key: cord-0803221-wxhr1o3i
authors: Yang, Myeon-Sik; Park, Min-Jung; Lee, Junhyeong; Oh, Byungkwan; Kang, Kyung Won; Kim, Yeonhwa; Lee, Sang-Myeong; Lim, Je-Oh; Jung, Tae-Yang; Park, Jong-Hwan; Park, Seok-Chan; Lim, Yun-Sook; Hwang, Soon B.; Lyoo, Kwang-Soo; Kim, Dong-il; Kim, Bumseok
title: Non-invasive administration of AAV to target lung parenchymal cells and develop SARS-CoV-2-susceptible mice
date: 2022-01-07
journal: Mol Ther
DOI: 10.1016/j.ymthe.2022.01.010
sha: 4538312b4ddbee4260a2b225bd36e80d4c545537
doc_id: 803221
cord_uid: wxhr1o3i

Adeno-associated virus (AAV)-mediated gene delivery holds great promise for gene therapy. However, the non-invasive delivery of AAV for lung tissues has not been adequately established. Here, we revealed that the intratracheal administration of an appropriate amount of AAV2/8 predominantly targets lung tissue. AAV-mediated gene delivery that we used in this study induced the expression of the desired protein in lung parenchymal cells, including alveolar type II cells. We harnessed the technique to develop severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-susceptible mice. Three kinds of immune function-relevant gene knockout (KO) mice were transduced with AAV encoding human angiotensin-converting enzyme 2 (hACE2) and then injected with SARS-CoV-2. Among these mice, type I interferon receptor (IFNAR) KO mice showed increased viral titer in the lungs compared to that in the other KO mice. Moreover, nucleocapsid protein of SARS-CoV-2 and multiple lesions in the trachea and lung were observed in AAV-hACE2-transduced, SARS-CoV-2-infected IFNAR KO mice, indicating the involvement of type I interferon signaling in the protection of SARS-CoV-2. In this study, we demonstrate the ease and rapidness of the intratracheal administration of AAV for targeting lung tissue in mice and this can be potentially used to study diverse pulmonary diseases.

Adeno-associated virus (AAV)-mediated gene delivery is promising for the treatment of human 43 diseases as it triggers only a mild immune response, integrates into the host genome with a very low 44 frequency, and can effectively infect human tissues. 1 Consequently, three AAV-based gene therapy 45 drugs, namely Glybera, Luxturna, and Zolgensma, have been approved for clinical use, and dozens of 46 drugs are being investigated under clinical trials. 2 47

Lungs are a part of the lower respiratory system that is responsible for the exchange of gasses in the 54 body. The lung parenchyma consists of a large number of alveoli composed of multiple types of cells, 55

including the alveolar epithelial cells, vascular endothelial cells, fibroblast, pericytes and so on. 6 Among 56 these, alveolar epithelial cells are the most important cell type for maintaining the alveolar-capillary 57 barrier function of the lungs. Alveolar epithelium is composed of alveolar type I (AT1) cells and alveolar 58 type II (AT2) cells. AT1 cells are squamous cells that cover 95% alveolar surface and provide the surface 59 area for gas exchange. 7 In contrast, AT2 cells are cuboidal cells and secrete the surfactant required to 60 reduce the surface tension for preventing alveolar collapse. 8 In addition, AT2 cells also serve as alveolar 61 stem cells that can differentiate into AT1 cells, suggesting the pivotal role of AT1 and AT2 cells in lung 62

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current 64 coronavirus disease 2019 pandemic, and to combat it, an unprecedented number of studies are being 65 conducted to better understand SARS-CoV-2. The SARS-CoV-2 genome is known to share 79% 66 homology with SARS-CoV, which emerged in the Guangdong Province, Southern China in 2002, and 67 SARS-CoV-2, like SARS-CoV, uses the human angiotensin-converting enzyme 2 (hACE2) as a receptor 68 to trigger cell entry. [10] [11] [12] However, one of the obstacles in SARS-CoV-2 research is that mice are not 69 susceptible to SARS-CoV-2, since murine ACE2 does not support the binding to SARS-CoV-2. 13 The 70

Perlman group have developed hACE2 transgenic mice that are susceptible to SARS-CoV in 2007. 14 71

However, despite the numerous advantages of using transgenic mice in terms of convenience and 72 economic efficiency, it takes a long time to build a cohort and/or adopt hACE2 to mice with a different 73 transgenic background. 74 invasive intratracheal delivery compared to that of the invasive intratracheal method, which is a widely 108 used delivery method for the lungs. GFP expression in the lungs of non-invasively administered mice 109 was comparable to those of invasively administered ones (Supplementary Figure S1e ). In addition, to 110 determine if non-invasive intratracheal delivery of AAV would work in other rodents, Syrian hamsters 111 were intratracheally injected with either AAV2/8-GFP or AAV2/9-GFP. Similar to mice, GFP expression 112 was observed in the lungs, but not in the liver, of the hamsters injected with AAV (Supplementary Figure  113 S1f). 114

Besides tissue tropism, AAV vectors also show cell-type tropism. The lung tissue is comprised of 115 various cells, including two types of parenchymal cells, namely AT1 and AT2. Thus, we looked into the 116 cellular distribution of GFP after intratracheal administration of AAV. Using confocal microscopy, GFP 117 expression was found in the alveolar squamous cells (AT1) stained with Aquaporin5 (AQP5), as well as 118 found to colocalize with AT2 cells stained with prosurfactant protein-C (proSPC; Figure 1c ), while GFP 119 expression was not merged with the vascular endothelial cells stained with von Willebrand factor (VWF). 120

These results indicate that intratracheal delivery of AAV2/8 targets both AT1 and AT2 cells but not 121 vascular endothelial cells. We further analyzed the AAV2/9 cell-type tropism in the lung tissue 122 (Supplementary Figure S2a) . Like AAV2/8, AAV2/9 showed similar cell-type tropism. The efficiency for 123 targeting AT2 cells was found to be similar between the two serotypes (Supplementary Figure S2b) . 124 125 Susceptibility of AAV-hACE2 transduced mice to SARS-CoV-2 is enhanced by the deficiency of 126

Based on the finding that intratracheal injection of AAV2/8 vector specifically targets lung parenchyma, 128 we attempted to use it to develop SARS-CoV-2-susceptible mice. For this, hACE2 with a FLAG tag at 129

To investigate the factors that can affect SARS-CoV-2 infection, we employed three kinds of immune 137 function-relevant gene knockout (KO) mice, which were namely knockouts for the nucleotide-binding 138 oligomerization domain 2 (NOD2), toll like receptor 7 (TLR7) and type I interferon receptor (IFNAR). 139

Mice were transduced with hACE2 or GFP using AAV (AAV-hACE2/NOD2 KO, AAV-hACE2/TLR7 KO, 140 and AAV-hACE2/IFNAR KO mice), followed by intranasal administration of SARS-CoV-2 two weeks 141 post AAV infection (Figure 2e ). When mice were transduced with AAV-hACE2, the SARS-CoV-2 titer in 142 the lungs of AAV-hACE2/IFNAR KO mice was robustly and significantly increased compared to that in 143 the AAV-hACE2 wild-type (WT) mice, while the viral titers in AAV-hACE2/NOD2 KO and AAV-144 hACE2/TLR7 KO mice were comparable to that in the AAV-hACE2/WT mice ( Figure 2f ). Expression 145 levels of hACE2 protein among the groups were similar (Supplementary Figure S3a) . SARS-CoV-2 was 146 not detected in the lungs of GFP transduced mice regardless of genotype, indicating that hACE2 is a 147 genuine receptor for SARS-CoV-2 ( Figure 2f ). These results suggest the relevance of type I interferon 148 signaling in protecting SARS-CoV-2 infection. 149

As AAV-hACE2/IFNAR KO mice were susceptible to SARS-CoV-2, we further analyzed the effect of 150 AAV serotypes and invasiveness of intratracheal injections on viral replication. The viral titer was not 151 influenced by AAV2/8 and AAV2/9 serotypes (Supplementary Figure S3b) . This result was supported 152 by those obtained by confocal microscopy that both serotypes target AT2 cells with similar efficiency 153 (Supplementary Figure S2b ). The administration method (non-invasive versus invasive) did not alter 154 the viral titer significantly as well (Supplementary Figure S3b) . 155

We also investigated how long hACE2 could be maintained in the lungs of AAV-hACE2 injected mice. 156

Considering that the lifespan of AT1 cells and AT2 cells are 120 days and 28-35 days, respectively, 16, 17 To further analyze the relevance of type I interferon signaling in SARS-CoV-2 infection, we randomly 164 divided IFNAR KO mice into four groups; AAV-GFP/IFNAR KO mice treated with DMEM (GFP+media), 165 AAV-hACE2/IFNAR KO mice treated with DMEM (hACE2+media), AAV-GFP/IFNAR KO mice treated 166 with SARS-CoV-2 (GFP+CoV2), and AAV-hACE2/IFNAR KO mice treated with SARS-CoV-167 2(hACE2+CoV2) (Figure 3a ). These mice were sacrificed at 2, 4, or 7 days DPI of SARS-CoV-2. Several efforts have been made to target AAV-mediated gene delivery to the lung parenchyma. vein injection of AAV2-ESGHGYF, which is of serotype 2 with a modified ESGHGYF peptide in the 192 capsid protein, has been shown to specifically target lung tissue. 18 However, the infected cells mainly 193 merged with CD31-expressing cells, which are supposed to be vascular endothelial cells. Although Non-invasive intratracheal administration employed in the present study is expected to be similarly 198 efficient when compared to the intubation method, but takes less than one minute to inject per mouse 199 and can be conducted under mild anesthesia without any invasive surgery. This implies that simply 200 altering the administration method could be efficient enough to target lung parenchyma. The method 201

we used here also targeted the lungs of hamsters, suggesting its potential application in a diverse range 202 of experimental animals. 203

Intranasal injection of AAV induced levels of GFP expression in the lung similar to that seen with 204 intratracheal injection. However, intranasal administration also led to the infection of the upper 205 respiratory tract (Supplementary Figure S1d) . Therefore, although intratracheal injection more 206 specifically targets the lung parenchyma, intranasal injection would be presumably more useful in 207 generating some other animal models. For instance, intranasal administration could be more effective 208

in studying a protein that has protective functions against infectious diseases in either the upper 209 respiratory tract and/or whole respiratory system, probably with minor adjustments in viral 210 concentrations and/or volume. Besides, like the present study, intranasal administration of AAV vector 211 would be useful to express viral receptors in the upper respiratory tract. 212

The lungs are composed of multiple lobes. For western blotting, we used the right-inferior lobe, which 213 is most distal from the trachea. The strong expression of GFP in the inferior lobe indicates that a single 214 injection of 4 × 10 11 VP of AAV in 25 μl volume is enough to infect all parts of the lung. Injection of a bloodstream. Based on our results, 4 × 10 11 VP of AAV in 25 μl volume is suitable to target the 219 expression of GFP in the lungs of 8-week-old male C57BL/6 mice. The instillator with a micro camera, 220 used in this study, was initially developed for pulmonary delivery of irritants like fine dust. We found it is 221 also very useful to deliver a small volume of virus inside a trachea, and the efficacy of target protein 222 expression using this system was comparable to that using an invasive method (Supplementary Figure  223 S3b). We, however, believe employing a machine is not essential to deliver the virus, since a few 224 publications have reported using a manual method without an automatic instillator, suggesting that 225 intratracheal injection is a non-invasive and easy method that can widely be used. 22-24 226 Intratracheal injection could be widely adopted in various fields of research. In the present study, we 227 revealed that this system can be used to generate animal models susceptible to infectious disease. 228

Previous studies reported that adenovirus-mediated hACE2 transduced mice are susceptible to SARS-229

CoV-2 infection. 25,26 However, unlike the AAV vector, administration of adenoviral vector causes severe 230 innate inflammatory responses and may be toxic. 27 Recently, Israelow et al. showed that AAV serotype 231 9-mediated hACE2 transduced WT and IFNAR KO mice are more susceptible to SARS-CoV-2, and this 232

finding is similar to our study, although with a different administration method. 28 In their study, AAV was 233 invasively injected into the trachea during surgery, implying that the procedure might hinder the recovery 234 of mice, especially IFNAR KO mice which are also immunocompromised. Interestingly, Israelow et al. 235

observed the viral titer in the lung of IFNAR KO mice were comparable to that of WT mice. 28 In contrast, 236 our study showed a robust increase of SARS-CoV-2 viral titer with microscopic lesions in the lung of 237 IFNAR KO mice. The discrepancy may come from the differences in administration procedures (non-238 invasive versus invasive), hACE2 expression amount (AAV2/8 versus AAV9), and/or SARS-CoV-2 239 strain (NCCP-43331 versus NR-52281). However, in the present study, we found that non-invasive and 240 invasive delivery of AAV-hACE2 caused similar levels of SARS-CoV-2 replication, and serotypes 241 AAV2/8 and AAV2/9 did not influence the viral titer as well (Supplementary Figure S3b) . Given the 242 importance of type I interferon response against SARS-CoV-2 infection, 29-31 increased SARS-CoV-2 243 virus in the background of IFNAR deficiency is not surprising. In line with the increased viral titer and 244 NP levels, significant lesions in the trachea and lung were observed in the AAV-hACE2/IFNAR KO mice 245 infected with SARS-CoV-2 virus in the present study. We expect that IFNAR KO mice transduced with We also investigated the relevance of TLR7 and NOD2 in SARS-CoV-2 pathogenesis. Since SARS-248

CoV-2 is a single-stranded RNA, we predicted it might be recognized by TLR7 effectively. 32, 33 The role 249 of NOD2 in the activation of innate immune antiviral response is well characterized. 34 Moreover, TLR7 250 and NOD2 are potently relevant to type I interferon signaling. 35, 36 However, SARS-CoV-2 viral titer was 251 found to increase only in the IFNAR KO mice and not in TLR7 KO or NOD2 KO mice. These results 252 may suggest that TLR7 and NOD2 are not implicated in the replication of SARS-CoV-2, and further 253 studies would be needed. 254

Interestingly, despite the lack of NP in the trachea and lung at 4 DPI and 7 DPI, the most marked 255 lesions were observed at 7 DPI (Figure 3 and 4) . We speculate that the increase in cytokine release 256 induced by hyperactivation of infiltrated inflammatory cells is the main cause of the lesions. 37 257

Interestingly, the pathological symptoms of SARS-CoV-2 infection in AAV2/8-hACE2 mice were not so 258 severe as those in K18-hACE2 or HFH4-hACE2 transgenic mice, 38,39 probably because of 259 neuroinvasion of SARS-CoV-2 in the aforementioned transgenic mice. 39, 40 We should mention that 260 AAV2/8-hACE2 mice express hACE2 mainly in the lung tissue (Figure 1d) . 261

In the present study, we revealed that intratracheally administered AAV infects lung parenchymal cells, 262 including AT2, which is highly susceptible to SARS-CoV-2. 41 Given the vector we used, GFP expression 263 is controlled by the CAG promoter, which is a ubiquitously active promoter for constitutive expression. 264

If we replace the CAG promoter with a Aqp5 promoter or Sftpc promoter, 42,43 which is specifically 265 activated in AT1 or AT2, respectively, the more specific cell-type expression could be achieved by 266 intratracheal delivery of AAV2/8. Given that SARS-CoV-2 primarily infects AT2 cells highly expressing 267 ACE2, 44 mice transducing hACE2 under the Sftpc promoter would be a more suitable model for SARS-268

CoV-2 studies. Future studies would be required to address the promoter-dependent specific 269 expression in AT1 and AT2 cells. In this study, we reveal that the system is suitable for inducing protein 270 overexpression in the pulmonary parenchyma. However, this system can be also adapted for 271 knockdown or knockout of gene expression. If AAV containing small hairpin RNA under U6 or H1 272 promoter, instead of GFP under CAG promoter, was used, it could knockdown the expression of the 273 endogenous mRNA of the target gene. Otherwise, guide RNA containing AAV could be intratracheally parenchymal cells. 276

In conclusion, we found that intratracheal administration of AAV2/8 targets pulmonary parenchymal cells, 277 including AT2 cells. This approach specifically targets the lung tissue. We employed this system for the 278 delivery of hACE2 to the lung tissue of various immune function-relevant gene KO mice. Following 279 SARS-CoV-2 infection to these mice, we identified type I interferon signaling, but not NOD2 and TLR7, 280

to have a protective effect against SARS-CoV-2 pathogenesis. Since this method is easy and quick to 281 apply, we speculate that it will be widely used to study diverse pulmonary diseases. 

SARS-CoV-2 (NCCP-43331) was provided by the National Culture Collection for Pathogens, Korea. 293

The virus was cultured in Vero E6 cells and all experimental procedures associated with virus handling 294 were conducted in a biosafety level 3 (BSL-3) facility, KOZRI, Jeonbuk National University. Viral stocks 295 were prepared by propagating in Vero E6 cells cultured in Dulbecco's Modified Eagle Medium (DMEM) 296 supplemented with 2% fetal bovine serum, 1% penicillin-streptomycin, and HEPES (Invitrogen). Viral 297 titers were determined by estimating the median tissue culture infectious dose. 298

293T cells were transfected with pcDNA3-hACE2-FLAG (or empty vector) construct using

AAVs were generated by co-transfection of pAAV vector containing either GFP or hACE2 gene, 

In this study we used six to eight weeks old, wild-type (WT), toll like receptor 7 (TLR7) knockout (KO), and lung tissues were harvested for analysis. At 2, 4, and 7 DPI, mice were sacrificed. Exsanguination 336 was performed by cutting a caudal vena cava, followed by careful isolation of tracheal and lung tissue. 337

Non-invasive injection: Mice were lightly anesthetized using an isoflurane vaporizer chamber. In 339 ventrodorsal recumbency, AAV-hACE2 or control (AAV-GFP) mice were injected intratracheally with 340 approximately 4 × 10 11 viral particles (VP) per mouse under a micro camera. After six hours, mice were 341 intranasally inoculated with 4 × 10 11 VP of AAV-hACE2 or AAV-GFP using a pipette. 342

Invasive injection (Tracheotomy and intratracheal instillation of AAV-hACE2): Zoletil (25 mg/kg; 343 mixture of zolazepam and tiletamine, Zoletil 50, Virbac, Carros, France) was administered via 344 intramuscular injection for anesthesia. After shaving the throat region with a razor blade, the site was 345 scrubbed with 10% povidone-iodine solution. Pinnal-pedal reflex was used to assess the depth of 346 anesthesia. Head and four limbs were fixed with plaster strips. To expose the cervical trachea, a 1.5 cm 347 long ventral cervical midline incision was made from the larynx to the sternum. Thyroid gland, 348 sternohyoid muscles, and peritracheal tissues were bluntly separated and pulled aside carefully to avoid 349 traumatizing the nerves, vessels, and esophagus. After full exposure of the ventral side of trachea, 4 × 350 10 11 VP of AAV-hACE2 or AAV-GFP was injected using an insulin syringe (31 Gauge needle). After 351 infection, the skin incision line was washed with sterile saline and closed using a surgeon's knot with 352 6/0 non-absorbable suture. To protect the wound and prevent contamination, the throat region was

One week after AAV vector transduction, mice under light isoflurane anesthesia were intranasally 358 inoculated with 1 × 10 6 plaque-forming unit (PFU) of SARS-CoV-2 in 30 μl DMEM using a pipette in an 359 ABSL3 facility. 360

Tissues were homogenized and cells were lysed using radioimmunoprecipitation buffer (150 mM NaCl, Gold containing DAPI (P36941, Thermo Fisher, MA, USA), and images were acquired using a confocal 379 microscope (LSM 900, Carl Zeiss, Oberkochen, Germany). seeded in a 12-well plate at 2 × 10 5 cells/well and cultured overnight in a CO2 incubator. The supernatant 383 of homogenized tissues was serially ten-fold diluted (from 10 1 to 10 6 ) and applied to the culture plate. 384

After one hour of incubation, the supernatant was removed. Each well was washed with PBS and 1.5 385 ml of media was overlaid. The culture plate was incubated for another three days in a CO2 incubator. 386

Cells were then fixed with 1 ml of 4% formalin and stained with 0.4% of crystal violet in 70% methanol 387 in PBS. Viral titer was calculated by counting the number of plaques. 388

Formalin-fixed samples were routinely processed and embedded in paraffin wax (Surgipath Paraplast, 390

Leica Biosystems Inc., IL, USA). Formalin-fixed paraffin-embedded tissue blocks were sectioned at 4 391 μm-thick sections using a standard rotary microtome (HM-340E, Thermo Fisher, MA, USA). Tissue 392 sections were stained with hematoxylin and eosin. 393

Abnormalities in the tracheal and pulmonary tissue were scored using representative microscopic 394 lesions. Scoring criteria ranged from 0 to 3 based on severity or proportion of lesioned tissue: 0, non-395 to-rare or under 10%; 1, mild or 10% to 40%; 2, moderate or 40% to 70%; and 3, severe or over 70%. 396

Tracheal abnormalities were scored using the following criteria: 1, inflammation of lamina propria; 2, 397 cellular exudates in tracheal lumen; and 3, tracheal epithelium damage. Lung abnormalities were scored 398 using the following criteria: 1, inflammation of peribronchiolar region; 2, inflammation of perivascular 399 region; 3, cellular exudates in bronchiolar lumen; 4, bronchiolar epithelium damage; 5, thickening of the 400 alveolar wall (interstitial pneumonia); and 6, hemorrhage. While scoring, care was taken because 401 lesions were not evenly distributed across the entire tissue and showed various patterns. The final score 402 was the sum of scores under each criterion; a high score indicated a higher degree of microscopic 403 damage. The criteria used for scoring are categorized and summarized in Supplementary Table 1 . 404

For immunohistochemistry, a silane-coated slide was used for its strong adhesiveness. To re-establish 405 immunoactivity, antigen retrieval was conducted using citrate buffer (pH 6.0) at 95 °C for 30 minutes 406 and at room temperature for 20 minutes. Sections were then incubated overnight at 4 °C with a SARS-407 horseradish peroxidase-conjugated anti-rabbit and anti-rat IgG antibody 411 Vector Laboratory, CA, USA) were used. The antibody was visualized with 3,3'-diaminobenzidine (SK-412 4105, Vector Laboratory, CA, USA) at the concentration recommended by the manufacturers. Tissues 413 were counterstained with methyl green. All slides were examined (BX53, Olympus, Tokyo, Japan) and 414 imaged (DP80, Olympus, Tokyo, Japan) microscopically using a light microscope. All histopathologic 415 examinations were conducted in a double-blind fashion with trained pathologists. To quantify 416 immunohistochemical results, image analysis was performed using TS Auto 5.1 (Olympus, Tokyo, 417 Japan). The percentage of immunopositive area was analyzed at a defined magnification and area (400 418 magnification field, 0.144 mm 2 ). 419

For immunofluorescence staining, we employed the method of intratracheal instillation of the fixative. ImmunoResearch, PA, USA). After washes with PBS for 90 minutes, samples were mounted with All data are expressed as mean ± standard deviation. One-way analysis of variance was used to test 439 for statistical significance among experiment groups followed by Duncan's multiple range test for 440 multiple comparisons as a post hoc test. Asterisk indicates significant differences among the groups (* 441 P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001). All statistical analyses were carried out using 

Adeno-associated virus vector as a platform for gene 464 therapy delivery

Engineering adeno-associated virus vectors for gene therapy

Analysis of AAV serotypes 1-468 9 mediated gene expression and tropism in mice after systemic injection

Novel 471 adeno-associated viruses from rhesus monkeys as vectors for human gene therapy

Adenovirus-475 associated virus vector-mediated gene transfer in hemophilia B

Epithelial progenitor cells in lung development

The development and plasticity of alveolar type 482 1 cells

Alveolar epithelial type II cell: defender of the alveolus revisited

Structural basis of receptor recognition by SARS-CoV-2

Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

Functional assessment of cell entry and receptor 500 usage for SARS-CoV-2 and other lineage B betacoronaviruses

Lethal infection of K18-504 hACE2 mice infected with severe acute respiratory syndrome coronavirus

SARS-CoV-2 entry 508 factors are highly expressed in nasal epithelial cells together with innate immune genes

Cell cycle kinetics in the alveolar epithelium

Alveolar type I cells: molecular phenotype and development

Evasion of Type I Interferon by SARS-CoV-2

Activation and evasion of type I interferon responses by SARS-CoV-2

Antiviral activities 556 of type I interferons to SARS-CoV-2 infection

Recognition of single-stranded RNA viruses by Toll-like receptor 7

Recognition of nucleic 562 acid and nucleic acid analogs by Toll-like receptors 7, 8 and 9

Activation of innate immune antiviral responses by Nod2

NOD2, RIP2 and IRF5 play a critical role in the type 569 I interferon response to Mycobacterium tuberculosis

Toll-like receptor 7-572 mediated type I interferon signaling prevents cholestasis-and hepatotoxin-induced liver fibrosis

Cytokine Storm

Pathogenesis of SARS-CoV-2 in Transgenic Mice Expressing 581

Human Angiotensin-Converting Enzyme 2

Severe acute 583 respiratory syndrome coronavirus infection causes neuronal death in the absence of 584 encephalitis in mice transgenic for human ACE2

SARS-CoV-2 Receptor ACE2 Is

Stimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell 588 Subsets across Tissues

Cell-specific expression of aquaporin-5 (Aqp5) in alveolar epithelium 591 is directed by GATA6/Sp1 via histone acetylation

Multiple stromal populations contribute to pulmonary fibrosis without 595 evidence for epithelial to mesenchymal transition

Single-cell RNA-seq data 598 analysis on the receptor ACE2 expression reveals the potential risk of different human organs 599 vulnerable to 2019-nCoV infection

nucleocapsid protein of SARS-CoV-2 at 2, 4, 7 days post-infection. (b) Scale bar = 50 μm

bar = 100 μm. (c,e) Percentages of stained area were scored. All values are presented as mean ± 633 standard deviation

SARS-CoV-2 causes multiple lesions in the trachea and lungs of AAV-hACE2/SARS-636

Histopathology of IFNAR KO mice expressing GFP or hACE2 in the 637 respiratory system after injection of either SARS-CoV-2 or media. Images of tracheal and lung tissue 638 (parenchyma, bronchiole, and blood vessel) of the mice at 2, 4, 7 days pot infection

Scale bar = 50 μm. (b) Scale bar = 200 μm. (c,d) Scale bar = 100 μm. (e,f) The extent of 640 lesions and severity were quantified and summation of the histopathology scores. All values are 641 presented as mean ± standard deviation