Comprehensive single cell analysis of pandemic influenza A virus infection in the human airways uncovers cell-type specific host transcriptional signatures relevant for disease progression and pathogenesis

AbstractRespiratory viruses, such as the 2009 pandemic strain of influenza A virus (IAV, H1N1pdm09), target cells found in the human respiratory epithelium. These cells, which form a pseudostratified epithelial layer along the airways, constitute the first line of defence against respiratory pathogens and play a crucial role in the host antiviral response. However, despite their key role in host defence, it remains unknown how distinct cell types in the respiratory epithelium respond to IAV infection and how these responses may contribute to IAV-induced pathogenesis and overall disease outcome. Here, we used single cell RNA-sequencing (scRNA-seq) to dissect the host response to IAV infection in its natural target cells. scRNA-seq was performed on human airway epithelial cell (hAEC) cultures infected with either wild-type pandemic IAV (WT) or with a mutant version of IAV (NS1R38A) that induced a robust innate immune response. We then characterized both the host and viral transcriptomes of more than 19,000 single cells across the 5 major cell types populating the human respiratory epithelium. For all cell types, we observed a wide spectrum of viral burden among single infected cells and a disparate host response between infected and bystander populations. Interestingly, we also identified multiple key differences in the host response to IAV among individual cell types, including high levels of pro-inflammatory cytokines and chemokines in secretory and basal cells and an important role for luminal cells in sensing and restricting incoming virus. Multiple infected cell types were shown to upregulate interferons (IFN), with type III IFNs clearly dominating the antiviral response. Transcriptional changes in genes related to cell differentiation, cell migration, and tissue repair were also identified. Strikingly, we also detected a shift in viral host cell tropism from non-ciliated cells to ciliated cells at later stages of infection and observed major changes in the cellular composition. Microscopic analysis of both WT and NS1R38A virus-infected hAECs at various stages of IAV infection revealed that the transcriptional changes we observed at 18 hpi were likely driving the downstream histopathological alterations in the airway epithelium. To our knowledge, this is the first study to provide a comprehensive analysis of the cell type-specific host antiviral response to a respiratory virus infection in its natural target cells – namely, the human respiratory epithelium.

Download Full-text

Rickettsia rickettsii Infection of Human Macrovascular and Microvascular Endothelial Cells Reveals Activation of Both Common and Cell Type-Specific Host Response Mechanisms

Infection and Immunity ◽

10.1128/iai.01335-09 ◽

2010 ◽

Vol 78 (6) ◽

pp. 2599-2606 ◽

Cited By ~ 29

Author(s):

Elena Rydkina ◽

Loel C. Turpin ◽

Sanjeev K. Sahni

Keyword(s):

Endothelial Cells ◽

Host Response ◽

Cell Types ◽

Microvascular Endothelial Cells ◽

Cell Type ◽

Rickettsia Rickettsii ◽

Specific Host ◽

Cell Type Specific ◽

Cox 2 ◽

Microvascular Endothelial

ABSTRACT Although inflammation and altered barrier functions of the vasculature, due predominantly to the infection of endothelial cell lining of small and medium-sized blood vessels, represent salient pathological features of human rickettsioses, the interactions between pathogenic rickettsiae and microvascular endothelial cells remain poorly understood. We have investigated the activation of nuclear transcription factor-kappa B (NF-κB) and p38 mitogen-activated protein (MAP) kinase, expression of heme oxygenase 1 (HO-1) and cyclooxygenase 2 (COX-2), and secretion of chemokines and prostaglandins after Rickettsia rickettsii infection of human cerebral, dermal, and pulmonary microvascular endothelial cells in comparison with pulmonary artery cells of macrovascular origin. NF-κB and p38 kinase activation and increased HO-1 mRNA expression were clearly evident in all cell types, along with relatively similar susceptibility to R. rickettsii infection in vitro but considerable variations in the intensities/kinetics of the aforementioned host responses. As expected, the overall activation profiles of macrovascular endothelial cells derived from human pulmonary artery and umbilical vein were nearly identical. Interestingly, cerebral endothelial cells displayed a marked refractoriness in chemokine production and secretion, while all other cell types secreted various levels of interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) in response to infection. A unique feature of all microvascular endothelial cells was the lack of induced COX-2 expression and resultant inability to secrete prostaglandin E2 after R. rickettsii infection. Comparative evaluation thus yields the first experimental evidence for the activation of both common and unique cell type-specific host response mechanisms in macrovascular and microvascular endothelial cells infected with R. rickettsii, a prototypical species known to cause Rocky Mountain spotted fever in humans.

Download Full-text

Coagulation Factors IX and X Enhance Binding and Infection of Adenovirus Types 5 and 31 in Human Epithelial Cells

Journal of Virology ◽

10.1128/jvi.02562-08 ◽

2009 ◽

Vol 83 (8) ◽

pp. 3816-3825 ◽

Cited By ~ 29

Author(s):

Mari I. Jonsson ◽

Annasara E. Lenman ◽

Lars Frängsmyr ◽

Cecilia Nyberg ◽

Mohamed Abdullahi ◽

...

Keyword(s):

Epithelial Cells ◽

Adenovirus Type ◽

Coagulation Factors ◽

Cell Types ◽

Body Fluids ◽

Target Cells ◽

Natural Target ◽

Adenovirus Receptor

ABSTRACT Most adenoviruses bind directly to the coxsackie and adenovirus receptor (CAR) on target cells in vitro, but recent research has shown that adenoviruses can also use soluble components in body fluids for indirect binding to target cells. These mechanisms have been identified upon addressing the questions of how to de- and retarget adenovirus-based vectors for human gene and cancer therapy, but the newly identified mechanisms also suggest that the role of body fluids and their components may also be of importance for natural, primary infections. Here we demonstrate that plasma, saliva, and tear fluid promote binding and infection of adenovirus type 5 (Ad5) in respiratory and ocular epithelial cells, which corresponds to the natural tropism of most adenoviruses, and that plasma promotes infection by Ad31. By using a set of binding and infection experiments, we also found that Ad5 and Ad31 require coagulation factors IX (FIX) or X (FX) or just FIX, respectively, for efficient binding and infection. The concentrations of these factors that were required for maximum binding were 1/100th of the physiological concentrations. Preincubation of virions with heparin or pretreatment of cells with heparinase I indicated that the role of cell surface heparan sulfate during FIX- and FX-mediated adenovirus binding and infection is mechanistically serotype specific. We conclude that the use of coagulation factors by adenoviruses may be of importance not only for the liver tropism seen when administering adenovirus vectors to the circulation but also during primary infections by wild-type viruses of their natural target cell types.

Download Full-text

Single-nucleus RNA-seq reveals dysregulation of striatal cell identity due to Huntington’s disease mutations

10.1101/2020.07.08.192880 ◽

2020 ◽

Author(s):

Sonia Malaiya ◽

Marcia Cortes-Gutierrez ◽

Brian R. Herb ◽

Sydney R. Coffey ◽

Samuel R.W. Legg ◽

...

Keyword(s):

Huntington's Disease ◽

Huntington’S Disease ◽

Neurodegenerative Disorder ◽

Cell Types ◽

Transcriptional Networks ◽

Rna Seq ◽

Cell Type ◽

Cell Identity ◽

Transcriptional Changes ◽

Cell Type Specific

ABSTRACTHuntington’s disease (HD) is a dominantly inherited neurodegenerative disorder caused by a trinucleotide expansion in exon 1 of the huntingtin (Htt) gene. Cell death in HD occurs primarily in striatal medium spiny neurons (MSNs), but the involvement of specific MSN subtypes and of other striatal cell types remains poorly understood. To gain insight into cell type-specific disease processes, we studied the nuclear transcriptomes of 4,524 cells from the striatum of a genetically precise knock-in mouse model of the HD mutation, HttQ175/+, and from wildtype controls. We used 14-15-month-old mice, a time point roughly equivalent to an early stage of symptomatic human disease. Cell type distributions indicated selective loss of D2 MSNs and increased microglia in aged HttQ175/+ mice. Thousands of differentially expressed genes were distributed across most striatal cell types, including transcriptional changes in glial populations that are not apparent from RNA-seq of bulk tissue. Reconstruction of cell typespecific transcriptional networks revealed a striking pattern of bidirectional dysregulation for many cell type-specific genes. Typically, these genes were repressed in their primary cell type, yet de-repressed in other striatal cell types. Integration with existing epigenomic and transcriptomic data suggest that partial loss-of-function of the Polycomb Repressive Complex 2 (PRC2) may underlie many of these transcriptional changes, leading to deficits in the maintenance of cell identity across virtually all cell types in the adult striatum.

Download Full-text

Single-Cell RNA-Seq of Cisplatin-Treated Adult Stria Vascularis Identifies Cell Type-Specific Regulatory Networks and Novel Therapeutic Gene Targets

Frontiers in Molecular Neuroscience ◽

10.3389/fnmol.2021.718241 ◽

2021 ◽

Vol 14 ◽

Author(s):

Ian A. Taukulis ◽

Rafal T. Olszewski ◽

Soumya Korrapati ◽

Katharine A. Fernandez ◽

Erich T. Boger ◽

...

Keyword(s):

Single Cell ◽

Regulatory Networks ◽

Stria Vascularis ◽

Cell Types ◽

Cellular Level ◽

Transcriptional Analysis ◽

Rna Seq ◽

Transcriptional Responses ◽

Transcriptional Changes ◽

Cell Type Specific

The endocochlear potential (EP) generated by the stria vascularis (SV) is necessary for hair cell mechanotransduction in the mammalian cochlea. We sought to create a model of EP dysfunction for the purposes of transcriptional analysis and treatment testing. By administering a single dose of cisplatin, a commonly prescribed cancer treatment drug with ototoxic side effects, to the adult mouse, we acutely disrupt EP generation. By combining these data with single cell RNA-sequencing findings, we identify transcriptional changes induced by cisplatin exposure, and by extension transcriptional changes accompanying EP reduction, in the major cell types of the SV. We use these data to identify gene regulatory networks unique to cisplatin treated SV, as well as the differentially expressed and druggable gene targets within those networks. Our results reconstruct transcriptional responses that occur in gene expression on the cellular level while identifying possible targets for interventions not only in cisplatin ototoxicity but also in EP dysfunction.

Download Full-text

Thapsigargin at Non-Cytotoxic Levels Induces a Potent Host Antiviral Response that Blocks Influenza A Virus Replication

Viruses ◽

10.3390/v12101093 ◽

2020 ◽

Vol 12 (10) ◽

pp. 1093

Author(s):

Leah V. Goulding ◽

Jiayun Yang ◽

Zhimin Jiang ◽

Hongyu Zhang ◽

Daniel Lea ◽

...

Keyword(s):

Virus Infection ◽

Influenza A Virus ◽

Influenza A ◽

Unmet Need ◽

Virus Production ◽

Antiviral Response ◽

Interferon Response ◽

Attractive Alternative ◽

Type I ◽

Host Antiviral Response

Influenza A virus is a major global pathogen of humans, and there is an unmet need for effective antivirals. Current antivirals against influenza A virus directly target the virus and are vulnerable to mutational resistance. Harnessing an effective host antiviral response is an attractive alternative. We show that brief exposure to low, non-toxic doses of thapsigargin (TG), an inhibitor of the sarcoplasmic/endoplasmic reticulum (ER) Ca2+ ATPase pump, promptly elicits an extended antiviral state that dramatically blocks influenza A virus production. Crucially, oral administration of TG protected mice against lethal virus infection and reduced virus titres in the lungs of treated mice. TG-induced ER stress unfolded protein response appears as a key driver responsible for activating a spectrum of host antiviral defences that include an enhanced type I/III interferon response. Our findings suggest that TG is potentially a viable host-centric antiviral for the treatment of influenza A virus infection without the inherent problem of drug resistance.

Download Full-text

Histone Deacetylase 2 Is a Component of Influenza A Virus-Induced Host Antiviral Response

Frontiers in Microbiology ◽

10.3389/fmicb.2017.01315 ◽

2017 ◽

Vol 8 ◽

Cited By ~ 6

Author(s):

Prashanth T. Nagesh ◽

Mazhar Hussain ◽

Henry D. Galvin ◽

Matloob Husain

Keyword(s):

Histone Deacetylase ◽

Influenza A Virus ◽

Influenza A ◽

Antiviral Response ◽

Histone Deacetylase 2 ◽

Host Antiviral Response

Download Full-text

Antioxidant Regulation of Cell Reprogramming

Antioxidants ◽

10.3390/antiox8080323 ◽

2019 ◽

Vol 8 (8) ◽

pp. 323 ◽

Cited By ~ 2

Author(s):

Yuichiro J. Suzuki ◽

Nataliia V. Shults

Keyword(s):

Transcription Factors ◽

New Technologies ◽

Cell Types ◽

Cell Reprogramming ◽

Target Cells ◽

Direct Reprogramming ◽

Heart Muscle Cells ◽

Multiple Cell ◽

Cell Type Specific ◽

Induced Pluripotent

Discovery of induced pluripotent stem cells (iPSCs) has revolutionized regeneration biology, providing further mechanistic insights and possible therapeutic applications. The original discovery by Yamanaka and co-workers showed that the expression of four transcription factors in fibroblasts resulted in the generation of iPSCs that can be differentiated into various cell types. This technology should be particularly useful for restoring cells with limited proliferative capacities such as adult heart muscle cells and neurons, in order to treat diseases affecting these cell types. More recently, iPSCs-mediated cell reprogramming has advanced to new technologies including direct reprogramming and pharmacological reprogramming. Direct reprogramming allows for the conversion of fibroblasts into cardiomyocytes, neurons or other cells by expressing multiple cell type-specific transcription factors without going through the production of iPSCs. Both iPSC-mediated reprogramming as well as direct reprogramming can also be promoted by a combination of small molecules, opening up a possibility for pharmacological therapies to induce cell reprogramming. However, all of these processes have been shown to be affected by reactive oxygen species that reduce the efficacies of reprogramming fibroblasts into iPSCs, differentiating iPSCs into target cells, as well as direct reprogramming. Accordingly, antioxidants have been shown to support these reprogramming processes and this review article summarizes these findings. It should be noted however, that the actions of antioxidants to support cell reprogramming may be through their ROS inhibiting abilities, but could also be due to mechanisms that are independent of classical antioxidant actions.

Download Full-text

Contributions of Ubiquitin and Ubiquitination to Flaviviral Antagonism of Type I IFN

Viruses ◽

10.3390/v13050763 ◽

2021 ◽

Vol 13 (5) ◽

pp. 763

Author(s):

Erika Hay-McCullough ◽

Juliet Morrison

Keyword(s):

Immune Response ◽

Host Response ◽

Type I Interferon ◽

Antiviral Response ◽

Cellular Protein ◽

Type I ◽

Type I Ifn ◽

Cellular Regulation ◽

Host Antiviral Response

Flaviviruses implement a broad range of antagonism strategies against the host antiviral response. A pivotal component of the early host response is production and signaling of type I interferon (IFN-I). Ubiquitin, a prevalent cellular protein-modifying molecule, is heavily involved in the cellular regulation of this and other immune response pathways. Viruses use ubiquitin and ubiquitin machinery to antagonize various steps of these pathways through diverse mechanisms. Here, we highlight ways in which flaviviruses use or inhibit ubiquitin to antagonize the antiviral IFN-I response.

Download Full-text

Innate Immune Evasion of Porcine Epidemic Diarrhea Virus through Degradation of F-box and WD repeat domain-containing 7 protein via Ubiquitin-proteasome Pathway

Journal of Virology ◽

10.1128/jvi.00889-21 ◽

2021 ◽

Author(s):

Mingwei Li ◽

Yang Wu ◽

Jianfei Chen ◽

Hongyan Shi ◽

Zhaoyanag Ji ◽

...

Keyword(s):

Antiviral Response ◽

Innate Immune ◽

Target Cells ◽

Diarrhea Virus ◽

Ubiquitin Proteasome Pathway ◽

Repeat Domain ◽

Ubiquitin Proteasome ◽

Proteasome Pathway ◽

Porcine Epidemic Diarrhea ◽

Host Antiviral Response

Porcine epidemic diarrhea virus (PEDV) causes a porcine disease associated with swine epidemic diarrhea. Different antagonistic strategies have been identified, and the mechanism by which PEDV infection impairs the production of interferon (IFN) and delays the activation of the IFN response to escape host innate immunity has been determined, but the pathogenic mechanisms of PEDV infection remain enigmatic. Our preliminary results revealed that endogenous F-box and WD repeat domain-containing 7 (FBXW7), the substrate recognition component of the SCF-type E3 ubiquitin ligase, is downregulated in PEDV-infected Vero E6 cells, according to the results from an isobaric tags for relative and absolute quantification (iTRAQ) analysis. Overexpression of FBXW7 in target cells makes them more resistant to PEDV infection, whereas ablation of FBXW7 expression by small interfering RNA (siRNA) significantly promotes PEDV infection. In addition, FBXW7 was verified as an innate antiviral factor capable of enhancing the expression of RIG-I and TBK1, and it was found to induce interferon-stimulated genes (ISGs), which led to an elevated antiviral state of the host cells. Moreover, we revealed that PEDV nonstructural protein 2 (nsp2) interacts with FBXW7 and targets FBXW7 for degradation through the K48-linked ubiquitin-proteasome pathway. Consistent with the results proven in vitro , FBXW7 reduction was also confirmed in different intestinal tissues from PEDV-infected specific-pathogen-free (SPF) pigs. Taken together, the data indicated that PEDV has evolved with a distinct antagonistic strategy to circumvent the host antiviral response by targeting the ubiquitin-proteasome-mediated degradation of FBXW7. Our findings provide novel insights into PEDV infection and pathogenesis. IMPORTANCE To counteract the host antiviral defenses, most viruses, including coronaviruses, have evolved with diverse strategies to dampen host IFN-mediated antiviral response, wither by interfering with or evading specific host regulators at multiple steps of this response. In this study, a novel antagonistic strategy was revealed showing that PEDV infection could circumvent the host innate response by targeted degradation of endogenous FBXW7 in target cells, a process that was verified to be a positive modulator for the host innate immune system. Degradation of FBXW7 hampers host innate antiviral activation and facilitates PEDV replication. Our findings reveal a new mechanism exploited by PEDV to suppress the host antiviral response.

Download Full-text