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Computer Science
Medicine

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Systems Biology-Based Platforms to Accelerate Research of Emerging Infectious Diseases.

Emerging infectious diseases (EIDs) pose a major threat to public health and security. Given the dynamic nature and significant impact of EIDs, the most effective way to prevent and protect against them is to develop vaccines in advance. Systems biology approaches provide an integrative way to understand the complex immune response to pathogens. They can lead to a greater understanding of EID pathogenesis and facilitate the evaluation of newly developed vaccine-induced immunity in a timely manner. In recent years, advances in high throughput technologies have enabled researchers to successfully apply systems biology methods to analyze immune responses to a variety of pathogens and vaccines. Despite recent advances, computational and biological challenges impede wider application of systems biology approaches. This review highlights recent advances in the fields of systems immunology and vaccinology, and presents ways that systems biology-based platforms can be applied to accelerate a deeper understanding of the molecular mechanisms of immunity against EIDs.

INTRODUCTION
Emerging infectious diseases (EIDs) are defined as newly appearing infections in a population or diseases that have previously existed, but are rapidly emerging in incidence or geographic range. 1,2 EIDs are often initiated by pathogens with a change in pathogenicity, and these emerging pathogens are highly likely to be zoonotic, with a broad range of hosts or vector-borne. [3][4][5] New human pathogens are especially likely to continue emerging, mainly from mammals and birds, for the foreseeable future. For these reasons, an effective global surveillance system for novel pathogens is needed. Due to the diverse nature of antigenic variation of emerging pathogens and their various strategies to evade the immune system, EIDs continue to pose difficult challenges to clinicians, public health profes-sionals, and researchers globally.
An understanding of EID pathogenesis begins with the investigation of the immune response to emerging pathogens. An immune response is the consequence of an intricate and elaborate network of a large number of specialized cells and organs operating over space and time. In response to pathogens, an immune system must orchestrate the complex interactions and activities of a large number of diverse cell types, thereby recognizing and eliminating the pathogens. The ability to respond quickly and effectively to control invading pathogens is achieved by both the innate and adaptive immune systems. Innate immunity provides a fast and immediate sensing of pathogens, whereas adaptive immunity is responsible for a late and long-lasting protective immune memory that is able to respond quickly to future infections. The major goal of vaccines is to mimic these naturally induced memory responses by training the immune system with altered forms of a pathogen or components of pathogens that do not cause illness. Therefore, the success of vaccination largely depends on the quality of vaccine-induced immunogenicity.
Thus far, vaccines have been prepared by traditional methods, involving isolating infectious agents, attenuating or inactivating them, and exposing them to the immune system. 6 Although this traditional approach has proven very efficient ag-ainst pathogens with relatively low antigen variability, such as smallpox, polio, measles, mumps, and rubella, these approaches are not optimal to provide adequate protection against recently appearing EIDs, such as avian influenza virus infection, Ebola hemorrhagic fever, and dengue fever. 7,8 Thus, novel systemic approaches are required to predict protective immune responses from high throughput sequencing and bioinformatics analysis and to identify potential antigenic targets to guide and accelerate vaccine development.
In recent years, systems biology has rapidly emerged as a new discipline. 9 As researchers move from a traditional reductionist approach to a systemic approach, there are rapidly increasing amounts of novel omics data and other relevant data available. Recent advances in next-generation sequencing (NGS) and high-throughput technologies have enabled researchers to comprehensively assess whole genomes, transcriptomes, proteomes, and metabolomes of cells and tissues. These systemic approaches are being applied to the field of immunology and vaccine development, since the integration of genomics, transcriptomics, and metabolomics can give a global picture of nearly 30000 genes, proteins, and cells participating in the immune response to pathogens or vaccination. [10][11][12][13] In this review, we highlight the major EIDs that have had a significant global impact and for which vaccines do not currently exist, and we summarize recent EIDs in South Korea. Moreover, we discuss various systems biology approaches and examine how these strategies offer significant potential for discovering new biomarkers of pathogenicity or predictors of protective immunity. Overall, we suggest that a systems biology approach would likely enhance our understanding of the pathogenesis of newly emerging pathogens and accelerate the development of next generation vaccines against EIDs.

EMERGING INFECTIOUS DISEASES
Recent global outbreaks of EIDs, such as Ebola or Zika virus (ZIKV) diseases, reminded the world of the imminent threat of unexpected pathogens. EIDs can result from infectious agents that have recently appeared or have existed, but are rapidly increasing in incidence. 14,15 Major pathogens include highly pathogenic avian influenza (HPAI) virus, severe fever with thrombocytopenia syndrome (SFTS) virus (SFTSV), ZIKV, dengue virus (DENV), and Middle East respiratory syndrome coronavirus (MERS-CoV).
Southeast Asia has been the epicenter of many emerging diseases in the past decade. 16 Table 1 shows the characteristics of recently occurred or current EIDs occurring in Southeast Asia in the 21st century. Such diseases pose a major threat to public health and have the potential to cause a high incidence of fatality among the global community. Therefore, further investigation into the emergence and pathogenesis of EIDs is required for public health and safety.
There are several factors contributing to the emergence of new pathogens: microbial adaptation and genomic change, increased frequency of international travel and commerce, and climate change. 17 Given that microbial adaptation is driven by the evolution of microbes to survive in diverse environments, microbes have evolved to change their genomes. RNA viruses carry RNA as their genetic material and can quickly adapt to and exploit different hosts because of the high error rates of the viral enzymes (RNA-dependent RNA polymerases) during their replication. 18 The segmented nature and faulty replication mechanisms of Orthomyxoviruses promote genetic variability and frequent re-assortment events, resulting in genetically diverse and novel genomic constellations in infected hosts. 19 As an example, hemagglutinin surface proteins of the influenza A virus undergo antigenic drift and shift, acquiring new infectivity and evading recognition by the immune system. 20 Another important consequence of the unstable genomic make-up of RNA viruses is the potential for interspecies transmission between animal hosts and humans. Accordingly, the recent emergence of avian influenza viruses could be devastating in an immunologically naïve population and pose a serious threat to public health. 21 Another major factor responsible for EIDs is an increased incidence of international travel and commerce. 22,23 The frequent use of air travel has enabled increased globalization, and international tourism has expanded 1.5 times in the last decade. Many travelers can be infected by and become carriers of EID pathogens without their knowledge. A viral incubation period can frequently last more than 2 weeks; thus, it is impossible to successfully monitor travelers with EIDs at an early stage of infection. 24 Surveillance of travelers with respiratory illness is especially difficult, because respiratory viruses can propagate easily and cause outbreaks in non-endemic disease areas. For example, in 2015, South Korea experienced a MERS outbreak, which was initiated by a man who had traveled from Bahrain, which is located in the Middle East. 25 Despite South Korea being a non-endemic area for MERS, 186 cases were reportedly infected with MERS-CoV during the 2015 outbreak, and this number was the highest among MERS non-endemic areas. 26 Lastly, climate change can contribute to ecological changes and, thus, contribute to the emergence of EIDs. Over the last 10 years, the temperature of the earth has increased by approximately 0.4°C, and this global warming has contributed to climate change throughout the world. A warmer climate can provide a more favorable environment for the survival and completion of the vector life cycle. 27 In particular, the mosquito population has been widely established across Europe, as well as tropical regions, such as Africa and South Asia. 28 As an example, prior to 1970, only nine countries had experienced dengue fever, which is transmitted by mosquitoes and occurs mostly in tropical regions. However, today, cases of DENV-associated disease can be found in more than 125 countries. 29 Moreover, climate change is frequently invoked as a primary cause of expansions in incidences of tick-borne diseases, particularly Lyme disease. 30 A wide distribution of tick vectors increased the prevalence of tick-borne pathogens, given that rising temperatures accelerate the cycle of development, the production of eggs, and the density and distribution of the tick population. 31 Therefore, it is necessary to explore how climate change will alter the distribution, dynamics, and risk of vector-borne diseases.

Highly pathogenic avian influenza infection
In South Korea, there are currently several major EIDs, such as HPAI infection, SFTSV infection, MERS-CoV infection, and DENV/ZIKV-associated diseases, that could pose great risks to public health in the near future. Since 2003, outbreaks of HPAI virus have been alarming, because they have caused significant economic loss and public health concerns. 32 The HPAI virus can undergo rapid evolution by gene mutation, reassortment, and homologous recombination in avian species and vertebrate reservoir hosts. 33 Although there is no evidence thus far to suggest a direct transmission of HPAI virus to humans in Korea, concerns remain due to the potential for avian influenza viruses circulating in poultry to become transmissible between species and to directly infect humans. In addition, due to the recent increase in zoonotic infections in poultry and persistent human infections in China, influenza A (H7N9) virus has remained a public health threat. Moreover, in February 2013, cases of human infection with a novel, lowly pathogenic H7N9 virus were reported in the Anhui and Shanghai regions of eastern China, and as of April 2017, the total number of H7N9 cases has exceeded 1344, with 511 deaths. 34,35 Severe fever with thrombocytopenia syndrome virus infection SFTS infection is an emerging tick-borne hemorrhagic fever that was first described in rural areas of China. The causative agent, SFTSV, is a novel phlebovirus in the Bunyaviridae family. SFTS cases were first reported in China (2009), Japan (2012), and Korea (2013). [36][37][38][39] The major clinical signs and symptoms of SFTS include high fever, gastrointestinal symptoms, thrombocytopenia, leukocytopenia, and multi-organ dysfunction, with an average case-fatality rate of 10-30%. Moreover, bone marrow suppression and hemophagocytic histiocytes are commonly found in Korean patients with SFTS infection. 40 Despite no available treatment for SFTS infection yet, early plasma exchange implementation has proved to be beneficial. 41 Moreover, SFTSV is believed to be transmitted through tick bites; however, there are a few suspected cases of human-to-human transmission in China and Korea. [42][43][44][45] Although human-to-human transmission is considered very rare, it is urgently necessary to develop effective diagnostic and preventative tools to protect against SFTSV and maintain continuous surveillance, because the virus is thought to spread via blood or mucosal secretions.

Middle East respiratory syndrome coronavirus infection
MERS-CoV infection is a respiratory disease caused by MERS-CoV, a positive single stranded RNA virus. 46 Although the detailed mode of transmission and period of virus shedding from infected patients are not fully understood, a nosocomial infection process is anticipated as the main transmission process. 47 MERS-CoV was first identified in the Middle East where dromedary camels were found to carry MERS-CoV without any respiratory symptoms. 48 The fatality rate of MERS-CoV infection in humans is approximately 20−50%, and its symptoms include fever, cough, and breathing difficulties. 48 Since 2012, about 30 countries, including Saudi Arabia, China, Malaysia, and Jordan, have reported MERS outbreaks, with Saudi Arabia reporting approximately 80% of all outbreaks. 49 Surprisingly, South Korea, which is a non-endemic area for MERS, experienced MERS outbreaks in 2015. 26 It is thought that a traveler returning from Bahrain carrying the MERS virus caused an outbreak of 186 cases and 36 deaths, which had a substantial impact on the South Korean economy. 26 Given the continuum of emergence and the large magnitude of MERS outbreaks in various regions, further studies are required to bolster capabilities for timely detection and effective control and prevention of MERS-CoV.

ZIKV/DENV-associated infection
As a consequence of global warming, ZIKV/DENV-associated diseases are potential EIDs that could arise in South Korea. DENV and ZIKV are mainly transmitted by Aedes albopictus and Aedes aegypti mosquitoes, respectively. Dengue fever is a major public health concern in many parts of the tropics and subtropics, and four different serotypes are currently co-circulating in most dengue-endemic areas. 50 The pathogenesis of dengue fever is very unique in regards to antibody-dependent immune enhancement associated with DENV. Whereas primary infection usually confers durable, if not life-long, protection against re-infection by a homologous DENV serotype, secondary infection by viruses of a heterologous DENV serotype can frequently occur and result in severe pathology of the host. 51,52 Thus, the unique complexity of DENV pathogenesis and its effect on immune enhancement pose a great challenge to the development of a safe and effective DENV vaccine. Even though South Korea is not a region where dengue is epidemic, there have been many imported DENV infection cases in South Korea, and their number is increasing due to increased traveling to Southeast Asia. 53,54 Likewise, ZIKV is an another emerging mosquito-borne pathogen which belongs to the family Flaviviridae and is closely related to other pathogens of public health importance, including Yellow Fever virus, DENV, Japanese Encephalitis virus, and West Nile virus. The first case of human infection by ZIKV was reported in Uganda in 1952. 55 Usually, ZIKV infection results in asymptomatic clinical features, but in some cases, ZIKV infection can lead to fatal microcephaly in infants and Guillain-Barre syndrome in adults. 56 With increasing cases of ZIKV infection in America, many Asian countries, including India, the Philippines, Taiwan, and Vietnam, have reported ZIKV outbreaks, as of 2016. 57 Although South Korea is considered a non-endemic region for Aedes aegypti-transmitted diseases, the first patient infected with ZIKV was reported in South Korea in 2016. The patient had stayed in Brazil for 3 weeks, and upon arrival in South Korea, complained of various symptoms, including fever, eyeball pain, and maculopapular rash. 58 Given that Asia is endemic for ZIKV infection and the climate of South Korea is changing each year, it will be only be a mat-ter of time before mosquito-borne EIDs become endemic in South Korea. Thus, we need to prepare for protection against mosquito-borne EIDs.
As summarized above, in addition to virus genetic variation, complex environmental factors, including ecological and behavioral influences, can be important contributing components of the recent emergence of viral diseases. Recent Ebola and Zika outbreaks have shown that the world is unprepared to detect local outbreaks and respond quickly enough to prevent them from becoming global pandemics. Given the great concern for public health owing to the speed and increased volume of global transportation, the ability to rapidly develop and deliver vaccines when new 'unknown' diseases emerge offers our best hope to outpace EID outbreaks, save lives, and avert disastrous economic consequences.

LEARNING FROM SYSTEMS BIOLOGY APPROACHES
As mentioned above, due to ecological, environmental, and demographic factors that increase exposure to emerging pathogens, EID cases are increasing in prevalence globally. Together with the ongoing evolution of viral and microbial variants and selection for drug resistance, these factors will contribute to the continued emergence of EIDs. Therefore, there is an urgent need for effective surveillance and control strategies, as well as rapid vaccine development. Early warning of EIDs depends on the ability to identify the unusual as early as possible. Information is, therefore, essential and this can be achieved using systems biology tools.
Systems biology is an approach to understanding living systems that focuses on modeling diverse types of high-dimensional interactions to develop a more comprehensive understanding of complex phenotypes manifested by the system. 10 Systems biology tools enable us to develop and validate predictive models of infectious disease initiation, progression, and outcomes. These models can be derived from the study of the architecture and dynamics of systems-wide host/pathogen molecular interaction networks during infection. Ideally, integrated datasets generated from a combination of high-throughput multi-omics data (transcriptomics, proteomics, metabolomics, lipidomics, etc.) can be used to construct predictive models of the networks and dynamic interactions between the biological components of the complex pathogen-host system. 59 In particular, through years of dedicated effort, high-throughput RNA sequencing (RNA-seq) technology, which is a powerful way to profile the transcriptome with great efficiency and high accuracy, has been employed in various viral infections and diseases. [60][61][62][63][64][65][66] These studies proved that RNA-seq technology has the potential to reveal the dynamics of pathogen genome alteration and systemic changes in host and pathogen gene expression during the process of infection. Thus, if this approach is applied to EID research, information can help to accelerate uncovering the pathogenesis of novel infections and the interaction mechanism of emerging pathogens. As a notable example, several groups have taken advantage of RNAseq technology to provide novel insights into how ZIKV infection may cause microcephaly in a short period of time. Nowakowki, et al. 67 using single-cell RNA-seq and immunohistochemistry, first reported that AXL, a viral receptor for ZIKV, is highly expressed in human radial glial cells, astrocytes, endothelial cells, and microglia in the developing human cortex and in progenitor cells in the developing retina. Additionally, Onorati, et al. 68 analyzed ZIKV-infected neuronal progenitor cells to identify transcriptional changes caused by the virus. Using techniques including single-cell RNA-seq, they described the derivation and characterization of neocortical and spinal cord neuroepithelial stem cells to model early human neurodevelopment and ZIKV-related neuropathogenesis. These data may lead to significant advances in efforts to develop a vaccine against ZIKV.
As discussed above, more extensive knowledge of the factors underlying EID pathogenesis can help with the development of more effective prevention strategies. If we are to protect ourselves against EIDs, the essential first step is to establish effective global disease surveillance to give an early warning of emerging infections. This can be achieved by rapid, portable, and inexpensive high-throughput screening of EID pathogens. Furthermore, systems-level informatics will not only reduce the time required for experimental immunology and vaccinology, but also promote identification and rational design of novel vaccine candidates and the determinants of vaccine-induced immunity. This will prompt the escalation of control measures where necessary and facilitate outbreak tracking.

SYSTEMS VACCINOLOGY APPROACHES
Vaccines are one of the greatest successes in the history of mankind. However, despite a global effort to develop vaccines against major infectious diseases, the correlates of protection are still poorly defined for many existing vaccines. Moreover, pathogens can evade and suppress immune responses, and human genetic variability can contribute to host susceptibility to infectious diseases or vaccine responsiveness. Although the eradication of smallpox is one of the many successes of global immunization programs, many vaccine challenges remain, especially for the design of effective vaccines for major killers, such as AIDS, dengue fever, and malaria. 69 Furthermore, there is an urgent need to develop vaccines for EIDs, including the diseases mentioned above and neglected diseases, such as those caused by Chikungunya, Hendra, and Nipah viruses.
Recent studies have used systems biology approaches to identify molecular networks that orchestrate immunity to vaccinations in humans and potentially define correlates of pro-tection. 10,11,[70][71][72][73][74][75][76][77][78][79][80] The first attempt at this approach was made with one of the most successful vaccines ever developed, the yellow fever YF-17D vaccine. 70,81 As a live attenuated virus vaccine, a single shot of the yellow fever vaccine can confer protection in more than 90% of vaccinees, and it results in long-lasting immunity, a strong neutralizing antibody response, and a robust T cell response. Two independent groups have simultaneously published gene expression profiling data of blood samples from vaccinees within a few days after vaccination. 70,82 These molecular signatures helped researchers predict the magnitude of vaccine-induced immunity and provided novel insights into the dynamic nature of the innate and adaptive immune responses to YF-17D vaccine. In particular, Querec, et al. 70 reported that EIF2AK4 (also known as GCN2), a known sensor of low amino acid levels inside the cell, was frequently found in the blood of vaccinees and was strongly correlated with the magnitude of the later CD8 T cell response to YF-17D vaccine.
Subsequently, systems biology approaches have been extended to influenza and other vaccine studies. Identifying molecular mechanisms of influenza vaccine-induced immunity was more challenging than the yellow fever vaccine study, given that the variation in antigenic match between the vaccine and circulating influenza strains can contribute to vaccine efficacy. Nakaya, et al. 71 compared transcriptomic data for those given either the trivalent influenza vaccine (TIV) or the live attenuated influenza vaccine (LAIV). Both vaccines induced the expression of genes involved in innate immune pathways, although LAIV induced higher expression of several interferon-related genes, similar to another live attenuated vaccine, YF17D. Therefore, these data demonstrated that live and dead influenza vaccines clearly result in differential vaccine-induced immunity.
Among many factors that determine the efficacy of vaccines, the age of vaccinees is the most important. In fact, older adults and children are most vulnerable to severe and often lethal influenza virus infection, due to decreased vaccine efficacy. Thus, the use of adjuvants can be helpful to improve the efficacy of influenza virus vaccines for children and older adults. [83][84][85] In support of this idea, Nakaya, et al. 75 conducted blood transcriptome analysis in children less than 2 years old to compare the immunogenicity of TIV with and without MF59 adjuvant, an oil-in-water adjuvant known to increase the magnitude of the antibody response to inactivated vaccines. Interestingly, the more potent antibody responses generated in subjects receiving MF59-adjuvanted vaccines correlated with strong transcriptional signatures of interferon signaling network in blood samples.
In addition to transcriptome analysis, other systems-level approaches, such as proteomics, metabolomics, and lipidomics, can provide greater depth in evaluating correlates of vaccine-induced immunity. The latest research by Dr. Pulendran's group analyzed vaccine-induced immunity in multi-scale ways in different age groups of healthy adults immunized with shin-gles vaccine, Zostavax. 77 The authors presented an integration data of peripheral blood mononuclear cells (PBMC) transcriptomics, flow cytometry of blood cell populations, plasma cytokine analysis, and metabolomics. The integration of these data revealed transcriptomic association with metabolic correlates of vaccine immunity. Interestingly, this analysis revealed sterol regulatory binding protein-1 and its targets as potentially key integrators of antibody and follicular helper T cell responses. Therefore, these data suggest that blood cells potentially regulate gene transcription in response to metabolic cues, suggesting the value in integration of diverse systems-level data.
Given that a vaccine usually initiates a broad, polyfunctional, and persistent immune response integrating all effector cells of the immune system, it is important for scientists to understand the complexity and dynamic and diverse quality of the immune response. 86 Therefore, innovative tools, such as using single cell technologies to 'omics' approaches, have enabled researchers to quantitatively examine entire classes of molecules at a single cell level, and should further facilitate our understanding of the functional diversity of the immune response. As shown in Fig. 1, using systems biology tools for in- Fig. 1. The process of systems vaccinology approaches. Systems biology approaches applied to clinical trials can lead to the generation of new hypotheses that can be tested and ultimately lead to better vaccine development. For example, correlates of vaccine-induced immunity in clinical trials can be profiled in detail with high-throughput technologies, such as RNA-sequencing, proteomics, and metabolomics. The high-throughput data thus generated can be integrated using bioinformatics tools and used to create hypotheses about the biological mechanisms underlying vaccine efficacy and immunogenicity. Such hypotheses can then be tested with animal models or in vitro human systems. The insights gained from experimentation can then guide the identification of biomarker and the design and development of new vaccines. Thus, systems vaccinology approaches can provide translational solutions for novel and enhanced vaccine development.
creased understanding of vaccine-mediated immunity can help scientists be better positioned to identify correlates of vaccine immunogenicity and make progress towards developing new and enhanced vaccines and therapeutics.

CURRENT LIMITATIONS AND CHALLENGES
Thus far, we have reviewed recent advances in the fields of systems immunology and vaccinology, and presented how systems biology-based platforms can be applied to accelerate a deeper understanding of molecular mechanisms of immunity against EIDs. Despite recent advances, there remain computational and biological challenges regarding systems biology approaches. Because the immune response to a vaccine can be affected by a wide range of confounding factors, including cellular, population, molecular, individual, and technical factors, scientists face many challenges and potential limitations on the analysis of systems-level informatics. These challenges and limitations are described in Fig. 2.
Before conducting a large-scale systems biology approach, one should consider the following limitations. Although previous studies involving mathematical modeling of the dynamics of immune responses can provide clues for optimal experimental design, systems-level experiments should be very carefully designed to control for all variables in terms of time points. Furthermore, given that an immune response occurs as a consequence of coordinated events among many immune cells over space and time, it is important to understand how immune cells respond at both the cell population and the single cell level. Single-cell sequencing allows researchers to examine the genomes or transcriptomes of individual cells, thus providing a high-resolution view of cell-to-cell variation. This technique can be used to assess the individual contribution of single cells in complex tissues by profiling the transcriptome in an unbiased manner. In addition, technological problems during systems biology approaches can be resolved by the solutions shown in Table 2.
Additionally, despite the high costs of high throughput analysis and limited numbers of human samples, to minimize confounding factors, it is best to maximize sample sizes to achieve appropriate statistical power. Besides sample size, another major confounding variable from a systems vaccinology approach is that the observed transcriptomic changes may result from new induction of gene expression or may simply reflect the changing cellular composition of PBMC compartments. Given that it is impossible to obtain cellular or tissue samples from lymph nodes or spleens of healthy human subjects post vaccination, vaccine efficacy tests must be performed on PBMCs drawn pre-and post-vaccination. This limits the optimal understanding of vaccine-induced protective immune responses. One should keep in mind that PBMCs include mixed and dynamic cell populations, and thus, there may be dynamic events occurring within lymphoid compartments of PBMCs. 87,88 Therefore, it will be more informative and precise to determine the transcriptome in sorted cell subsets, such as B cells, T cells, natural killer cells, macrophages, and dendritic cells.

REVERSE VACCINOLOGY-BASED VACCINE DESIGN
Emerging technologies, such as structure-based antigen design and synthetic vaccinology, have heralded in a new era of vaccine discovery and development. As an example, a reverse vaccinology process was recently applied, based on the notion that protective antigens could be identified by determining the antibody repertoire from infected or vaccinated individuals. 89,90 Fig. 3 demonstrates the identification of novel and the most effective epitopes using NGS and high-throughput robotic crystallization platforms to enable more precise prediction of epitope targets for efficient antibody generation. Webbased epitope databases, vaccine design resources, and protein structure comparison resources can help to improve our understanding of immune responses and the escape and evolution of pathogens. 91 As an example, there have been massive antibody engineering efforts to improve the breadth and potency of anti-HIV-1 antibodies. 92 Given the importance of neutralizing antibodies for vaccine protection, the dissection of antibody variable regions using reverse vaccinology tools is a rational approach to design vaccines and will ultimately lead to more effective vaccines for EIDs.

SUMMARY AND FUTURE DIRECTIONS
Recent advances in systems biology-based technologies have enabled scientists to investigate the immune responses induced by pathogens and vaccines at a greater depth than ever before. More importantly, these advances are facilitating the identification of robust molecular and cellular signatures of protective immunity, which can help to generate diagnostic tools that reduce the length and cost of current clinical trials.

Fig. 2.
Potential biases for systems vaccinology data analysis. The application of systems biology approaches to the fields of immunology and vaccinology faces many challenges and potential limitations. The potentially challenging factors for systems vaccinology analysis can be divided into different categories, such as cellular, population, molecular, individual, and technical aspects.  Fig. 3. Use of reverse vaccinology tools in vaccine design. The workflow of vaccine design using reverse vaccinology is shown. First, B cells or plasma cells can be isolated from infected or vaccinated individuals and PCR amplification of antibody gene heavy and light chains can lead to human B cell repertoire analysis, which enables the identification of protective antibodies. Using sequence information of antibody variable regions, the interaction of antibodies with their target antigen can be structurally characterized and used to predict the protective epitope using crystallography. The protective epitope can then be engineered to produce an optimized immunogen with enhanced delivery format, such as nanoparticle technologies or adjuvants. Finally, this new reverse vaccinology-based antigen can be tested in humans.
When these systems approaches are applied to EIDs, they will enable more rapid and rational design of vaccine development. Thus, in this developing field, vaccinologists, immunologists, bioinformaticians, and systems biologists must work hand-in-hand to advance our understanding of the molecular mechanisms by which vaccines induce protective immunity and help drive development of the next generation of vaccines to prevent EIDs.


ERAP1/ERAP2 and RUNX3 polymorphisms are not associated with ankylosing spondylitis susceptibility in Chinese Han

Summary Previous studies show that endoplasmic reticulum‐associated aminopeptidase (ERAP1/ERAP2) and runt‐related transcription factor 3 (RUNX3) gene polymorphisms are associated with AS (ankylosing spondylitis) in European Caucasians. However, contradictory results were reported in different Asian populations. The purpose of this study was to determine whether eleven candidate single nucleotide polymorphisms (SNPs) in ERAP1/ERAP2 and six in RUNX3 genes confer susceptibility to AS with or without acute anterior uveitis (AAU) [AS+AAU+ or AS+AAU–] in Chinese Han. Therefore, a case–control association study was performed in 882 AS+AAU–, 884 AS+AAU+ and 1727 healthy controls. Genotyping was performed using the iPLEXGold genotyping assay. A meta‐analysis was performed to assess the association of polymorphisms of ERAP1 with AS susceptibility in Asian populations. No association was found between SNPs of ERAP1/ERAP2/RUNX3 and susceptibility of AS with or without AAU. A case–control study between patients with human leucocyte antigen HLA‐B27‐positive and healthy controls also failed to demonstrate an association of the tested SNP with AS with or without AAU. Moreover, a meta‐analysis showed that there was no association of rs30187, rs27037, rs27980, rs27434 and rs27582 in ERAP1 with AS in Chinese Han. Taken together, 17 SNPs in ERAP1/ERAP2 and RUNX3 genes did not confer disease susceptibility to AS in Chinese Han.

Introduction
Ankylosing spondylitis (AS), a particular subtype of spondyloarthritis, is a progressive chronic disorder primarily affecting the spine. Peripheral arthritis, enthesitis and iritis are the common complications of AS [1]. The incidence of AS is 0Á24% in Chinese Han [2] and 0Á55% in Europeans [3]. Acute anterior uveitis (AAU), an inflammation usually affecting the iris and ciliary body of the eye, is the most common form of ocular involvement. Earlier studies show that 12% of patients experience AAU when diagnosed with AS, and the incidence can increase up to 25% after 20 years and 60% after more than 50 years follow-up [4,5]. Our previous study showed that AAU patients with AS show a more common bilateral eye involvement and a higher frequency of fibrinous exudates in the anterior chamber compared with AAU without AS patients in human leucocyte antigen HLA-B27 positive carriers [6].
The pathological mechanisms leading to AS remain uncertain. Twin and family studies showed that genetic factors play a very important role in the occurrence of AS [3,7]. Recently, genomewide association studies (GWAS) in European Caucasians show that gene polymorphisms in endoplasmic reticulum-associated aminopeptidase (ERAP1/ERAP2) and runt-related transcription factor 3 (RUNX3) are associated significantly with AS susceptibility [8][9][10].
The ERAP1 gene is the second most important gene that has been shown to be related closely to AS after HLA-B27. ERAP1 and ERAP2, which are localized in the endoplasmic reticulum, play an important role in trimming peptides prior to or after their binding onto major

95
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. histocompatibility complex (MHC) class I molecules [11]. The MHC-peptide complex is presented by antigenpresenting cells and can be recognized by specific T cell receptors as well as natural killer cell receptors [12,13]. RUNX3, a member of the RUNX transcription factor family, plays a key role in T cell line development and in regulation of the transforming growth factor (TGF)-b signalling pathway in dendritic cells [14]. RUNX3 affects multiple types of immune cells [15], including regulatory T cells and natural killer (NK) cells, but seems to be particularly involved in the differentiation and development of CD8 1 T cells [16,17]. This latter lymphocyte subpopulation is presumed to play a vital role in the development of AS [18][19][20]. ERAP1/ERAP2 and RUNX3 interact with each other in the formation of peptide repertoires and T cell development and differentiation, respectively [21,22]. The peptide spectrum formed after ERAP1/ERAP2 cleavage affects the type and quantity of activated CD8 1 T cells directly [23], and it was therefore likely that both ERAP1/ERAP2 and RUNX3 could be involved in the development of AS.
Contradictory results have, however, been reported in several studies on the genetic association of gene polymorphisms in ERAP1/ERAP2 and RUNX3 with AS susceptibility worldwide [9,10,[24][25][26]. The aim of our study was therefore to re-evaluate the association of polymorphisms in ERAP1/ERAP2 and RUNX3 with AS susceptibility in a large cohort of Chinese Han patients.  Table 1. The SNPs rs27037, rs27980 and rs27582 were reported to be in strong linkage disequilibrium (LD), as well as the SNPs rs30187 and rs27434 and the SNPs rs2549782 and rs2248374. Based on conflicting results in several studies [9,10,[24][25][26], all sites were selected for the current study (Fig. 1).

Case and control cohorts
The iPLEX Gold Genotyping Assay technique was used for genotyping analysis on a Sequenom MassArray System (Sequenom, San Diego, CA, USA). MassArray Assay Design software (Sequenom, San Diego, CA, USA) was applied for primer design. The study was performed strictly following the standard procedures of the manufacturer (Agena Bioscience, San Diego, CA, USA).

Statistical analysis
The v 2 test was applied for Hardy-Weinberg equilibrium (HWE) analysis in all tested SNPs. Allele and genotype frequency were calculated with the Sequenom MassArray System platform. SPSS (SPSS Inc., Chicago, IL, USA) version 19.0 was used to calculate 95% confidence intervals (CI) and odds ratios (ORs). The Bonferroni correction was employed to correct multiple comparisons. A corrected P-value (Pc) of less than 0Á05 was considered to be statistically significant.

Meta-analysis
A literature search was performed using the PubMed database for studies on the associations of polymorphisms in ERAP1/ERAP2 or RUNX3 with AS susceptibility. Ankylosing spondylitis (AS), acute anterior uveitis (AAU), RUNX3, ERAP1 (ARTS1), ERAP2 and polymorphisms were searched in the PubMed database (up to May 2017). The following inclusion criteria were used: (i) case-control studies on AS susceptibility examining the role of ERAP1/ ERAP2 or RUNX3 polymorphisms (ii) containing data of minor allele frequencies (MAF) and 95% CI and OR (iii) showing numbers of cases and controls. Exclusion criteria were: (i) duplicate data in references, (ii) replication studies and (iii) twins and family studies. The author, year of publication, ethnicity, numbers of controls and cases and MAF of polymorphisms in the ERAP1/ERAP2 gene or RUNX3 gene were collected from each study. RevMan version 5.3 (http://ims.cochrane.org/revman/download) software was applied for the meta-analysis. Both the fixed-and randomeffect models were used to account for pooled ORs (I 2 statistics). A list of the included studies is shown in the Supporting information, Table S1.

Case-control study
Clinical features of AS 1 AAUand AS 1 AAU 1 patients are shown in Table 2. The proportion of male patients in the AS 1 AAUand AS 1 AAU 1 groups are similar (68-69%).
The average age of AS patients also having experienced AAU is older than that of the AS patients without AAU. The ratio of HLA-B27 was 97Á17% in AS 1 AAUand 82Á92% in AS 1 AAU 1 patients. Seventeen SNPs (eleven located in or near the ERAP1/ERAP2 gene, six in the RUNX3 gene) were genotyped in 1766 AS patients with or  without AAU (AS 1 AAU -882, AS 1 AAU 1 884) and 1727 healthy controls. All the tested polymorphisms in ERAP1/ ERAP2 genes and RUNX3 gene are shown in the Supporting information, Table S2. Genotype frequencies of all SNPs were in line with the HWE (Fisher's P-value > 0Á05).
Our results showed that some SNPs (Supporting information, Table S2) (rs10044354, rs1065407, rs27434, rs27582 and rs27037) showed no association of polymorphisms with risk of AS 1 AAUafter correction for multiple comparisons. This was also the case for SNPs rs27434, rs30187 and rs27037 polymorphisms with risk of AS 1 AAU 1 . A case-control study performed on HLA-B27positive patients and non-typed healthy controls also showed similar results (data not shown). Haplotype analysis was performed on the ERAP1gene using Haploview version 4.2 software. None of the haplotypes showed an association with AS with or without AAU in our Chinese Han population (Supporting information, Table S3).

Meta-analysis
A literature search in the PubMed database resulted in the identification of 22 studies; 12 studies met the study inclusion criteria, including four studies from European populations and eight studies from Asian populations (two from Korean, six from Chinese populations), including the present study [9,25,26,30,31,34,35,[37][38][39][40]. Ten studies were excluded (seven studies due to lack of data and three studies having duplicate data). A literature search in the PubMed database showed that there were no reports concerning the association of ERAP1/ERAP2 or RUNX3 gene polymorphisms with AS susceptibility in Japanese individuals. At the same time, we found that ERAP1/ ERAP2 or RUNX3 gene polymorphisms were a risk factor for AS in Korean and Taiwanese patients, but this was not obvious in Chinese mainland patients. Therefore, Chinese Han were divided into two subgroups in this meta-analysis (Chinese, including data from the Chinese mainland and China Taiwan Island; Chinese1, including data only from the Chinese mainland). The meta-analysis results of the association of ERAP1/ERAP2 polymorphisms with risk of AS is shown in Table 3. Insufficient reports were available for a meta-analysis of the RUNX3 gene.
Heterogeneity was found among all the studies. No obvious heterogeneity was found in studies on the association of rs30187, rs27434 and rs27044 with AS susceptibility in European populations (I 2 5 0%, I 2 5 0%, I 2 5 18%) (Supporting information, Figs S1-S3). However, heterogeneity was obvious in the studies from Asian populations (I 2 5 0$89%) (Supporting information, Figs S1-S6), even in Chinese populations (I 2 5 0$84%) (Supporting information, Figs S1-S6). The funnel plot of the loci showed no significant asymmetry. The meta-analysis was thus considered not to have a significant publication bias in each group of included studies. Forest plots of the meta-analysis of those loci are shown as Supporting information, Figs S1-S6.

Discussion
In the present study, known AS-associated SNPs were assayed in a large Chinese Han population to evaluate the association of polymorphisms in ERAP1/ERAP2 and RUNX3 genes with AS in Chinese Han. Our study demonstrated a trend of several polymorphisms for AS or AS with AAU in Chinese Han, including one SNP in ERAP2 and five in ERAP1. However, all these polymorphisms lost statistical significance after correction for multiple comparisons. Furthermore, a meta-analysis of the existing literature supported our findings that there was no association of polymorphisms in these genes with AS in Chinese Han. Subgroup analysis indicated that geographical factors might be the important causes of heterogeneity. South Koreans, Chinese Han and Taiwanese belong to the East Asian population. However, the studies from the peninsula and inland China are quite different, suggesting that environmental factors may play a role.
Aminopeptidases encoded by ERAP1/ERAP2 have two major functions presumed to be related to inflammatory and immune responses. One function is that of a 'molecular ruler' , trimming peptides along with ERAP2 prior to or after loading them onto MHC class I molecules. SNPs in ERAP1/ ERAP2 may affect the peptide repertoire, possibly resulting in an aberrant CD8 1 T cell response [20,23]. ERAP1 gene polymorphisms have been confirmed to be associated with AS in Caucasians [8][9][10]. However, our meta-analysis of all data reported on the association of polymorphisms in these genes with AS indicated that population and geographical differences might be one of the causes of heterogeneity. Even in Asian populations, the association of ERAP1 gene polymorphisms with AS differs depending on the studied populations [25,30,34]. Results of studies from Korea and Taiwan were similar, but different from data from mainland China. Our study showed no association of ERAP1 gene polymorphisms with AS susceptibility, which was in agreement with an earlier GWAS by Lin et al. [25]. Results from our metaanalysis also showed that data from Taiwan and Korea were similar. Little is known about the association of ERAP1gene polymorphisms with AS in Japanese. This may be due to the fact that HLA-B27 is rare in the Japanese population and functional ERAP abnormalities are probably seen only in relation with HLA-B27. Another function of aminopeptidases encoded by ERAP1/ERAP2 is presumed to be related to inflammatory and immune responses in regulating inflammation via the shedding of membrane-bound cytokine receptors, such as tumour necrosis factor receptor 1 (TNF-R1) [41], interleukin (IL)-1RII [42] and IL-6Ra [43]. Whether this activity is involved in AS pathogenesis seems unlikely, as earlier studies showed that there were no differences in the levels of IL-6R, IL-1R2 and TNF-R1 in cell culture supernatants or serum when comparing AS patients with controls [8,36]. In this study, we found no association between ERAP1 gene polymorphisms and AS susceptibility in Chinese Han, which might be due to the following three reasons. First, the vast territory of mainland China has different climate zones as well as a variety of different natural environments, all of which might play a role. Secondly, we only collected patients from two affiliated hospitals, which might not be sufficient to represent the huge Chinese population and widespread distribution over the country. Thirdly, ERAP1 gene polymorphisms and AS susceptibility may vary ethnically, and the SNPs in ERAP1 found to be associated with AS in European Caucasians might not be the susceptibility loci for Chinese Han.
The MHC gene HLA-B27 has been shown to be associated strongly with ankylosing spondylitis susceptibility, and confers 20-30% of the genetic risk [1]. The combined risk of ERAP1/ERAP2 and HLA-B27 in AS is up to 70% in European populations [52,53], and these two factors are now presumed to be the main risk alleles for AS. As mentioned above, combining earlier data from Lin et al. [25] with our results showed that there is no significant association of ERAP1/ERAP2 polymorphisms and the risk of AS in Chinese Han. The reason for this discrepancy is unclear, and may be due to as yet unknown environmental factors.
Our study has several limitations. First, samples included in the study came from only two affiliated hospitals. In view of the large population of Han Chinese in mainland China, a selection bias for AS and AS with AAU may be present. Thus, multi-centre studies are needed in future studies. Secondly, geographical factors were not taken into account for sample collection. Environmental differences are known to exist between northern and southern, inland, coastal and peninsular areas of China. Thirdly, the metaanalysis confirmed our result and also indicated that geographical factors may be the main reason of high heterogeneity, but the high value of I 2 indicates that part of the data should be subject of further analysis.
In conclusion, the present study shows that genetic polymorphisms of ERAP1/ERAP2 and RUNX3 genes may not be involved in AS susceptibility in Chinese Han.

Supporting information
Additional Supporting information may be found in the online version of this article at the publisher's web-site: As shown in (e), data from our study and genome-wide association study (GWAS) of Lin, I 2 5 36% and P 5 0Á11. . As shown in (d), data from our study and Zhang, I 2 5 0% and P 5 0Á11. Fig. S3. Forest plots for endoplasmic reticulum-associated aminopeptidase (ERAP1): rs27044 and susceptibility of ankylosing spondylitis (AS) of all studies [(a) studies from both Europe and Asia, (b) studies from Europe, (c) studies from Asia (Chinese mainland, China Taiwan Island, Korea)]. As shown in (c), data from Asia, I 2 5 88% and P 5 0Á05.    Table S1. Included and excluded studies in the metaanalysis Table S2. Allele and genotype frequencies of single nucleotide polymorphisms (SNPs) in Chinese Han patients with ankylosing spondylitis (AS) and AS with AAU Table S3. Haplotype analysis of 11 SNP sites of ERAP1 in Chinese Han ankylosing spondylitis (AS) patients with or without acute anterior uveitis (AAU)


extfieldsofstudy: Medicine Chemistry

s2fieldsofstudy: Biology Chemistry Medicine

text:
Cistanche tubulosa phenylethanoid glycosides induce apoptosis in H22 hepatocellular carcinoma cells through both extrinsic and intrinsic signaling pathways

Background Cistanche tubulosa (Schenk) R. Wight is a traditional Chinese medicine that parasitizes the roots of the Tamarix plant and has been used to treat male impotence, sterility, body weakness, and as a tonic. However, its antitumor effect on hepatocellular carcinoma is still elusive. Here, we investigated the antitumor effect of C. tubulosa phenylethanoid glycosides (CTPG) on H22 hepatocellular carcinoma cells both in vitro and in vivo and its mechanisms. Methods The morphology, viability, apoptosis, cell cycle and mitochondrial membrane potential (Δψm) of H22 cells were analyzed by inverted microscopy, MTT assay and flow cytometry, respectively. The expression and activation of proteins in apoptosis pathway were detected by Western blot. The in vivo antitumor effect was evaluated in tumor mouse model established using male Kunming mice. Results CTPG treatment significantly suppressed H22 cell growth in a dose- and time-dependent manner, which was correlated with the increased apoptosis and cell cycle arrest at G0/G1 and G2/M phases. Moreover, the chromosomal condensation was observed in CTPG-treated H22 cells. CTPG treatment significantly increased Bax/Bcl-2 ratio, reduced Δψm and enhanced the release of cytochrome c. The levels of cleaved caspase-8 and caspase-9 in both extrinsic and intrinsic signaling pathways were significantly increased that sequentially activated caspase-7 and -3 to cleave PARP. Finally, CTPG inhibited the growth of H22 cells in mice and improved the survival rate of tumor mice. Conclusions These results suggested that CTPG suppressed H22 cell growth through both extrinsic and intrinsic apoptosis pathways.

Background
Liver cancer ranked sixth for cancer incidence and fourth for cancer deaths worldwide. Moreover, it ranked fourth for cancer incidence and first for cancer mortality in countries with low sociodemographic index [1]. In China, liver cancer is the third leading cause of cancer-related death in 2015 [2]. More than 90% of primary liver cancers are hepatocellular carcinoma (HCC) in the world [3]. Currently, hepatic resection is the main option for the therapy of HCC. However, less than 30% of patients with HCC met the criteria of curative hepatic resection and the overall 5-year survival rate is still as low as 35-50% due to the high recurrence rate [4,5]. The availability of treatment options for the patients with intermediate to advanced HCC is very limited. Sorafenib, a molecular targeted drug, has been approved by FDA as the first-line treatment for advanced HCC. However, sorafenib only prolongs about 3 months of survival and the response rate is less than 4% [6,7]. It is urgent to develop new drugs or strategies against HCC.
Traditional Chinese medicine (TCM) alone or combined with other strategies has been used to treat HCC and shown the clinical benefits including prolonged survival time, improved life quality, reduced adverse reactions, and so on [8,9]. Cistanche, a kind of TCM, has various biological functions, such as anti-oxidation, anti-inflammation, anti-aging and neuroprotection [10,11]. Phenylethanoid glycosides have been considered the major active components of Cistanche, which have diverse activities including antioxidation, anti-inflammation, hepatoprotection and neuroprotection [12][13][14][15]. Our group has reported that Cistanche tubulosa phenylethanoid glycosides (CTPG) could induce apoptosis in melanoma B16-F10 cells and inhibited the growth of tumor in mice [16]. In this study, we measured the antitumor effect of CTPG on HCC H22 cells both in vitro and in vivo and investigated its mechanisms. We found that CTPG induced apoptosis in H22 cells through both extrinsic and intrinsic signaling pathways and suppressed the growth of H22 tumor in mice.

Cell line
The mouse H22 hepatocellular carcinoma cells were obtained from the Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, Xinjiang University (Urumqi, Xinjiang, China) and cultured in RPMI 1640 medium (Gibco) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin, and 10% heat-inactivated fetal bovine serum (Gibco) at 37°C in a humidified atmosphere of 5% CO 2 .

Analysis of cell cycle
H22 cells were inoculated in 60 mm culture dishes and treated with different concentrations of CTPG (0, 100, 200, 300 and 400 μg/ml) or 0.3% DMSO for 24 h. All cells were collected and washed twice with PBS. Cells were fixed in 70% ice-cold ethanol at − 20°C for 2 h and washed twice with PBS, then re-suspended in 300 μl Propidium iodide/RNase staining buffer (BD Biosciences). After 10 min at room temperature, samples were collected by flow cytometry (BD FACSCalibur, USA) and cell cycle distribution was analyzed with the ModFit LT 3.0 software.

Hoechst 33,258 staining
The morphological changes of H22 cell nuclei were analyzed by membrane-permeable DNA-binding dye Hoechst 33,258 staining. H22 cells were seeded in 6-well plate at the concentration of 1 × 10 5 cells/well in 2 ml medium. After 60%~70% confluence, the cells were treated with CTPG (0, 100, 200, 300 and 400 μg/ml) for 24 h. The cells were collected and fixed with 4% ice-cold Paraformaldehyde at 4°C for 10 min. After washing with PBS, cells were stained with Hoechst 33,258 (Beyotime, China) at 4°C for 10 min. Samples were observed by inverted fluorescence microscope (Nikon Eclipse Ti-E, Japan).
H22 cells were treated with different concentrations of CTPG (0, 100, 200, 300 and 400 μg/ml) or 0.3% DMSO for 24 h. Cells were collected and lysed with the Cell Lysis Solution RIPA (Beijing ComWin Biotech Co., Ltd) for 30 min on ice. Samples were spun down (12,000 g for 15 min at 4°C) to collect the supernatants and protein concentrations were measured by BCA Kit (Thermo Fisher Scientific, USA). Equal amount of protein in each sample was isolated by 12% SDS-PAGE and transferred to PVDF membranes (Biosharp, China). After blocking with TBST buffer contained 5% nonfat milk, membranes were incubated with corresponding primary antibodies and secondary antibodies conjugated to horseradish peroxidase (HRP), respectively. After washing with TBST, the target proteins were detected by ECL assay kit (Beyotime, China).

Animals and ethics statement
6-8 weeks old male Kunming mice were purchased from Animal Laboratory Center, Xinjiang Medical University (Urumqi, Xinjiang, China). Mice were kept in a standard temperature-controlled, light-cycled animal facility of Xinjiang University. All animal studies were carried out according to the guidelines of the Animal Care and Use

Tumor mouse study
For induction of tumor mouse model, male Kunming mice were subcutaneously injected with 1 × 10 6 H22 cells in 100 μl PBS into the right flank. After 3 days, mice were randomly divided into 3 groups (7 mice/ group). Control group was injected with 0.1 ml DMSO subcutaneously around tumor. CTPG-200 and CTPG-400 groups were subcutaneously injected with 200 or 400 mg/kg CTPG in 0.1 ml DMSO around tumor. Mice were treated every 2 days for up to 21 days. Tumor sizes were measured using calipers up to 25 days and tumor volume was calculated according to the formula: tumor volume (mm 3 ) = (length×width 2 )/2. After 25 days, survival of tumor mice was monitored every day until the end of this study.

Statistical analysis
Statistical significance was calculated by one-way analysis of variance among the treatment and control groups. All data were expressed as the mean ± standard deviation (S.D.). p < 0.05 was considered statistically significant.

CTPG reduced the viability of H22 cells in vitro
In order to explore antitumor effect of CTPG on HCC, H22 cells were treated with different concentrations of CTPG (0, 100, 200, 300 and 400 μg/ml) in vitro. After 24 h, the morphology of H22 cells was observed using inverted microscope. We found that the morphology of H22 cells was dramatically changed by CTPG treatment. With increasing CTPG concentration, cells became small and round and cell number was also greatly reduced (Fig. 1a). MTT assay was used to analyze the viability of H22 cells after CTPG treatment for 24, 48 and 72 h, respectively. CTPG significantly reduced H22 cell viability in a dose-dependent and time-dependent Total protein was isolated to analyze the expressions of Bax and Bcl-2 by Western blot. ** p < 0.01; *** p < 0.001 compared to control manner (Fig. 1b). CTPG at 300 μg/ml arrived at the best inhibitory rate (Fig. 1c). The values of IC 50 of CTPG for H22 cells are 236 μg/ml at 24 h and 169.8 μg/ml at 48 h.

CTPG induced apoptosis in H22 cells
To investigate whether the decreased viability of H22 cells is mediated by the induction of apoptosis, H22 cells were treated with different concentrations of CTPG (0, 100, 200, 300 and 400 μg/ml) for 24 h and stained with PI and Annexin V. The flow cytometry results showed that CTPG significantly induced apoptosis of H22 cells (including early and late apoptosis) in a dose-dependent manner (Fig. 2a). Although the high dose of CTPG also significantly increased necrosis of H22 cells, necrosis plays a minor role in the inhibition of H22 cell growth due to its lower proportion (8.3%) compared to that of apoptosis (52.6%). Further, total proteins of H22 cells were isolated after CTPG treatment and the expressions of anti-apoptotic B cell lymphoma 2 (Bcl-2) and proapoptotic BCL-2-associated X protein (Bax) were detected by Western blot. Grayscale scanning data showed that the expression levels of Bax and Bcl-2 were increased and decreased, respectively. The Bax/Bcl-2 ratio was significantly increased (Fig. 2b). These results suggest that CTPG induces apoptosis in H22 cells.

CTPG induces chromosomal condensation and cell cycle arrest in H22 cells
It has reported that DNA damage and cell cycle arrest induced by drugs can inhibit tumor cell growth and cause apoptosis in tumor cells [17,18]. To detect the morphology of nuclei in H22 cells after CTPG treatment for 24 h, H22 cells were stained by Hoechst 33,342 and observed using inverted fluorescent microscopy. CTPGtreated cells showed a dose-dependent increase of brightly condensed chromatin of nuclei, while the untreated cells showed the homogeneously stained nuclei (Fig. 3a). Cell cycle distribution in H22 cells was further analyzed by PI staining after CTPG treatment for 24 h. As shown in Fig. 3b, CTPG treatment significantly increased the proportion of G0/G1-and G2/M-phase cells and significantly decreased the proportion of S-phase CTPG decreased mitochondrial membrane potential and increased the release of cytochrome c Mitochondrial-dependent pathway plays an important role in the induction of apoptosis [19,20]. The changes of mitochondrial membrane potential (Δψm) can be monitored by JC-1 staining due to JC-1 aggregate (red fluorescence) can disintegrate into monomer (green fluorescence) with the reduction of Δψm [21]. After CTPG treatment for 24 h, H22 cells were stained by JC-1 dye. The flow cytometry data showed that the red fluorescence in FL-2 channel and green fluorescence in FL-1 channel were significantly decreased and increased upon CTPG treatment. The proportion of PE − FITC + cells were significantly increased (Fig. 4a), suggesting that CTPG reduced the Δψm in H22 cells. This is consistent with the increased Bax/Bcl-2 ratio. Consequently, we observed the release of cytochrome c was significantly increased upon CTPG treatment (Fig. 4b). These results indicated that CTPG might partially induce apoptosis in H22 cells via mitochondrial-dependent (intrinsic) pathway.

CTPG activated caspase pathway and prevented DNA repair
Next, the activation of caspase induced by CTPG via both extrinsic and intrinsic signaling pathways was analyzed. After CTPG treatment for 24 h, total proteins were isolated from H22 cells and the levels of pro-and cleaved-caspases were detected by Western blot. Compared with the untreated or the DMSO control, CTPG treatment significantly up-regulated not only the level of cleaved caspase-8 (extrinsic pathway) but also the level of cleaved caspase-9 (intrinsic pathway) (Fig. 5). Sequentially, activated caspase-8 and -9 cleaved the downstream pro-caspase-3 and -7 that was observed in Fig. 5. Activated caspase-3 cleaved the DNA repair enzyme of poly (ADP-ribose) polymerase (PARP) to prevent DNA repair and accumulate DNA damage as observed in Fig. 3a. These results indicated that CTPG induced apoptosis in H22 cells through both extrinsic and intrinsic signaling pathways.
CTPG suppresses the growth of H22 HCC in vivo and improves the survival rate of tumor mice Finally, the antitumor effect of CTPG on HCC was evaluated in tumor mouse model, which was established by subcutaneous injection of H22 cells. After 3 days of H22 cell injection, tumor mice were treated with CTPG for 8 times. The body weight of mice and tumor sizes were monitored at indicated time points. As shown in Fig. 6a, the body weight of mice in each group has no significant difference, suggesting that the selected doses of CTPG have no obvious side effect.
Interestingly, the tumor growth in mice treated with both 200 mg/kg and 400 mg/kg of CTPG was significantly inhibited (Fig. 6b). Moreover, the two doses of CTPG treatment greatly improved the survival of tumor mice (3/7, 3/7) compared with control group (0/7) at the end of the experiment (Fig. 6b). We also found that CTPG significantly enhanced the proliferation of splenocytes isolated from male Kunming mice in a dose-dependent manner (Fig. 6c), suggesting that CTPG has immunostimulatory effect.

Discussion
TCM has been used to treat various diseases including cancers for a long history. It has been reported that TCM can induce apoptosis in different types of tumor Fig. 5 The levels of cleaved-caspases and cleaved-PARP. H22 cells were treated with different concentrations of CTPG for 24 h. Total protein was isolated to detect the levels of cleaved-caspases and cleaved-PARP by Western blot cells through both extrinsic (death receptor-mediated) and intrinsic (mitochondria-dependent) signaling pathways to exert antitumor effects [22][23][24][25]. The two pathways can activate caspase-8 and -9, respectively [24,26]. Here, we found that CTPG significantly suppressed H22 cell growth through induction of apoptosis and cell cycle arrest. The levels of cleaved caspase-8 and -9 were significantly up-regulated by CTPG treatment, suggesting that both extrinsic and intrinsic signaling pathways were involved in the induction of apoptosis. Our previous study showed that CTPG induced apoptosis in melanoma B16-F10 cells by mitochondria-dependent pathway that increased the level of cleaved caspase-9 but not caspase-8 [16]. CTPG might activate distinct signaling pathways in different types of tumor cells.
Mitochondrial membrane integrity is tightly regulated by the members of the BCL-2 protein family including Bax and Bcl-2 [27,28]. The ratio of Bax to Bcl-2 plays a critical role in mitochondria-dependent apoptosis pathway [29]. In H22 cells treated by CTPG, Bax/Bcl-2 ratio was significantly up-regulated, which might cause the reduction of Δψm and the release of cytochrome c observed in this study. Consequently, pro-caspase-9 was cleaved and activated. Finally, the initiators of active caspase-8 and -9 activated the executioner of caspase-3 to cleave PARP to prevent DNA repair. Taken together, these results suggested that CTPG induced apoptosis in H22 cells through both extrinsic and intrinsic signaling pathways.
In tumor mouse model, CTPG significantly suppressed the growth of H22 HCC and greatly improved the survival of tumor mice. Interestingly, CTPG dose-dependently promoted the proliferation of splenocytes from Kunming mice, which is consistent with our previous study [16]. These results suggested that CTPG might suppress the growth of H22 HCC in mice through both direct antitumor effect and indirect immune enhancement.

Conclusions
CTPG suppressed the growth of H22 cells both in vitro and in vivo and induced apoptosis in H22 cells through both extrinsic and intrinsic signaling pathways. These data indicated that CTPG might be a potential candidate for the treatment of HCC.

Availability of data and materials
The raw data for this study are available upon reasonable request to the corresponding author.
Authors' contributions JL and JL designed the experiments. PY, JL, AA and YY performed the experiments. LX, XW and YL analyzed the data. PY, JL and JL wrote the manuscript. All authors contributed and approved the final manuscript.

Ethics approval
The animal study was approved by the Committee on the Ethics of Animal Experiments of Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, Xinjiang University.