PART Ⅰ：SARS-CoV-2 infection affects tissues including the heart and kidney
Mar 18, 2022
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PART Ⅰ：SARS-CoV-2 infection in the Syrian hamster model causes inflammation as well as type I interferon dysregulation in both respiratory and non-respiratory tissues including the heart and kidney
Magen Ellen Francis, Una Goncin & et al.
Introduction
SARS-CoV-2 (severe acute respiratory coronavirus 2) which causes COVID-19 (coronavirus 2019)emerged in late 2019 with subsequent massive public health and economic impacts[1,2]. SARS-CoV-2 is a positive-sense, single-stranded RNA virus of the family Coronoviridae, genus Betacoronavirus[3], and since its emergence has led to over 170 million confirmed cases and 3.5 million deaths worldwide as of June 1, 2021 [4]. Although coronavirus infections in humans were previously thought to only cause mild,cold-like symptoms, the more recent coronaviruses to emerge, SARS-CoV(severe acute respiratory coronavirus) and MERS-CoV (Middle East respiratory syndrome coronavirus), are associated with a wide array of respiratory and non-respiratory symptoms[5,6], ranging from mild to severe, leading to organ failure and death [Z. Asymptomatic cases have also been reported[8]. Given the burden on public health, as well as the range of disease severity, developing animal models in order to study SARS-CoV-2 pathogenesis and identify countermeasures is of high importance. In vivo assessment of host response to viral infection is essential to identify host factors regulating severe disease as well as biomarkers that may help inform patient supportive care measures.
Assessment of host response to SARS-CoV-2 has led to valuable insight into disease progression and possible immune mechanisms leading to severe disease. It has now been widely shown that severe cases of COVID-19 in humans are often accompanied by increased expression of inflammatory genes, including Interleukin-6(IL-6), Interleukin-1 beta(IL-1beta), and Tumour Necrosis Factor (TNF) [9-11]. Prior to SARS-CoV-2, it was known that coronaviruses were capable of regulating the type I interferon response[12,13]. Several studies have found that type I interferon responses are significantly blunted during SARS-CoV-2 infection suggesting this may also contribute to poor COVID-19 outcomes[14,15]. These clinical data suggest potential markers of pathogenesis for further experimental investigations.
Syrian hamsters were one of a handful of animal species identified early during the pan-demic to be productively infected with SARS-CoV-2 and of potential use for preclinical development and in vivo analysis[l6]. Hamsters were originally determined to be susceptible to SARS-CoV [17] and since have been shown to be a model for SARS-CoV-2[18]. Hamsters are able to support SARS-CoV-2 replication within the lower respiratory tract[18-20] with some similarities to the infection and disease dynamics as that of humans although the infection time course seems to be shorter in hamsters[21]. Ferrets have not been shown to support infection within the lung as the virus has only been detected in the upper respiratory tract suggesting this model may be useful for transmission studies[22]. Additionally, mice have not been shown to be susceptible to the original Wuhan SARS-CoV-2 virus. Hamsters have also been shown to mount an effective antibody response and have been utilized for both COVID-19 vaccine and treatment studies[19,20,23]. As with other non-traditional animal models, there is a lack of hamster-specific biological reagents for characterizing immune responses and the mechanisms of pathogenesis[24].
Here, we investigated the immune response and pathogenesis of SARS-CoV-2 infection in hamsters to determine disease mechanisms and the involvement of specific organs such as the lungs, heart, and kidney since COVID-19 is a multisystem disease.SARS-CoV-2 inoculated Syrian hamsters were sampled over a 15-day time course for virological and immunological analysis by qRT-PCR, transcriptome analysis, virus titration assays, and immunohistochemistry (IHC). The respiratory tract had a high burden of replicating the virus in both the upper and lower regions with specific blunting of the type I interferon response. While we found little evidence of infectious virus outside the respiratory tract, viral RNA was present in several organs including the kidneys, heart, lymph node, and liver with concomitant expression of inflammatory mediators such as IL-6, TNF, and IL-1beta. The lung, as well as extrapulmonary organs, showed signs of inflammatory cell infiltration leading to tissue damage. Taken together, we have defined the hamster model of immunopathogenesis in the context of COVID-19 which can be used to identify correlates of protection and disease, for evaluation of medical COuntermeasures.
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Results
SARS-CoV-2-inoculated hamsters have weight loss that correlated with viral load, infectious virus in the respiratory tract, and viral RNA in non-respiratory tissues
Syrian hamsters are susceptible to infection with SARS-CoV-2 suggesting they may be valuable for the investigation of immunopathology and mechanisms of disease[20]. Hamsters are aged 6-8 weeks were inoculated intranasally with SARS-CoV-2 at 10° TCIDs0. The animals were monitored for temperature and weight throughout the course of the infection. As typical during viral infection in small animals, hamsters began showing a decrease in temperature by day 1 post-inoculation(pi)(S1A Fig)which returned to baseline between day 3 and 7 (S1B Fig). Inoculated hamsters began losing weight on day 2 pi, with weights being significantly different from baseline on days 3 to 7. Weight loss was most severe on day 6 pi where the average body weight of the inoculated animals was 88.26% of the original weight (Fig 1A). After day 6, there was a steady increase in weight over the remaining time course on average recovering to baseline by day 12. Individual animals showed two different trajectories of weight loss: mild (~4%weight loss) and severe(>15% weight loss)(Fig1B). No animals lost more than 20% of their original weight and also reached other criteria for the humane endpoint as defined in the methods during the study. Animals were removed from the study at pre-determined end days for sample collection, therefore individual weights for all animals are not shown.
COVID-19 affects several organ systems in humans including the heart, kidneys, liver, spleen, and large intestine[25,26]. Therefore, we were interested in determining the tropism of the virus and associated immunopathology throughout the body of infected hamsters to gain insight into the disease. Animals were removed from the study on days 0,2,5,8,12 and 15pi to assess tissue-specific responses for necropsy and analysis of the immunopathology. Blood, nasal washes, and tissues (respiratory and non-respiratory) were collected. Viral tropism was assessed by qRT-PCR for the SARS-CoV-2 envelope gene(E)and virus titration with Tissue Culture Infectious Dose 50(TCIDs0)assays (Fig2). E gene expression analysis is represented by brown circles on the graphs. Analysis of the respiratory tract revealed vRNA present in the nasal turbinates, trachea, and all four tested lung lobes(right cranial, right middle, right caudal, and accessory) on days 2,5 and 8 pi. Viral RNA was still detected in the right middle lung lobe and the accessory lung lobe of one animal on day 12 pi. Live viral titer analysis, represented by the grey circles, indicated that infectious virus was present in the nasal turbinates on days 2 (7.5 TCIDs0/mL(Logl0))and 5 (6 TCIDs0/mL(Logl0))pi. Infectious virus peaked in the lungs on day 2, at~9 TCIDs/mL(Log10)in the right middle lung and was present until day 5. The trachea had the lowest virus levels of all the respiratory tissue which was only present on day 2(~3 TCIDso/mL(Logl0)).vRNA was also detected in non-respiratory tissues including the heart, kidney, large intestine, and the mediastinal lymph node at low to moderate levels. The mediastinal lymph node had the highest level of vRNA,~5 TCIDs(Log10), and was the only non-respiratory tissue to be positive for infectious virus.
Fig 1. Intranasal SARS-CoV-2 infection in hamsters leads to significant weight loss. Syrian hamsters were intranasally inoculated with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at 105 TCID50. Weight (A and B) was recorded for 15 days post-inoculation. Average results (A) show the mean. Individual results represent each animal (B). indicates a p-value <0.05 determined by ANOVA comparing hamsters on the day's post-inoculation to baseline (day 0). Error bars indicate +/- standard error (SE).
To determine if virus resolution was due to elicitation of neutralizing antibodies, we next quantified the neutralizing antibody titers in infected hamsters throughout the time course (Eig 3A). Plasma was isolated from blood collected at necropsy to be used for neutralization assays at 100 TCIDs per reaction of SARS-CoV-2. Neutralizing antibodies were detected as early as day 8 pi, at an endpoint titer of approximately 63 (geometric mean). This increased to a geometric mean of 160 on day 12 and peaked on day 15 at a geometric mean of 587. These observations suggested that hamsters are able to elicit high titers of neutralizing antibodies after SARS-CoV-2 infection. Nasal washes were collected throughout the study to determine viral shedding from the upper respiratory tract.vRNA was detected in the nasal washes up to 12 days after inoculation with live virus observed on days 2 and 5, peaking at day 2 at just over 6 TCID5o/mL (Log10). We compared the live viral titer found in the nasal washes of all animals on day 5 to their respective weight loss on that day(Fig 3B). A Pearson correlation for normally distributed data was utilized for this analysis to determine if a linear relationship exists between the weight of infected animals and viral titer in the nasal washes on day 5 post-inoculation with day5being a day in which some animals begin to clear the virus. We found a Pear-son correlation of-0.68 such that as the viral titer in the nasal washes increased, animals had an increased likelihood of more severe weight loss(p-value =0.00242). Additionally, we assessed the relationship between lung viral titer and weight loss on day 5 pi. Since only 3 animals were collected each day the data sets to assess for a correlation are limited by our sample size. Although this number of data sets does not meet the desired number of measurements, we performed a Pearson correlation and found a negative, although the non-significant, correlation between lung viral titer and weight loss on day 5 post-inoculation. Based on the viral titer from the respective lung lobes assessed compared to animal weight, the right cranial lung had an R-value of -0.91(p-value=0.27), the right middle lung had an R-value of-0.92(p-value=0.25)and the right caudal lung had an R-value of -0.58 (p-value=0.61)(S2 Fig). Additional assessment on more animals should be done in order to gain confidence in the relationship between SARS-CoV-2 lung viral titer and whole animal weight loss.
Fig 2. Live SARS-CoV-2 virus is confined to respiratory tissues and mediastinal lymph nodes while viral RNA is present in extrapulmonary organs. Animals were removed from the study on day 0 prior to infection as well as days 2, 5 8, 12, and 15 post-inoculation. respiratory tissues collected included the nasal turbinate, trachea, and lung (right cranial lobe, right middle lobe, right caudal lobe, accessory lobe). Extrapulmonary organs analyzed included the mediastinal lymph node, heart, kidney, liver spleen, and large intestine. All tissues were harvested to quantify virus by qPCR detection of the SARS-CoV-2 E gene in TCID50/mL equivalents based on a standard curve of viral RNA using RNA extracted from a stock of SARS-CoV-2 with a known concentration (TCID50/mL) (Brown). The infectious virus was quantified by a Tissue Culture Infectious Dose Assay and expressed in TCID50/mL (Grey). TCID50/mL was calculated using the Reed and Muench method. The dotted line represents the limit of detection in the live viral load assay, based on the lowest dilution of the sample. The limit of detection for qPCR detection of the E gene was 101 TCID50/mL. Data points represent individual animals and the line represents the average.
Pneumonia and lung pathology are often the defining feature of respiratory viral infection, resulting in significant morbidity and mortality. To determine the extent of damage to the lungs in SARS-CoV-2 infected hamsters, and the ability of hamsters to recapitulate the severe respiratory involvement in human COVID-19 patients, we examined disease pathogenesis within the left lung of hamsters throughout the course of infection (Fig 4). Microscopic analysis of H&E staining indicated an increase in damage in the lung over the course of the infection, with the most severe pathology noted on day 8 pi which partially resolved by day 15(Eig 4A). Uninfected hamsters had clear alveolar space without evidence of inflammatory cell infiltration or hemorrhage. By day 2 pi, mononuclear cell infiltration was present in the alveolar and peribronchial spaces (yellow arrow). Cell infiltration continued on day 5 with increased infiltration. On day 8, the peribronchial space was characterized by the presence of inflammatory mononuclear cells. Epithelial cell sloughing in the bronchioles was noted (black arrow)and hemorrhage could be observed throughout the tissue (red arrow). Hemorrhage was resolved by day 12 although hyperplasia was evident(green arrow). By day 15 hyaline membrane formation(blue arrow) was observed with concomitant reduction of inflammatory cell infiltration.
Fig 3. Viral shedding of SARS-CoV-2 increases with the severity of clinical outcomes but diminishes as neutralizing antibodies are detectable in animal plasma. Nasal washes were collected to quantify the virus by qPCR detection of the SARS-CoV-2 E gene (Brown) and expressed in TCID50/ml equivalents. Live viral load assays (Grey) were performed to quantify infectious viruses. Infectious titers of the nasal washes were determined by tissue culture infectious dose titration assays and the resulting TCID50/mL was calculated using the Reed and Muench method. LOD = limit of detection for live viral load assays. Virus neutralization titers were determined by standard neutralization assays using plasma collected throughout the time course (Black) (A). Live viral titer in the nasal washes negatively correlated with weight loss on day 5 post-infection. R-value = -0.68 (p-value = 0.00242) (B).
To validate viral load results in the lung, as well as visualize the location of the virus, we performed IHCstaining of the left lung lobe to detect SARS-CoV-2 spike protein. No viral protein was detected in uninfected hamsters suggesting the specificity of the antibody used. On day2 pi, the left lung had significant staining (black arrow)suggesting accumulation of SARS-CoV-2 spike antigen (Eig 4B). Staining was most prominent in the epithelial cells of the bronchiole, with diffuse staining throughout the epithelial cells of the alveolar wall. On day 5 light staining (blue arrows) was present through the airways and alveolar cells. No staining was detected on day 8 pi where the images appeared similar to images of the uninfected bronchiole.
Fig 4. Inoculated hamsters have significant inflammation in the lung coinciding with SARS-CoV-2 Spike antigen staining. The left lung was collected from each animal at necropsy and perfused with 10% formalin prior to paraffin block embedding and H&E staining. Red arrows indicate hemorrhage; green arrows indicate hyperplasia; yellow arrows indicate mononuclear cell infiltration; black arrows represent cell sloughing; blue arrow represents hyaline membrane formation (A). The lung was stained for the presence of SARS-CoV-2 spike protein by IHC using an anti-spike rabbit monoclonal antibody. Black arrows indicate high amounts of viral antigen staining and blue arrows indicate the presence of low amounts of viral antigen staining (B). The entire lung was weighed at necropsy and lung gross pathology was represented as a percentage of total animal body weight (C). At least 3 animals per time point were analyzed. indicates a p-value less<0.05 determined by ANOVA comparing hamsters on the day's post-inoculation to baseline (day 0). Error bars indicate +/- SE. Slides were visualized and imaged using the Leica DMI100 Brightfield microscope and DMC5400 20 MP color CMOS camera. Images were captured and 10X and 40X. The images shown are representative of 3 animals per group, per time point.
To quantify inflammation and inflammatory cell infiltration as well as edema in the lung, the weights of the lungs at necropsy were analyzed. The entire lung was weighed at necropsy and a ratio to the animal's body weight was calculated and then expressed as a percentage(Fig 4C).On days 0 and 2 pi, the lung was less than one percent of hamster body weight. This increased to a significant peak on day 5 at just under 4 percent of body weight on average. Lung weight decreased to 1 percent and remained statistically significant from baseline throughout the remaining time course.
Host gene expression and immunopathology profiling indicates a pronounced inflammatory response with blunted type I interferon signaling in the respiratory tract
Since the human data investigated systemic cytokine levels in the blood, we were interested in determining the immune responses at the site of infection as well as other affected organs. We investigated the levels of type I interferon response-related (IFN(Interferon)-beta, STAT(signal transducer and activator of transcription)2,IRF (interferon regulatory factor)1, IRF3,and TLR(Toll-like receptor)3),type II interferon response-related (IFN-gamma,IRF2, STAT1, CXCL10),and innate/adaptive markers and mediators(CD3, CD4,CD8A, CD19,IL-2, TGF (transforming growth factor)-beta,IL-21,IL-10,IL-4,IL-5,IL-5R,IL-13,IL-12,CD14,PKR, CCR3, CCL20, CCL22,IL-1beta, IL-6, TNF).
Human clinical reports have indicated that SARS-CoV-2 infection leads to a dysregulation of the interferon response[14]. We investigated the type I and I IFN responses by evaluating the expression of IFN-beta, STAT2, IRF1, IRF3, and TLR3 for the type I response and IFN-gamma, IRF2, STAT1, and CXCL10 for the type II response(Fig5). IFN-beta was significantly downregulated in both the nasal turbinates and right cranial lung with the greatest inhibition on day 2.Downstream regulators of the type I IFN response were also decreased, including STAT2 and IRF1 which reached maximum inhibition later in the time course, as well as IRF3 and TLR3 which showed no increase in gene expression. We also examined the type II IFN response, as well as genes whose corresponding proteins act in both IFN pathways. IFN-gamma, the type II IFN, showed upregulation in the nasal turbinates, and most prominently in the lung (Fig 5).In both tissues, there was an increase to a peak followed by resolution by day 15 pi. Nasal turbinate levels of IFN-gamma peaked on day 8 at a fold-change of 12, while levels in the lung peaked on day 5 at a fold-change of 17. In the respiratory tissues, type I IFN antagonist IRF2 showed significant increases, peaking on day 8 in the nasal turbinate and day 2 in the right cranial lung, at fold-changes of 24 and 25, respectively. General IFN response molecules showed significant increases as well.STAT1 showed strong upregulation in both tissues on days 2 and 5, while CXCL10 peaked at day 2 in the nasal turbinates at a fold-change of close to 70. In the lung, CXCL10 peaked on day 8 at a fold-change of 30 but did not resolve back down to baseline and remained significantly increased relative to naive animals on days 12 and 15 pi. From this data, it appears that while the type II IFN response remains intact, hamsters inoculated with SARS-CoV-2 show a dysregulated type IIFN response in respiratory tissues.
Fig 5. Gene expression analysis in the respiratory tissues of SARS-Cov-2 infected hamsters reveals significant interferon dysregulation with concomitant increases in inflammatory and immune mediators. qRT-PCR was performed on RNA extracted from nasal turbinate and right cranial lung tissue of SARS-CoV-2 inoculated hamsters. Samples were assessed for type I interferon response-related (IFN-beta, STAT2, IRF1, IRF3, TLR3), type II interferon response-related (IFN-gamma, IRF2, STAT1, CXCL10), and innate/adaptive mediators (CD3, CD4, CD8, CD19, IL-2, TGF-beta, IL-21, IL- 10, IL-4, IL-5, IL-5R, IL-13, IL-12, CD14, CCR3, PKR, CCL20, CCL22, IL-1beta, IL-6, TNF) using primers specific to hamster genes (Table 1). Foldchange was calculated via ΔΔCt against baseline (Day 0) with BACT as the housekeeping gene. At least three animals were analyzed for each time point.
Additionally, we also assessed innate and adaptive gene regulation. Overall, these genes were upregulated in both the nasal turbinates and right cranial lung, with nasal turbinates typically showing higher expression(Fig 5). The nasal turbinates had peak gene expression of IL-2, TGF-beta, IL-21, IL-4, IL-5R, IL-13, and CCR3 on day 5 pi, with the highest expression coming from TGF-betal at a fold-change of 97, and IL-13 at 45.IL-10 expression peaked on day2 at a fold-change of 38 but remained upregulated throughout the time course, significantly on days 5 and 10.IL-5 expression peaked on day 12 at a fold-change of 21. Within the lung, the highest expression per gene varied: TGF- betal, IL-21, and IL-13 showed the highest upregulation on day 5; IL-10, IL-4, and IL-5 on day 8; and IL-2 on day 12.IL-4 and IL-13 showed the greatest upregulation, at 25 and 18 on their respective peak days. T cell-specific markers were highest in the nasal turbinates, with peak gene expression on day 12 and remaining high on day 15. This suggests an involved T cell response which was further supported by the upregulation of Th2 cytokines(IL-4, IL-5, and IL-13), The cytokines(shown previously IL-12 and IFN-gamma), as well as T regulatory and Th17 cytokines (IL-10 and TGF-betal), pointing to an overall T cell response to SARS-CoV-2 infection. Therefore, we performed IHCto determine the presence of CD3+ and CD20+cells within the respiratory tract (S3 Fig).CD3+staining was concentrated to blood vessels (red arrows) in the uninfected lung tissue with no staining within the alveolar spaces. The presence of CD3+ cells (S3A Fig, black arrows) increased in the alveolar space throughout the time course reaching maximum staining on day 8 post-inoculation and then decreasing in intensity out today 15. CD20 staining was not detected in uninfected tissue. Small numbers of CD20+cells were detected on days 2,5, and 8post inoculation (S3B Fig, denoted by black arrows) which diminished on days 12 and 15 as indicated by grey arrows.
Analysis of the inflammatory markers within the nasal turbinates indicated significant transient increases of all inflammatory targets resolving by days 12 and 15.IL-1beta reached maxi-mum levels on day 2 pi at a fold-change of 40 relatives today 0uninfected animals(Fig 5).IL-6, IL-12, and TNF reached apex day 5 pi(fold-changes of 66,20, and 26, respectively). Corresponding significance can be found in the S1 Table. In contrast, these transcripts reached apex between days 2 and 5, but at a lower fold-change compared to the lung. In both the nasal turbinates and in the lung, several inflammatory cytokines remained elevated compared to baseline until day 12 pi. Taken together, the sites of infection showed a clear increase in inflammatory markers, especially IL-6 in the nasal turbinates and TNF in the lung.
