4⁺ cells differentiation could be explained partially by the elevated occupancy of PAX8 on target genes.

Figure 3.

Genome-wide occupancy of PAX8 in Th1 cells treated by Nr-CWS. (A) Heatmap shows the genome-wide occupancy of PAX8 at all annotated gene promoters determined by ChIP-seq. Average PAX8 enrichment measured by log2 (peak p values) in 200-bp bins is shown within genomic regions covering 2 kb up- and downstream of coding genes. (B) Volcano plots show the genes with significantly 412 reduced and 14 elevated peaks of CBX4 enrichments compared with the control and Nr-CWS group. (C) Pearson correlation analysis shows the positive correlation (r = 0.462, p = 0.002) between fold changes of transcription and the difference of PAX8 enrichment of target genes.

PI3 K/Akt signaling pathway is required for PAX8 by Nr-CWS

In the presence of increased PAX8 induced by Nr-CWS in Th1 cells, we investigated the mechanism of regulating the expression of PAX8. In GO (Biological function) and KEGG analysis, PI3 K/Akt was indicated as one of the top regulatory signals modulated by Nr-CWS. We suspected that PI3 K/Akt might bridge between Nr-CWS and PAX8 up-regulation. Palomid 529 (Akt inhibitor) and 740Y-P (PI3 K activator) were added into Th1 cells respectively, we found that protein levels of phosphorylated Akt and PAX8 were both increased by Nr-CWS or 740Y-P, but compromised by Palomid 529 (Figure 4), suggesting that the activity of PI3 K/Akt signaling pathway indeed contributed to up-regulation of PAX8 in Th1 cells in vitro. In summary, we determined that Nr-CWS was able to activate PI3 K/Akt signaling pathway to facilitate the expression of PAX8, and the latter further strengthened the binding ability to the promoter of target genes to improve their transcription for Th1 cells differentiation.

Figure 4.

Activated PI3 K/Akt signaling pathway by Nr-CWS for PAX8 up-regulation. WB assay shows that phosphorylated Akt can be promoted by Nr-CWS. Palomid 529 (Akt inhibitor) and 740Y-P (PI3 K activator) are used as positive controls.

Discussion

Nr-CWS is composed of peptidoglycan and arabinogalactan which covalently bind to mycolic acids, and has been acknowledged as a promising immune enhancer for innate immune-stimulating activities and anti-tumor activities. More and more evidence shows that Nr-CWS can activate macrophages, natural killer cells, ²³ CD8^{+ 14} and CD4⁺ T cells, ¹⁶ and Nr-CWS has also been reported to be capable of inducing cytokines, such as IFN-γ, IL-1, CSF, and TNF, which can further activate immune system. ²⁴ However, the overall alteration of gene expression in target cells induced by Nr-CWS is not fully illuminated. Our transcriptomic study provides a valuable resource for approving the molecular impacts of Nr-CWS on CD4⁺ cells, particularly for Th1 cells differentiation and activation. From omics data, a large number of novel candidate genes and signaling pathways associated with Nr-CWS indicate that the RNA profiles of the Th1 subset population stimulated by Nr-CWS are largely different from the untreated cells (Figure 2B). The well-known genes including IGFBP2, IL2, IL4, RXRA, IFNL1 and IL17A all display significant changes, as well as a host of signaling pathways, are involved in anti-tumor effects on Th1 cells by Nr-CWS.

PAX8 as a novel target is found to be up-regulated by Nr-CWS in our study. PAX8 is initially identified as a paired-box gene that is essential for the embryogenesis and morphogenesis of the thyroid, Müllerian, and renal/upper urinary tracts. ²⁵ Meanwhile, PAX8 is largely dispensable for adult tissue homeostasis. The presence of robust expression of PAX8 in a large proportion of ovarian, endometrial and kidney carcinomas suggests that PAX8-targeted therapy has a promising future. ²⁶ However, there are scarcely any studies on PAX8 in immune cells. One previous study has reported that PAX8 can occupy the active enhancer elements and facilitate the activation of histone acetylation at enhancers or promoters of genes associated with various metabolic pathways.^27,28 Our results indicate that the elevated PAX8 induced by Nr-CWS distribute to more genome-wide regions, which seemingly contributes to the positive effects on gene expression. The epigenetic role of PAX8 in Th1 cells differentiation actually is still obscure and needs to be further investigated.

On the other hand, the results of this study determine the regulatory mechanism underlying the up-regulation of PAX8 by Nr-CWS. In limited works of literature, the main focus was on the produced cytokines and functional maturation, the binding receptors as well as intracellular signaling pathways responding to Nr-CWS are poorly reported. Thanks to the high throughput sequencing, multiple signals efficiently affected by Nr-CWS are suggested. But in terms of the composition of Nr-CWS, both of peptidoglycan^29,30 and arabinogalactan^31,32 have been pointed to be involved in PI3 K/Akt signaling pathway in previous studies. PI3 K/Akt signaling pathway plays a crucial role in regulating the migration of various cells,^33–36 and PI3 K/Akt pathway activation is determined to drive the expression of PAX8, indicating that phosphorylated Akt is one of the early events initiated by Nr-CWS. In addition to providing a regulatory relationship, the Nr-CWS/PI3 K/Akt/PAX8 axis also leaves more gaps for us to fill in a future study.

Overall, we determined that Nr-CWS activates PI3 K/Akt signaling pathway and facilitate the expression of PAX8. The up-regulated PAX8 further strengthened the binding ability to the promoter of target genes to improve their transcription for Th1 cells differentiation and maturation.