Dynamic gene network reconstruction from gene expression data in mice after influenza A (H1N1) infection

Abstract Background The immune response to viral infection is a temporal process, represented by a dynamic and complex network of gene and protein interactions. Here, we present a reverse engineering strategy aimed at capturing the temporal evolution of the underlying Gene Regulatory Networks (GRN). The proposed approach will be an enabling step towards comprehending the dynamic behavior of gene regulation circuitry and mapping the network structure transitions in response to pathogen stimuli. Results We applied the Time Varying Dynamic Bayesian Network (TV-DBN) method for reconstructing the gene regulatory interactions based on time series gene expression data for the mouse C57BL/6J inbred strain after infection with influenza A H1N1 (PR8) virus. Initially, 3500 differentially expressed genes were clustered with the use of k-means algorithm. Next, the successive in time GRNs were built over the expression profiles of cluster centroids. Finally, the identified GRNs were examined with several topological metrics and available protein-protein and protein-DNA interaction data, transcription factor and KEGG pathway data. Conclusions Our results elucidate the potential of TV-DBN approach in providing valuable insights into the temporal rewiring of the lung transcriptome in response to H1N1 virus

Systems biology and systems genetics—novel innovative approaches to study host–pathogen interactions during influenza infection

Influenza represents a serious threat to public health with thousands of deaths each year. A deeper understanding of the host–pathogen interactions is urgently needed to evaluate individual and population risks for severe influenza disease and to identify new therapeutic targets. Here, we review recent progress in large scale omics technologies, systems genetics as well as new mathematical and computational developments that are now in place to apply a systems biology approach for a comprehensive description of the multidimensional host response to influenza infection. In addition, we describe how results from experimental animal models can be translated to humans, and we discuss some of the future challenges ahead

A systems biology approach- quantification and molecular insights into influenza-A virus infection

Influenza A virus (IAV) circulating worldwide are highly contagious and can cause acute to severe respiratory disease. Annually, around 500 million individuals are infected by influenza, which causes about 500,000 deaths worldwide, including 5000-8000 deaths in Germany. In addition to,yearly epidemics, several pandemics are reported globally, recently in 2009 (influenza A/H1N1/pdm2009) which resulted in around 50 millions of deaths globally. The biological basis for the increased severity of some IAVs remains unclear. Unpredicted mutation which leads to intra-host evolution of quasi-species, and strong inflammation are important hallmarks of severe pandemic IAV infection. Understanding the differences in the pathogenicity of virus strains is an important aspect of influenza kinetic. Modeling influenza kinetics plays an integral role to understand the differences and potential mechanisms of a virulent strain compared to a less pathogenic one. Furthermore, investigating the molecular mechanisms of severe pandemic IAV (pdmIAV) is of great importance in controlling the complications and reducing the pulmonary damage. Comprehensive genome wide expression data involving both innate and adaptive immune response helps to understand molecular mechanisms of host response during severe influenza infection

Network and Algebraic Topology of Influenza Evolution

Evolution is a force that has molded human existence since its divergence from chimpanzees about 5.4 million years ago. In that same amount of time, an influenza virus, which replicates every six hours, would have undergone an equivalent number of generations over only a hundred years. The fast replication times of influenza, coupled with its high mutation rate, make the virus a perfect model to study real-time evolution at a mega-Darwin scale, more than a million times faster than human evolution. While recent developments in high-throughput sequencing provide an optimal opportunity to dissect their genetic evolution, a concurrent growth in computational tools is necessary to analyze the large influx of complex genomic data. In my thesis, I present novel computational methods to examine different aspects of influenza evolution. I first focus on seasonal influenza, particularly the problems that hamper public health initiatives to combat the virus. I introduce two new approaches: 1. The q2-coefficient, a method of quantifying pathogen surveillance, and 2. FluGraph, a technique that employs network topology to track the spread of seasonal influenza around the world. The second chapter of my thesis examines how mutations and reassortment combine to alter the course of influenza evolution towards pandemic formation. I highlight inherent deficiencies in the current phylogenetic paradigm for analyzing evolution and offer a novel methodology based on algebraic topology that comprehensively reconstructs both vertical and horizontal evolutionary events. I apply this method to viruses, with emphasis on influenza, but foresee broader application to cancer cells, bacteria, eukaryotes, and other taxa

마크로파지 매개 사이토카인 조절을 통한 바이러스 면역 치료제 개발 연구

학위논문(박사) -- 서울대학교대학원 : 의과대학 의학과, 2023. 2. 석승혁.Macrophages are an essential component of innate cellular immunity with flexible functions prominently involved in host defense and immunity against foreign microorganisms, including bacteria, viruses, and fungi. Many viruses target macrophages, and activated macrophages lead to phagocytosis and the release of pro-inflammatory cytokines and chemokines. However, excessive secretion of pro-inflammatory cytokines by macrophages contributes to local tissue damage and a dangerous systemic inflammatory response. Since monocytes and macrophages are the central cells that secrete pro-inflammatory cytokines, the efficient control of these cells can be used as a therapeutic target to regulate cytokines. In this study, I aimed to regulate influenza virus-mediated hyper-inflammation by targeting macrophages and then examined the immune mechanism regulating interleukin (IL)-12 in macrophages during dengue virus infection. First, in this study, I developed liposomes that are selectively delivered to macrophages. Also, I found that liposomal dexamethasone (DEX/lipo) significantly reduced the protein level of tumor necrosis factor-alpha (TNF-α), IL-1β, IL-6, and the C-X-C motif chemokine ligand 2 (CXCL2) as well as the number of infiltrated immune cells in the bronchoalveolar lavage fluids as compared to the control and free dexamethasone (DEX) in influenza virus-infected mice. Moreover, the intranasal delivery of DEX/lipo during disease progression reduced the death rate by 20%. Therefore, the intranasal delivery of DEX/lipo may serve as a novel promising therapeutic strategy for treating influenza virus-induced pneumonia (Chapter 1). Next, I revealed the cause of decreased IL-12 after severe dengue virus infection in macrophages through dengue virus binding receptor-mediated signaling. As a result, in type I interferon receptor knockout (IFNAR KO) mice infected with severe dengue virus, down-regulated IL-12 decreased vessel tight junction through increasing matrix metalloproteinase-9 (MMP-9). In addition, I found that mice treated with recombinant IL-12 rapidly regained body weight and attenuated dengue hemorrhagic fever. Based on a reliable model, I have developed a potential therapeutic strategy to attenuate dengue hemorrhagic fever (Chapter 2). Together, my results provide valuable insights into the development of immunotherapies for viral disease via regulating cytokines by targeting macrophages.마크로파지는 숙주의 방어와 세균, 바이러스, 진균을 포함한 외부 미생물에 대한 면역에 현저하게 관여하는 여러 기능을 가진 타고난 세포 면역의 중요한 구성 요소이다. 많은 바이러스가 마크로파지를 표적으로 하고, 활성화된 마크로파지는 식세포작용을 일으키며 염증성 사이토카인과 케모카인을 분비한다. 그러나 마크로파지에 의한 염증 유발 사이토카인의 과도한 분비는 국소 조직 손상과 위험한 전신 염증 반응에 기여한다. 단핵구와 마크로파지는 염증성 사이토카인을 분비하는 주요 세포이기 때문에 이러한 세포의 효율적인 제어는 사이토카인을 조절하는 치료 표적으로 사용될 수 있다. 본 연구에서는 마크로파지를 표적으로 하여 인플루엔자 바이러스 매개 염증 반응을 조절하고, 이후 뎅기 바이러스 감염 시 마크로파지에서 특이적으로 IL-12를 조절하는 면역 기전을 조사하고자 하였다. 첫번째로, 본 연구는 마크로파지에 선택적으로 전달이 되는 리포솜을 개발하였다. 이후 인플루엔자 바이러스 감염 마우스 모델에서 덱사메타손 리포솜을 전달 했을 때 TNF-α, IL-1β, IL-6, CXCL2와 같은 염증성 사이토카인, 케모카인이 감소하고, 침윤된 염증성 세포를 감소된다는 것을 밝혔다. 이로 인해 질병이 진행되는 동안 덱사메타손 리포솜 치료는 사망률을 20%까지 감소시켰다. 결과적으로 덱사메타손 리포솜의 비강 내 전달은 인플루엔자 바이러스 매개 폐렴의 치료를 위한 유망한 치료 전략으로 작용할 수 있음을 시사한다 (Chapter 1). 다음으로 마크로파지에 심각한 뎅기 바이러스 감염 후 IL-12가 감소하는 원인을 뎅기 바이러스 결합 수용체 매개 신호전달을 통해 밝혀냈다. 그 결과, IFNAR KO 마우스가 중증 뎅기 바이러스에 감염된 경우, 감소된 IL-12가 MMP-9를 증가시켜 혈관 투과성을 증가시킨다는 것을 밝혔다. 또한, 재조합 IL-12로 치료한 마우스가 체중을 빠르게 회복하고 뎅기출혈열을 완화시키는 것을 밝혔다. 본 연구에서는 뎅기출혈열을 억제할 수 있는 잠재적인 치료 전략을 제시하였다 (Chapter 2). 결론적으로 본 연구는 마크로파지를 표적으로 하여 사이토카인을 조절함으로써 바이러스에 대한 면역 요법의 치료적 접근에 중요한 근거 자료로 사용할 수 있을 것으로 기대된다.List of figures 1 List of abbreviations 3 Chapter 1 5 Introduction 6 Materials and Methods 9 Results 15 Discussions 27 Chapter 2 32 Introduction 33 Materials and Methods 36 Results 47 Discussions 73 References 79 Abstract in Korean 86박

Influenza A virus Genomic Reassortment and Packaging

Influenza A viruses (IAV) are a major human and environmental pathogen. IAV successfully infects a diverse host range and adaptation of new viral strains to humans may cause pandemic events with high morbidity and mortality. As a member of the Orthomyxoviridae family, IAV inherently possesses a segmented genome, which enables a process of segment transmission between viruses following cellular co-infection, a process termed reassortment. The high rate of IAV mutation and continued co-circulation of diverse viral strains in divergent host species leads to the persistent prospect for emergence of new IAV with pandemic potential. Therefore, it is of great importance to understand the viral and host factors that restrict and promote the generation of emergent virus strains, their potential for pathogenesis, and discover novel mechanistic countermeasures against IAV, including improved vaccination and targeted therapeutic strategies. Human and avian IAV co-circulate and occasionally co-infect the same host, leading to the potential for generation of novel genome constellations following reassortment. The specific host and viral molecular determinants that allow replication of reassortant progeny virus are not well defined. Here, I show that the viral genetic context and host cell in which reassortment occurs determine the potential for genetic diversity derived from multiple distantly related strains. Importantly, we identify single gene reassortants between a North American avian strain and the 2009 pandemic H1N1 virus that are capable of causing disease in mammals and replicate in a human cell line as well as induce the production of several pro-inflammatory cytokines linked to severe disease outcomes. Additionally, utilizing a different viral genetic background, I show that the reassortment potential is regulated by species and cell type specific differences in viral replication due to augmented viral polymerase function dependent on the identity of a single amino acid in the PA protein. Together, these studies provide evidence that context- dependent compatibility between both viral and host factors determine the possibility for generation of novel reassortant genome constellations and regulate their potential for replication and transmission in new host species. Reassortment between IAV strains is likely dictated by the functional compatibility of vRNA segments bound by IAV nucleoprotein during genome packaging. I hypothesized that nucleoprotein (NP) scaffolds specific RNA elements that are required for genome packaging and interaction between viral RNA (vRNA) genome segments. Therefore, I sought to determine the functional consequences of genome architecture on genome packaging and for the first time determine the nucleotide-resolution landscape of NP-vRNA interactions in infected cells. We utilized Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) coupled to next-generation sequencing to determine the specific interaction sites of vRNA bound by NP. We then interrogated the functional importance of regions of vRNA bound or unbound by NP and identified a number of potentially structured RNA features required for efficient genome packaging and virus propagation. These studies provide a framework for understanding the multifactorial restrictions of IAV reassortment and potential for generation of novel genome constellations with pandemic potential. Finally, these studies expand our understanding of how viral and host determinants shape the possible evolutionary trajectories of IAV through reassortment and required genetic elements needed for genome assembly

A Network Integration Approach to Predict Conserved Regulators Related to Pathogenicity of Influenza and SARS-CoV Respiratory Viruses

Respiratory infections stemming from influenza viruses and the Severe Acute Respiratory Syndrome corona virus (SARS-CoV) represent a serious public health threat as emerging pandemics. Despite efforts to identify the critical interactions of these viruses with host machinery, the key regulatory events that lead to disease pathology remain poorly targeted with therapeutics. Here we implement an integrated network interrogation approach, in which proteome and transcriptome datasets from infection of both viruses in human lung epithelial cells are utilized to predict regulatory genes involved in the host response. We take advantage of a novel "crowd-based" approach to identify and combine ranking metrics that isolate genes/proteins likely related to the pathogenicity of SARS-CoV and influenza virus. Subsequently, a multivariate regression model is used to compare predicted lung epithelial regulatory influences with data derived from other respiratory virus infection models. We predicted a small set of regulatory factors with conserved behavior for consideration as important components of viral pathogenesis that might also serve as therapeutic targets for intervention. Our results demonstrate the utility of integrating diverse 'omic datasets to predict and prioritize regulatory features conserved across multiple pathogen infection models

Identification of anti-inflammatory constituents in Hypericum species and unveiling the underlying mechanism in LPS-stimulated mouse macrophages and H1N1 influenza virus infected BALB/c mouse

Hypericum species are a large family of plants with potential medicinal value. To date, only H. perforatum has been thoroughly studied for its bioactivities due to its popularity among depression patients. Other than its anti-depression and anti-viral activities, H. perforatum also has anti-inflammatory activity, which is not well characterized. Previous studies by Hammer et al. (2007) evaluated the inhibitory effect of different H. perforatum extracts on lipopolysaccharide (LPS)-induced macrophage prostaglandin E2 (PGE2) production. The subsequent study also identified 4 synergistic anti-inflammatory constituents in a fraction of the H. perforatum extract, namely pseudohypericin, quercetin, amentoflavone, and chlorogenic acid (referred to as the 4 compounds). Lastly, the janus kinase - signal transducer and activator of transcription (JAK-STAT) pathway were proposed as molecular targets for the 4 compounds\u27 anti-inflammatory activity. The current study set out to test the central hypothesis that the 4 compounds contributed significantly to the anti-inflammatory activity of the H. perforatum extract by inducing suppressor of cytokine signaling 3 (SOCS3) both in vitro and in vivo. The first part of this study was to compare the chemical profiles and anti-inflammatory potential of extracts of H. perforatum, H. gentianoides, H. beanii, H. densiflorum, H. balearicum, H. forrestii, H.bellum, and H. patulum. At a concentration of 20 yg/mL, all nine extracts included had significant inhibitory effect on LPS-induced PGE2 and nitric oxide (NO) production in RAW 264.7 mouse macrophages. The extracts made from H. perforatum and H. gentianoides had distinctive chromatograms in LC-MS analysis and relatively stronger PGE2 and NO reducing efficacy. The 4 compounds accounted for a portion of the H. perforatum extract\u27s PGE2 inhibition and the majority of its NO and interleukin (IL)-1b reducing effects. LPS-stimulated tumor necrosis factor (TNF)-a production was only suppressed by the 4 compounds but not by the extract, suggesting the presence of counteractive constituents. Uliginosin A, one of the acylphloroglucinols found in the H. gentianoides extract, inhibited PGE2 and NO by more than 70% at 2 yM. Then, the importance of SOCS3 activation in the anti-inflammatory potential of the H. perforatum extract and the 4 compounds was investigated using SOCS3 knockdown RAW 264.7 macrophages. The results indicated that pseudohypericin was the major PGE2 and NO inhibiting constituent among the 4 compounds and required SOCS3 activation to exert the effect. At the same time, amentoflavone and quercetin accounted for the inhibition of pro-inflammatory cytokines TNF-a, IL-6, and IL-1b in a SOCS3 independent manner. Interestingly, although the 4 compounds\u27 PGE2 and NO inhibitory activities were compromised with SOCS3 knockdown, H. perforatum extract\u27s efficacy was not affected, suggested that components other than the 4 compounds inhibited these inflammatory mediators without activating SOCS3. Because a cell culture model cannot comprehensively reflect the complex nature of inflammation, influenza A infected-BALB/c mice were used to assess the in vivo immune-regulatory impact of H. perforatum. When the mice were infected with a high dose of H1N1 virus, H. perforatum oral treatment at 110 mg/kg body weight significantly increased viral titer in the lung 5 days post-infection. H. perforatum treatment also resulted in elevation of 18 pro-inflammatory cytokine and chemokine levels and increased the number of pro-inflammatory cells in the bronchoalveolar lavage, as compared to the 5% ethanol vehicle treatment. SOCS3 transcription in the lung was elevated after viral infection, and further potentiated by the H. perforatum extract. These results suggested that influenza might be a contraindication for H. perforatum, because SOCS3 elevation could impair the immune response against influenza virus infection, possibly through blocking type I interferon signaling. H. perforatum was applied to mice only during the later phase of influenza infection, in the hope that inflammatory tissue damage can be alleviated. But no significant improvement was found. Overall, the current study showed that the 4 compounds in H. perforatum partially depend on SOCS3 activation to exert their in vitro anti-inflammatory activity. However, the elevated SOCS3 by H. perforatum during influenza infection can be detrimental due to the impaired immune response

RECRUITMENT OF P63-EXPRESSING DEEP AIRWAY STEM CELLS FOR LUNG REPAIR FOLLOWING H1N1 INFLUENZA INFECTION IN MICE

Ph.DDOCTOR OF PHILOSOPH

The Cellular and Physiological Basis for Lung Repair and Regeneration: Past, Present, and Future.

The respiratory system, which includes the trachea, airways, and distal alveoli, is a complex multi-cellular organ that intimately links with the cardiovascular system to accomplish gas exchange. In this review and as members of the NIH/NHLBI-supported Progenitor Cell Translational Consortium, we discuss key aspects of lung repair and regeneration. We focus on the cellular compositions within functional niches, cell-cell signaling in homeostatic health, the responses to injury, and new methods to study lung repair and regeneration. We also provide future directions for an improved understanding of the cell biology of the respiratory system, as well as new therapeutic avenues