Abstract
The ongoing pandemic (also known as coronavirus disease-19; COVID-19) by a constantly emerging viral agent commonly referred as the severe acute respiratory syndrome corona virus 2 or SARS-CoV-2 has revealed unique pathological findings from infected human beings, and the postmortem observations. The list of disease symptoms, and postmortem observations is too long to mention; however, SARS-CoV-2 has brought with it a whole new clinical syndrome in “long haulers” including dyspnea, chest pain, tachycardia, brain fog, exercise intolerance, and extreme fatigue. We opine that further improvement in delivering effective treatment, and preventive strategies would be benefited from validated animal disease models. In this context, we designed a study, and show that a genetically engineered mouse expressing the human angiotensin converting enzyme 2; ACE-2 (the receptor used by SARS-CoV-2 agent to enter host cells) represents an excellent investigative resource in simulating important clinical features of the COVID-19. The ACE-2 mouse model (which is susceptible to SARS-CoV-2) when administered with a recombinant SARS-CoV-2 spike protein (SP) intranasally exhibited a profound cytokine storm capable of altering the physiological parameters including significant changes in cardiac function along with multi-organ damage that was further confirmed via histological findings. More importantly, visceral organs from SP treated mice revealed thrombotic blood clots as seen during postmortem examination. Thus, the ACE-2 engineered mouse appears to be a suitable model for studying intimate viral pathogenesis thus paving the way for identification, and characterization of appropriate prophylactics as well as therapeutics for COVID-19 management.
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Introduction
All over the world humans have been affected by the constantly emerging new coronavirus agent. Officially, the very first report was traced in Wuhan City of China during December 2019, and outbreaks are still being reported globally. Investigations are undergoing to the nature of its origin though [1, 2]. The causative infectious agent has been named as the severe acute respiratory syndrome-coronavirus 2019 (also known as SARS-CoV-2 or COVID-19, in short). Infected people exhibit symptoms such as fever, malaise, dry cough, and dyspnea, and are also diagnosed with varying degree of pneumonia [3]. Currently there are not many effective treatment modalities or the cure available; however, vaccines are highly effective in preventing the hospitalization, severe disease, and death. Researchers are working to understand disease mechanism(s) of SARS-CoV-2 infection so that they could design more effective drugs, and develop newer versions of the foolproof vaccines against COVID-19 to stop the ongoing pandemic.
While some viral agents such as poxviruses exhibit a wide host-range for transmissibility, and propagation including propensity to infect unrelated animal species but unfortunately the SARS-CoV-2 does not infect laboratory mouse unless the mouse has been engineered genetically to express human ACE-2 gene; the receptor employed by SARS-CoV-2 agent to enter inside the human cells [4]. In fact, laboratory mouse has served as the ‘workhorse’ for advancing biomedical research, and for devising newer therapies, and also to test, and validate underlying disease processes. The current study was designed using an engineered mouse to simulate some of the COVID-19 relevant disease symptoms, and to capture inflammatory signature markers that appear to be relevant for COVID-19 long-haulers. SARS-CoV-2 virus interacts with angiotensin converting enzyme II (ACE-2) receptor on cell surface. The receptor is present on various cell types throughout body, e.g., lungs, heart, stomach, liver, and kidney. The virion surface is coated with a spike protein (SP) that has two subunits: S1, and S2 (Fig. 1). The ‘S’ protein is known to induce both humoral, and cellular immune responses, and remains the target of vaccines that are based on full-length S protein, and its receptor-binding domain, including DNA, viral vector, and subunit-based vaccines. In addition, the peptides, antibodies, organic compounds, and short interfering RNAs (siRNAs) are additional therapeutics under development [5, 6]. Interestingly, the COVID-19 mRNA vaccines that are in use currently have been shown to induce neutralizing antibody response against the SARS-CoV-2 [7].
Once the S1 subunit attaches to cell, it is recognized by the ACE-2 receptor while the S2 subunit assists with fusion with cell membrane [8]. Virus then triggers an intense immune response [9,10,11,12,13,14,15]. The immune system detects the virus, and then cytokines, helper T-cells, and white blood cells become active [16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34]. This leads to the “cytokine storm” that contributes to multi-organ damage and can lead to death [35, 36]. In many cases the infection can be asymptomatic, or the virus just causes flu-like symptoms [37,38,39,40,41,42,43,44,45]. One of the symptoms is the shortness of breath due to extensive lung-cell death causing alveoli dysfunction [46]. Cells' death can also lead to edema, vessels clogging, and pneumonia. Individuals with co-morbidities, e.g., diabetes, hypertension, and cancer and people over the age of 65 are highly susceptible to developing pneumonia due to their compromised immune system [47].
Surprisingly, COVID-19 also causes clots inside the blood vessels of lungs, heart, kidney, and other vital organs, and these blood clots can induce additional medical emergencies like stroke or a heart attack, potentially resulting in death [48]. It is believed that clots are the results of SARS-CoV-2 induced damage in the lining of blood vessels. This damage can induce platelets recruitment to prevent the blood leaking out into the surrounding tissues. In fact, clots are formed to fix the damaged blood vessels; however, excessive clotting could block vessels though, thus disrupting the blood flow [48]. Research has shown that the number of available ACE-2 receptors can influence clots formation. In that context, more ACE-2 receptors can increase viral fusion events, thus potentially increasing blood vessels’ injury, hence paving the way for more clot formation. Unfortunately, excessive coagulation/coagulopathy could result in a “clotting cascade” leading to thrombosis (blood clot within a blood vessel). SARS-CoV-2 also causes acute cardiovascular injury. The proposed cause of cardiovascular injury is myocarditis because of the SARS-CoV-2 led systemic inflammation. Protein–protein interactions during infection lead to not only formation new virus particles but also cause tissue (blood vessel, myocardium, etc.) injury [49]. When spike protein binds to ACE-2 receptor in the heart, it alters cell-signaling process thus causing myocardial injury [48]. COVID-19 not only causes de novo myocardial injury but also puts individuals on a serious health risk trajectory who happen to have diabetes, are obese, have coronary artery disease, or heart failure, therefore, expediting myocardial injury further. In short, COVID-19 can severely affect heart’s potential long-term effects from myocarditis that essentially include “arrhythmia, heart failure, and increased risk of stroke or subsequent heart attacks”.
Another side effect of COVID-19 is excess fluid accumulation within body. When blood vessel encounters a foreign pathogen then endothelial cells react by changing from a squamous shape to a columnar shape that helps “adhesion molecules” attract cells such as leukocytes, and chemokines thus allowing the immune system to fight off the pathogen. When helper cells are recruited then shape of the endothelial lining is altered that can result in “thrombogenic basement membrane” leading the neutrophils to expand under the effects of cytokines, specifically IL-1a, and when this inflammatory process is further activated then endothelial lining gets disrupted. Furthermore, the endothelial cells containing metalloproteinases (MMPs) can destroy basement membrane of the arteries, and capillaries in the lungs causing fluid leakage [50]. It is important to remember that there are many variants of the SARS-CoV-2 such as alpha (B.1.1.7), beta (B.1.351), gamma (P.1), the commonest one delta (B.1.617.2), but very recently more newer variants, and sub-variants of “Omicron” and its progeny have been identified. The variants/sub-variants such as BA.1, BA.2, and their respective lineages are the modified forms of the original virion wherein mutations arise that raise public health concerns since they tend to spread easier and faster, causing worse symptoms, making testing less accurate, and that basically “escape” the immune surveillance provided by the COVID-19 vaccines or by natural infection [51,52,53,54]. The alpha, beta, gamma, and delta variants were first detected in the United Kingdom, South Africa, Brazil, and India, respectively. The delta along with other such variants/sub-variants are the current mutant virions that are present in the USA. These have been shown to be “more transmissible” than the alpha variant that had swept through the world [51,52,53,54,55]. New immune-evading Omicron variants such as BA.4, BA.5 are most likely present in many U.S. states [56].
There are currently not many known effective treatments or cure available for SARS-CoV-2, and if one gets infected there are only a few palliative measures that can be taken to decrease the symptoms. Convalescent sera, and the monoclonal antibodies have been shown to impart some protection during the early phase of the infection. Since there is no universal known treatment or cure, thus it is highly recommended that one gets vaccinated to decrease the chances of contracting COVID-19. In the present study, we treated the engineered ACE-2 mouse as well as human cells with SARS-CoV-2 spike protein (SP) and collected multiple data sets. The study paradigm turned out to be highly encouraging in understanding the COVID-19 in a much more elaborate way, and we believe that the results might help in devising better tools in diagnosing, treating, and preventing breakthrough infections, and managing COVID-19 symptoms in the long-haulers.
Materials and methods
Measurement of physiological parameters in animals
Male, and female transgenic mice expressing the human ACE-2 receptor (B6.Cg-Tg(K18-ACE2)2Prlmn/J, Genotype: Hemizygous genotype, Hemizygous for Tg(K18-ACE2)2Prlmn were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). The mice (in short, we will refer them as the ACE-2 mice) were housed in a pathogen-free environment under conditions of 20 °C ± 2 °C, 50% ± 10% relative humidity, 12 h light/dark cycles, and they were provided with food standard chow diet, and water ad libitum. The animal procedures were reviewed and subsequently approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Louisville School of Medicine, Louisville, Kentucky, USA. Further, the animal care and guidelines of the National Institutes of Health (NIH, USA) were also adhered to. The male, and female mice approximately of the same age (10–12 weeks) were recruited. The mice were anesthetized with Ketamine/Xylazine (50/10 mg/Kg), and then administered intranasally with the SARS-CoV-2 spike protein (ECD-His-tag, Genescript, Cat# Z03481), SP in short, and the followed by 100 μL air [57]. Post treatment mice were followed up to 5 days. Their body temperature, body weight, respiration rate, heart rate, systolic and diastolic pressure, and the intraocular pressure (IOP) were recorded in the SP treated and untreated mice groups as reported earlier in our published work [58,59,60]. Only measurements that were judged by data analytical system to be within the acceptable parameters were recorded, as valid.
Echocardiography of the SARS-CoV-2 spike protein (SP) treated ACE-2 mice versus untreated ACE-2 mice groups
Ultrasound was performed using Vevo 2100 imaging system; cardiac and aortic data were collected as described [61]. Mice were placed supine on a warm platform (37 °C) under isoflurane anesthesia. Using a MS550D (22–25 MHz) transducer, thoracic cavity was imaged. Aortic arch velocity, and cardiography function were assessed in pulse wave, and color Doppler modes. The transducer probe was placed on left hemithorax of the mice in the partial left decubitus position. Two-dimensionally targeted M-mode echocardiograms were obtained from a short-axis view of the left ventricle at or just below the tip of mitral-valve leaflet and were recorded. LV size, and the thickness of LV wall were also measured. Only the M-mode ECHO with well-defined continuous interfaces of the septum, and posterior wall were collected indicating the diastolic (longer), and systolic (shorter) chamber lengths of the ACE-2 mice treated with spike protein (SP) in comparison to the untreated control ACE-2 mice.
Creatine kinase isoform measurement
The blood levels of creatine kinase (CK) activity were also measured. In brief, the tissue-specific injury was determined by measuring the CK isoforms in serum samples from each group of mice. The CK-MM represents the cardiac and skeletal-muscle-specific isoform, while the CK-BB is primarily a nerve-specific and kidney-specific isoform, respectively. From each mouse, 10 µl of serum was mixed with 1 µl of activator, and loaded onto the CK gel as instructed by the manufacturer (QuickGel® CK Vis Isoenzyme Procedure; Helena Laboratories, TX, United States). The gels were run at 400 V for 4:15 min. The standard (ST) amounts of CK isoforms were also loaded in parallel to the samples [62, 63].
Experiments on human cells for the cytokine profiling, and Western blotting
Human umbilical vein endothelial cells (HUVEC), and human coronary artery endothelial cells (HCAEC) were treated either with SP or with freshly mixed poly(I:C) poly[I:C]-HMW, Invivogen, tlrl-pic) @ 2.5 mg/ml and SP (5–15 μg) in 10 μl sterile phosphate buffered saline (PBS). The respective control cells were treated with either @ 2.5 mg/kg poly (I:C) or PBS using the same volume, and cells were harvested at 6 h or 24 h post treatment. The relative expression profile of cytokines was performed using a proteome profiler antibody array (R&D Systems, ARY015; Minneapolis, MN) post 24 h of treatment. The arrays were hybridized with an equal amount of total protein from HUVEC treated and untreated SP, and control reagents. Assay was performed according to the manufacturer's protocol. For Western blotting, antibodies such as IL6 (Cat. #12153), IL8 (Cat. #94407), MIG (Cat. #30327, and uPAR (Cat. #12863) were purchased from Cell Signaling Technology (Danvers, MA) while CD147 antibody (Cat. #ab64616) and GAPDH (Cat. #SC-365062) were purchased from Abcam (Waltham, MA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. Anti-rabbit IgG-HRP conjugate and anti-mouse IgG-HRP conjugate, both were bought from Cell Signaling Technology (Cat. #7074, and Cat. # 7076, respectively). For GAPDH, primary antibody dilution used was 1:3000, and secondary antibodies with HRP conjugation, dilutions used were 1:5000, respectively. Protein was isolated using protein extraction buffer (RIPA lysis buffer, protease inhibitor cocktail and PMSF). Lysates were spun in extraction buffer for 12 h and then centrifuged at 12,000×g for 15 min. Supernatants at different time points from HUVEC and HCAEC were transferred to new tubes and protein concentrations were analyzed via Bradford protein estimation assay. Protein samples (a total protein of 50 µg) were run on a 10/12% sodium dodecyl sulfate (SDS)-polyacrylamide gel with Tris–glycine SDS buffer. Proteins from the gel were transferred electrophoretically overnight onto a PVDF membrane at 4 °C. Membranes were blocked with a 5% milk solution for 1 h. Primary antibodies were diluted at a concentration 1:1000 in TBST buffer and incubated on membrane overnight. All membranes were washed in TBST buffer 4 × and then incubated with secondary HRP conjugated antibody solution for 1 h at room temperature. Four TBST buffer washing steps followed before membranes were developed using a chemiluminescent substrate in a BioRad Chemidoc (Hercules, Calif., USA). Band intensities were determined using densitometry analysis. Relative optical densities of protein bands were analyzed using gel software Image Lab 3.0. Membranes were stripped and re-probed with GAPDH as the loading control. Expression levels of each protein were also quantified as shown in the respective bar charts, n = 3–5 petri dish/group. For molecules that were difficult to demonstrate via Western blotting, were subjected for an extra step of immunoprecipitation assay to before visualizing them on the blots.
Visceral organ observation, and histopathological investigation
Mice vital visceral organs were collected, and observed for their appearance after the experiments. The heart, lung, and kidney samples were also collected in 4% buffered paraformaldehyde for fixation, and were processed after embedding in paraffin. After that 5 µm-thick sections from each sample were cut, and stained with hematoxylin and eosin (H&E). The detailed methods for tissue processing, and staining have been described [64].
Statistical analysis
Data from mice, and human cells were collected, and statistically analyzed using the GraphPad Prism 9.0 (GraphPad Software, United States). Multiple comparisons were performed using one-way ANOVA with Bonferroni, as appropriate to analyze the difference between the groups, including a Tukey's post hoc analysis for the groups' comparison. The comparisons between two groups were performed by unpaired Student’s t-test. The *p < 0.05 was regarded as statistically significant. The data are reported as mean ± SEM, and error bars indicate SEM, n = 3–5 petri dish or 3-5 animals/group.
Results
Animals, especially transgenic strains such as mice have served excellent disease models in dissecting out the complex disease processes, and in testing new therapeutic compounds [65]. As per our “a priori” belief that binding of the SARS-CoV-2 virion’s spike protein (SP) to the host cell receptor, i.e., angiotensin converting enzyme 2 (ACE-2) in humans is associated with downstream cellular, and molecular signaling events. We could show many of the salient features that are generally seen in the COVID-19 patients in the clinic. To our knowledge, this study is one of the first disease modeling investigations in an experimental setting wherein we attempted to capture some of the clinical features, and postmortem observations that are seen in COVID-19 patients. In addition, we were able to collect data points both at whole organism level, as well as, under in vitro human cell culture conditions employing a range of tools such as cellular, biochemical, physiological, and histopathological approaches. While many studies have sought to simulate infection related observations; however, we are unaware of the similar attempts by others of using an engineered, and a humanized animal species, and other similar resources to specifically study a rage of parameters that are highly relevant to the actual COVID-19 clinical scenario. We believe that the findings from this study could help us learn further and gain newer insight(s) toward improving the efficacy of the currently available diagnostic, therapeutic, and prophylactic strategies to control the ongoing pandemic.
Measurement of physiological parameters, and echocardiography in mice
In some mice the body temperature post administration of the SARS-CoV-2 spike protein (SP) appeared to be little high but was not significant; however, during the next few days the temperature dropped down significantly in comparison to the control/untreated mice. Similarly, the body weight in the treated mice group was found to be less (Fig. 2A). Future work should focus whether temperature variation could potentially determine the disease outcome in models but in COVID-19 infected humans hypothermia displayed abnormal markers of coagulopathy thus clearly suggesting a hypercoagulable phenotype; however, hyperthermic slow resolvers did exhibit elevated inflammatory markers and the highest odds of mortality [66]. It is worth mentioning that COVID-19 is associated with clinically significant weight loss and risk of hospitalization in human subjects since the disease negatively impacts body weight and the nutritional status [67]. The respiration and heart rates were found to be not significantly affected in the SP treated ACE-2 mice in comparison to the untreated ACE-2 mice which contrasts with the observations in human patients (Fig. 2A) [68]. Likewise, the systolic and diastolic pressures were not affected much (Fig. 2B). This finding was in direct contrast to the clinical observation in human patients wherein COVID-19 increased both systolic, and diastolic blood pressures, and thus became a new onset of hypertension [69, 70]. Interestingly, the intraocular pressure (IOP) in SP treated ACE-2 mice was significantly affected than the untreated ACE-2 mice (Fig. 2B). COVID-19-related ocular hypertension has also been reported in human subjects [71].
More importantly, the echocardiography findings; however, did reveal alterations in cardiac functions as seen in the representative M‐mode echocardiography images from each group, i.e., SP administered, and control (saline) administered (CTL) indicating diastolic (longer) and systolic (shorter) chamber lengths in the ACE-2 mice treated with SP in comparison to the untreated control ACE-2 mice. The contraction and relaxation of the myocardium are found to be attenuated in the SP treated mice in comparison to the untreated control ACE-2 mice (Fig. 3). Clinical studies in human subjects have reported an association between COVID-19 and cardiovascular disease. Notably, the pre-existing cardiovascular disease appears to be strongly linked with worse outcomes such as death in patients with COVID-19. Nonetheless, COVID-19 itself can also induce cardiac injury, acute coronary syndrome, arrhythmia, and venous thromboembolism [72].
Creatine kinase assay
When the serum samples were subjected to assess the relative activities of various isoforms of the phospho-creatine kinase (CK) employing a gel-based assay from the SP treated ACE-2 mice, and untreated ACE-2 control mice groups, the tissue-specific injury was evident in the treated group as determined by the measurement of respective CK isoforms. For example, the muscle (CK-MM) injury was maximum, and significant followed by heart (CK-MB), and brain (CK-BB) (Fig. 4A and B). In fact, COVID-19 is accompanied by multiorgan failure in many patients, and that is strongly associated with increasing mortality rate [73, 74].
Protein array profiling, and Western blotting for cytokines/inflammatory molecules, on human cells
The expression profile of cytokines was captured by array analysis, and important protein targets were investigated via Western blotting either from cell culture supernatants or cell lysates that were treated with SP alone or with Poly: IC for different time points. Poly I:C is a synthetic polyinosinic-polycytidylic acid double-stranded RNA and has been used to stimulate release of cytokines and interferon-gamma production [57, 75]. The results revealed significant changes in the levels of cytokines and key protein molecules in the SP treated cells than the non-treated cells (Figs. 5, 6, and 7). Cytokines such as IL-6 have been considered as a potential COVID-19 early disease biomarker, and relevant prognostic tool for the development of fatal pneumonia in patients [76,77,78,79]. Interestingly, high CD47 levels seems to contribute to vascular disease, vasoconstriction, and hypertension thus predisposing individuals to serious complications like pulmonary hypertension, lung fibrosis, myocardial injury, stroke, and acute kidney injury [80, 81]. The role of urokinase plasminogen activator receptor (uPAR) has been suggested as one of the main orchestrators of fatal progression to pulmonary, kidney, and heart failure in COVID-19 patients. Newer drugs that could regulate uPAR system may help treat severe complications COVID-19 [82]. Because lack of therapeutic options for tackling acute respiratory distress syndrome (ARDS) in COVID-19 patients, attention has now focused on differentiating hyper- and hypo-inflammatory phenotypes of ARDS to help develop effective therapeutic interventions. In this regard, IL-8 which is a pro-inflammatory cytokine performs an important role in neutrophil activation and has been identified for the progression of COVID-19 disease [83, 84]. Furthermore, it has been reported that COVID-19 patients with severe outcome also display higher plasma levels of chemokines such as CXCL9/MIG, CXCL8/IL-8, and CXCL10/IP10 along with cytokines IL-6 and IL-10 than the patients with the milder form of the COVID-19 [85]. From our animal disease modeling study, it is apparent that molecules such as IL-6 and IL-8 can be used as potential biomarkers in COVID-19 patients and probably for COVID-19 disease prognosis also.
Visceral organ observation, and histopathological investigation
When mice visceral organs were collected, and observed for their appearance, it became abundantly clear that there was significant change in their appearance most likely because of the blood clots that have been often shown in patients suffering from COVID-19. More importantly, thrombi in the vasculature have also been reported in patients. The vital organs in our SP treated mice looked very dark in color (Fig. 8). In addition to gross observation of the organs, histological study on these vital organs employing hematoxylin and eosin (H&E) staining revealed a significant inflammatory phenotype more in the lung, and kidney than heart signifying extensive infiltrations of immune cells, e.g., neutrophils in the SP treated mice in comparison to the untreated control mice. Kidney, in fact, exhibited extensive tissue damage in the SP treated mice than the non-treated control mice (Fig. 9).
Discussion
In this study we show that upon SARS-CoV-2 virion spike protein (SP) treatment of the genetically engineered mice expressing the human ACE-2 receptor, and human cells led to the hyper-inflammatory state/phenotype relative to the untreated/control mice or human cells. The SP elicited secretome from inflamed targets (organs/cells), that is, from the in vivo (mice) or in vitro (human cells) systems causing an increased expression of the important proteins/targets such as cytokines/chemokines most likely mimicking the “cytokine storm” that is commonly observed in COVID-19 humans. It is well documented that excessive production of pro-inflammatory cytokines/chemokines is a severe clinical syndrome known to develop as a serious complication of infectious or inflammatory diseases such as during SARS-CoV-2 infection responsible for COVID-19. Evidence from clinical cases suggests that the occurrence of cytokine storm in severe acute respiratory syndrome secondary to SARS-CoV-2 infection is closely associated with a rapid deterioration of human health and high mortality in severe cases [86]. In our work, a significant increase in the levels of cytokines/chemokines or alterations in mice organs relative to untreated/control cells or mice confirm our hypothesis that biding of the SP to host cell is associated with downstream cellular processes/events that are highly detriment to host or its cells/organs. Such pathological events/processes are akin to the observations in the COVID-19 patients during SARS-CoV-2 viral pathogen replication in the target host cells.[87,88,89,90]. In people who recover from acute COVID-19 disease the pathology is still characterized and associated with mild form of cytokine storm that may or may not lead to long-term endothelial inflammation, microvascular thrombosis, and organ dysfunction but post COVID-19 related implications may still haunt some susceptible individuals for a foreseeable future [91,92,93,94].
Our findings support the hypothesis and corroborates some of the clinical observations of targeting SP as a COVID-19 preventing strategy for safeguarding human health against this deadly disease in susceptible human population. The same is true for the fact that therapeutically targeting the SP via specific monoclonal antibodies in the initial phase of the COVID-19 can prevent serious organ damage, and related health issues in the COVID-19 patients with alleviation of both the morbidity and mortality. Our results from the preclinical mouse model also suggest that creatinine kinase-based assays, and other blood biomarkers may be developed, and employed to not only protect other individuals who are vulnerable to adverse COVID-19 outcomes in whom there are increased chances of occurring serious COVID-19 symptoms but also in obese or numerous chronic diseases that affect individuals. In short, animal models such as genetically engineered ones may play important role(s) in studying “in-depth” disease mechanism(s) toward developing lifesaving therapeutics, and effective preventative measures. Finally, we hypothesize about the most plausible mechanisms(s) by which SARS-CoV-2 most likely induce the visceral organ damage in the infected host. Post internalization of the SARS-CoV-2 virions via the human angiotensin converting enzyme 2 (ACE-2) receptor, it causes a robust surge of inflammatory markers, epithelial barrier dysfunction, and multi-organ damage and congestive (cardio-pulmonary) heart failure (CHF) [36, 95]. Interestingly, the ACE-2 receptor is highly expressed in the renal tubular epithelial cells and podocytes. Studies have shown robust increase of neopterin (NPT) in COVID-19 patients. Interestingly, NPT is generated by IFN-γ-induced inflammatory macrophage (M1Q) in response to viral infection (Fig. 10). COVID-19 infection causes recruitment of inflammatory cells and a further robust surge of the inflammatory cytokines, epithelial barrier dysfunction, podocyte, and endothelial damage leading to acute kidney injury (AKI).
We strongly believe that pro-inflammatory macrophage (M1Q) activation leads to oxidative stress, and peroxynitrite/nitrosylation in cells/organs during COVID-19. Further, the resultant NLRP3 inflammasome formation may potentially activate the apoptosis pathway(s) leading to T cell lymphopenia (that is decrease in CD4+, and CD8+ cells) thus inciting the proximal tubular epithelial cell/podocyte injury and leakage (Fig. 10). It is known that the COVID-19 activates innate immune system causing AKI as reported in 27–40% of the ICU admissions [95,96,97,98,99,100,101,102,103,104,105,106,107,108,109]. More importantly, the humanized ACE-2 engineered mouse model can also be used to identify potential safety issues that may be associated with COVID-19 inhibitors that are being developed by pharmaceutical industry. We further hypothesize that newer version(s) of the modified approaches such as delivering beneficial molecules to the engineered mouse models or even to the cultured host cells via employing the protein transduction technology might reveal new disease target(s) in the coming future [110]. In the light of new emerging SARS-CoV-2 variants/sub-variants, it is somewhat difficult to predict whether we are going to have a peaceful future, COVID-wise, but it is certain that only the robust ‘cutting-edge’ tools, and technology might navigate us out of this deadly pandemic.
Limitation regarding extrapolation of mice experimental findings to human clinical observations: We do recognize that our work has limitations such as: (1) we did not use the actual infectious virus particles (virions) in our experiments, and (2) although we did use human cells in conducting in vitro experiments with SP alone or in combination with poly I:C, and a genetically engineered mouse model expressing the human angiotensin converting enzyme 2 (ACE-2) receptor to obtain the experimental data; however, despite above shortcomings, we were able to demonstrate many important features that seem to be similar, if not identical, to that of human COVID-19 as seen in real clinical settings.
In conclusion, we present a set of interesting evidence that interaction between the SARS-CoV-2 virion’s spike protein (SP) with that of the human angiotensin converting enzyme 2 (ACE-2) receptor leads to a robust cellular signaling cascade of events. If further research can validate or extend our findings then certainly such small, engineered animal models could serve as important tools in fighting, and winning this ongoing COVID-19 pandemic, and other related infectious diseases. As shown by others that a heightened pathological response in the form of increased cytokine storm, and multi-organ damage can lead to vital organ failure, and ultimately death in some COVID-19 patients as already revealed during the last > than ~ 2 years since the start of the pandemic [79, 111,112,113,114,115,116,117,118,119,120,121,122]. To dissect out further the physiological, and pathological implications of the SARS-CoV-2 induced changes, we carried out this important study to capture some of the initial/beginning phase of the intimate interaction(s) between the host cell receptor with the SARS-CoV-2 spike protein (SP) employing a genetically engineered mouse model expressing the human angiotensin converting enzyme 2 (ACE-2) receptor and the recombinant SARS-CoV-2 spike protein (SP) that was delivered via the intranasal route [123]. The SARS-CoV-2 spike protein (SP) binding to ACE-2 receptor did seem to amplify the susceptibility to COVID-19 virion-induced inflammation in various mice organs along with occurrence of the cytokine storm as elaborated in this study.
Data availability
The datasets generated and analyzed during this study are available upon request as per the data sharing policies of NIH.
References
Morens DM, Breman JG, Calisher CH, Doherty PC, Hahn BH, Keusch GT, Kramer LD, LeDuc JW, Monath TP, Taubenberger JK (2020) The origin of COVID-19 and why it matters. Am J Trop Med Hyg 103:955–959. https://doi.org/10.4269/ajtmh.20-0849
Umakanthan S, Sahu P, Ranade AV, Bukelo MM, Rao JS, Abrahao-Machado LF, Dahal S, Kumar H, Kv D (2020) Origin, transmission, diagnosis and management of coronavirus disease 2019 (COVID-19). Postgrad Med J 96:753–758. https://doi.org/10.1136/postgradmedj-2020-138234
Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, Zhang L, Fan G, Xu J, Gu X, Cheng Z, Yu T, Xia J, Wei Y, Wu W, Xie X, Yin W, Li H, Liu M, Xiao Y, Gao H, Guo L, Xie J, Wang G, Jiang R, Gao Z, Jin Q, Wang J, Cao B (2020) Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet 395:497–506. https://doi.org/10.1016/s0140-6736(20)30183-5
Singh M, Bhat PP, Mishra BP, Singh RK (1996) Biological transmissibility of buffalopox virus. J Appl Anim Res 9:79–88. https://doi.org/10.1080/09712119.1996.9706107
Du L, He Y, Zhou Y, Liu S, Zheng BJ, Jiang S (2009) The spike protein of SARS-CoV–a target for vaccine and therapeutic development. Nat Rev Microbiol 7:226–236. https://doi.org/10.1038/nrmicro2090
Jin DY, Zheng BJ (2009) Roles of spike protein in the pathogenesis of SARS coronavirus. Hong Kong Med J 15(Suppl 2):37–40
Jalkanen P, Kolehmainen P, Häkkinen HK, Huttunen M, Tähtinen PA, Lundberg R, Maljanen S, Reinholm A, Tauriainen S, Pakkanen SH, Levonen I, Nousiainen A, Miller T, Välimaa H, Ivaska L, Pasternack A, Naves R, Ritvos O, Österlund P, Kuivanen S, Smura T, Hepojoki J, Vapalahti O, Lempainen J, Kakkola L, Kantele A, Julkunen I (2021) COVID-19 mRNA vaccine induced antibody responses against three SARS-CoV-2 variants. Nat Commun 12:3991. https://doi.org/10.1038/s41467-021-24285-4
Huang Y, Yang C, Xu X-f, Xu W, Liu S-w (2020) Structural and functional properties of SARS-CoV-2 spike protein: potential antivirus drug development for COVID-19. Acta Pharmacol Sin 41:1141–1149. https://doi.org/10.1038/s41401-020-0485-4
Banji D, Alqahtani SS, Banji OJF, Machanchery S, Shoaib A (2021) Calming the inflammatory storm in severe COVID-19 infections: role of biologics—a narrative review. Saudi Pharm J 29:213–222. https://doi.org/10.1016/j.jsps.2021.01.005
Bozzano F, Dentone C, Perrone C, Di Biagio A, Fenoglio D, Parodi A, Mikulska M, Bruzzone B, Giacobbe DR, Vena A, Taramasso L, Nicolini L, Patroniti N, Pelosi P, Gratarola A, De Palma R, Filaci G, Bassetti M, De Maria A (2021) Extensive activation, tissue trafficking, turnover and functional impairment of NK cells in COVID-19 patients at disease onset associates with subsequent disease severity. PLoS Pathog 17:e1009448. https://doi.org/10.1371/journal.ppat.1009448
Connors JM, Levy JH (2020) COVID-19 and its implications for thrombosis and anticoagulation. Blood 135:2033–2040. https://doi.org/10.1182/blood.2020006000
Hasan A, Al-Ozairi E, Al-Baqsumi Z, Ahmad R, Al-Mulla F (2021) Cellular and humoral immune responses in covid-19 and immunotherapeutic approaches. Immunotargets Ther 10:63–85. https://doi.org/10.2147/itt.S280706
Hong R, Zhao H, Wang Y, Chen Y, Cai H, Hu Y, Wei G, Huang H (2021) Clinical characterization and risk factors associated with cytokine release syndrome induced by COVID-19 and chimeric antigen receptor T-cell therapy. Bone Marrow Transpl 56:570–580. https://doi.org/10.1038/s41409-020-01060-5
Liu T, Liu S, Zhou X (2021) Innate immune responses and pulmonary diseases. Adv Exp Med Biol 1304:53–71. https://doi.org/10.1007/978-3-030-68748-9_4
Lupu L, Palmer A, Huber-Lang M (2020) Inflammation, thrombosis, and destruction: the three-headed cerberus of trauma- and SARS-CoV-2-induced ARDS. Front Immunol 11:584514. https://doi.org/10.3389/fimmu.2020.584514
Azkur AK, Akdis M, Azkur D, Sokolowska M, van de Veen W, Brüggen MC, O’Mahony L, Gao Y, Nadeau K, Akdis CA (2020) Immune response to SARS-CoV-2 and mechanisms of immunopathological changes in COVID-19. Allergy 75:1564–1581. https://doi.org/10.1111/all.14364
Bergamaschi L, Mescia F, Turner L, Hanson AL, Kotagiri P, Dunmore BJ, Ruffieux H, De Sa A, Huhn O, Morgan MD, Gerber PP, Wills MR, Baker S, Calero-Nieto FJ, Doffinger R, Dougan G, Elmer A, Goodfellow IG, Gupta RK, Hosmillo M, Hunter K, Kingston N, Lehner PJ, Matheson NJ, Nicholson JK, Petrunkina AM, Richardson S, Saunders C, Thaventhiran JED, Toonen EJM, Weekes MP, Göttgens B, Toshner M, Hess C, Bradley JR, Lyons PA, Smith KGC (2021) Longitudinal analysis reveals that delayed bystander CD8+ T cell activation and early immune pathology distinguish severe COVID-19 from mild disease. Immunity 54:1257-1275.e8. https://doi.org/10.1016/j.immuni.2021.05.010
Conti P, Caraffa A, Gallenga CE, Ross R, Kritas SK, Frydas I, Younes A, Di Emidio P, Ronconi G, Toniato E (2020) IL-1 induces throboxane-A2 (TxA2) in COVID-19 causing inflammation and micro-thrombi: inhibitory effect of the IL-1 receptor antagonist (IL-1Ra). J Biol Regul Homeost Agents 34:1623–1627. https://doi.org/10.23812/20-34-4edit-65
Conti P, Caraffa A, Gallenga CE, Ross R, Kritas SK, Frydas I, Younes A, Ronconi G (2020) Coronavirus-19 (SARS-CoV-2) induces acute severe lung inflammation via IL-1 causing cytokine storm in COVID-19: a promising inhibitory strategy. J Biol Regul Homeost Agents 34:1971–1975. https://doi.org/10.23812/20-1-e
Gallerani E, Proietto D, Dallan B, Campagnaro M, Pacifico S, Albanese V, Marzola E, Marconi P, Caputo A, Appay V, Gavioli R, Nicoli F (2021) Impaired priming of SARS-CoV-2-specific naive CD8(+) T cells in older subjects. Front Immunol 12:693054. https://doi.org/10.3389/fimmu.2021.693054
Kalfaoglu B, Almeida-Santos J, Tye CA, Satou Y, Ono M (2020) T-cell hyperactivation and paralysis in severe COVID-19 infection revealed by single-cell analysis. Front Immunol 11:589380. https://doi.org/10.3389/fimmu.2020.589380
Lagadinou M, Zareifopoulos N, Gkentzi D, Sampsonas F, Kostopoulou E, Marangos M, Solomou E (2021) Alterations in lymphocyte subsets and monocytes in patients diagnosed with SARS-CoV-2 pneumonia: a mini review of the literature. Eur Rev Med Pharmacol Sci 25:5057–5062. https://doi.org/10.26355/eurrev_202108_26463
Lazzaroni MG, Piantoni S, Masneri S, Garrafa E, Martini G, Tincani A, Andreoli L, Franceschini F (2021) Coagulation dysfunction in COVID-19: the interplay between inflammation, viral infection and the coagulation system. Blood Rev 46:100745. https://doi.org/10.1016/j.blre.2020.100745
Li Z, Huang Z, Li X, Huang C, Shen J, Li S, Zhang L, Wong SH, Chan MTV, Wu WKK (2021) Bioinformatic analyses hinted at augmented T helper 17 cell differentiation and cytokine response as the central mechanism of COVID-19-associated Guillain-Barré syndrome. Cell Prolif 54:e13024. https://doi.org/10.1111/cpr.13024
Loo J, Spittle DA, Newnham M (2021) COVID-19, immunothrombosis and venous thromboembolism: biological mechanisms. Thorax 76:412–420. https://doi.org/10.1136/thoraxjnl-2020-216243
Mahmoudi S, Yaghmaei B, Sharifzadeh Ekbatani M, Pourakbari B, Navaeian A, Parvaneh N, Haghi Ashtiani MT, Mamishi S (2021) Effects of coronavirus disease 2019 (COVID-19) on peripheral blood lymphocytes and their subsets in children: imbalanced CD4(+)/CD8(+) T cell ratio and disease severity. Front Pediatr 9:643299. https://doi.org/10.3389/fped.2021.643299
Sami R, Fathi F, Eskandari N, Ahmadi M, ArefNezhad R, Motedayyen H (2021) Characterizing the immune responses of those who survived or succumbed to COVID-19: can immunological signatures predict outcome? Cytokine 140:155439. https://doi.org/10.1016/j.cyto.2021.155439
Tang Y, Sun J, Pan H, Yao F, Yuan Y, Zeng M, Ye G, Yang G, Zheng B, Fan J, Pan Y, Zhao Z, Guo S, Liu Y, Liao F, Duan Y, Jiao X, Li Y (2021) Aberrant cytokine expression in COVID-19 patients: associations between cytokines and disease severity. Cytokine 143:155523. https://doi.org/10.1016/j.cyto.2021.155523
Torres-Ruiz J, Pérez-Fragoso A, Maravillas-Montero JL, Llorente L, Mejía-Domínguez NR, Páez-Franco JC, Romero-Ramírez S, Sosa-Hernández VA, Cervantes-Díaz R, Absalón-Aguilar A, Nuñez-Aguirre M, Juárez-Vega G, Meza-Sánchez D, Kleinberg-Bid A, Hernández-Gilsoul T, Ponce-de-León A, Gómez-Martín D (2021) Redefining COVID-19 severity and prognosis: the role of clinical and immunobiotypes. Front Immunol 12:689966. https://doi.org/10.3389/fimmu.2021.689966
Townsend L, Dyer AH, Naughton A, Kiersey R, Holden D, Gardiner M, Dowds J, O’Brien K, Bannan C, Nadarajan P, Dunne J, Martin-Loeches I, Fallon PG, Bergin C, O’Farrelly C, Cheallaigh CN, Bourke NM, Conlon N (2021) Longitudinal analysis of COVID-19 patients shows age-associated T cell changes independent of ongoing ill-health. Front Immunol 12:676932. https://doi.org/10.3389/fimmu.2021.676932
Wang C, Xie J, Zhao L, Fei X, Zhang H, Tan Y, Nie X, Zhou L, Liu Z, Ren Y, Yuan L, Zhang Y, Zhang J, Liang L, Chen X, Liu X, Wang P, Han X, Weng X, Chen Y, Yu T, Zhang X, Cai J, Chen R, Shi ZL, Bian XW (2020) Alveolar macrophage dysfunction and cytokine storm in the pathogenesis of two severe COVID-19 patients. EBioMedicine 57:102833. https://doi.org/10.1016/j.ebiom.2020.102833
Wang W, Liu X, Wu S, Chen S, Li Y, Nong L, Lie P, Huang L, Cheng L, Lin Y, He J (2020) Definition and risks of cytokine release syndrome in 11 critically ill COVID-19 patients with pneumonia: analysis of disease characteristics. J Infect Dis 222:1444–1451. https://doi.org/10.1093/infdis/jiaa387
Yao C, Bora SA, Parimon T, Zaman T, Friedman OA, Palatinus JA, Surapaneni NS, Matusov YP, Cerro Chiang G, Kassar AG, Patel N, Green CER, Aziz AW, Suri H, Suda J, Lopez AA, Martins GA, Stripp BR, Gharib SA, Goodridge HS, Chen P (2021) Cell-type-specific immune dysregulation in severely ill COVID-19 patients. Cell Rep 34:108590. https://doi.org/10.1016/j.celrep.2020.108590
Yokota S, Miyamae T, Kuroiwa Y, Nishioka K (2021) Novel coronavirus disease 2019 (COVID-19) and cytokine storms for more effective treatments from an inflammatory pathophysiology. J Clin Med. https://doi.org/10.3390/jcm10040801
Hu B, Huang S, Yin L (2021) The cytokine storm and COVID-19. J Med Virol 93:250–256. https://doi.org/10.1002/jmv.26232
Tyagi SC, Singh M (2021) Multi-organ damage by covid-19: congestive (cardio-pulmonary) heart failure, and blood-heart barrier leakage. Mol Cell Biochem 476:1891–1895. https://doi.org/10.1007/s11010-021-04054-z
Shahriar S, Rana MS, Hossain MS, Karim A, Mredula TN, Nourin N, Uddin MS, Amran MS (2021) COVID-19: epidemiology, pathology, diagnosis, treatment, and impact. Curr Pharm Des 27:3502–3525. https://doi.org/10.2174/1381612827666210224142446
Boscolo-Rizzo P, Borsetto D, Spinato G, Fabbris C, Menegaldo A, Gaudioso P, Nicolai P, Tirelli G, Da Mosto MC, Rigoli R, Polesel J, Hopkins C (2020) New onset of loss of smell or taste in household contacts of home-isolated SARS-CoV-2-positive subjects. Eur Arch Otorhinolaryngol 277:2637–2640. https://doi.org/10.1007/s00405-020-06066-9
Cirillo N, Colella G (2021) Self-reported smell and taste alteration as the sole clinical manifestation of SARS-CoV-2 infection. Oral Surg Oral Med Oral Pathol Oral Radiol 131:e95–e99. https://doi.org/10.1016/j.oooo.2020.11.016
Dudine L, Canaletti C, Giudici F, Lunardelli A, Abram G, Santini I, Baroni V, Paris M, Pesavento V, Manganotti P, Ronchese F, Gregoretti B, Negro C (2021) Investigation on the loss of taste and smell and consequent psychological effects: a cross-sectional study on healthcare workers who contracted the COVID-19 infection. Front Public Health 9:666442. https://doi.org/10.3389/fpubh.2021.666442
Mazzatenta A, Neri G, D’Ardes D, De Luca C, Marinari S, Porreca E, Cipollone F, Vecchiet J, Falcicchia C, Panichi V, Origlia N, Di Giulio C (2020) Smell and taste in severe CoViD-19: self-reported vs testing. Front Med (Lausanne) 7:589409. https://doi.org/10.3389/fmed.2020.589409
Meunier N, Briand L, Jacquin-Piques A, Brondel L, Pénicaud L (2020) COVID 19-induced smell and taste impairments: putative impact on physiology. Front Physiol 11:625110. https://doi.org/10.3389/fphys.2020.625110
Mullol J, Alobid I, Mariño-Sánchez F, Izquierdo-Domínguez A, Marin C, Klimek L, Wang DY, Liu Z (2020) The loss of smell and taste in the COVID-19 outbreak: a tale of many countries. Curr Allergy Asthma Rep 20:61. https://doi.org/10.1007/s11882-020-00961-1
Struyf T, Deeks JJ, Dinnes J, Takwoingi Y, Davenport C, Leeflang MM, Spijker R, Hooft L, Emperador D, Domen J, Horn SRA, Van den Bruel A (2021) Signs and symptoms to determine if a patient presenting in primary care or hospital outpatient settings has COVID-19. Cochrane Database Syst Rev 2:CD013665. https://doi.org/10.1002/14651858.CD013665.pub2
Yan Q, Qiu D, Liu X, Guo X, Hu Y (2021) Prevalence of smell or taste dysfunction among children with COVID-19 infection: a systematic review and meta-analysis. Front Pediatr 9:686600. https://doi.org/10.3389/fped.2021.686600
Ganji R, Reddy PH (2020) Impact of COVID-19 on mitochondrial-based immunity in aging and age-related diseases. Front Aging Neurosci 12:614650. https://doi.org/10.3389/fnagi.2020.614650
Zaki N, Alashwal H, Ibrahim S (2020) Association of hypertension, diabetes, stroke, cancer, kidney disease, and high-cholesterol with COVID-19 disease severity and fatality: a systematic review. Diabetes Metab Syndr 14:1133–1142. https://doi.org/10.1016/j.dsx.2020.07.005
Biswas S, Thakur V, Kaur P, Khan A, Kulshrestha S, Kumar P (2021) Blood clots in COVID-19 patients: simplifying the curious mystery. Med Hypotheses 146:110371. https://doi.org/10.1016/j.mehy.2020.110371
Singh M, Shmulevitz M, Tikoo SK (2005) A newly identified interaction between IVa2 and pVIII proteins during porcine adenovirus type 3 infection. Virology 336:60–69. https://doi.org/10.1016/j.virol.2005.03.003
Libby P, Lüscher T (2020) COVID-19 is, in the end, an endothelial disease. Eur Heart J 41:3038–3044. https://doi.org/10.1093/eurheartj/ehaa623
Bowen JE, Sprouse KR, Walls AC, Mazzitelli IG, Logue JK, Franko NM, Ahmed K, Shariq A, Cameroni E, Gori A, Bandera A, Posavad CM, Dan JM, Zhang Z, Weiskopf D, Sette A, Crotty S, Iqbal NT, Corti D, Geffner J, Grifantini R, Chu HY, Veesler D (2022) Omicron BA.1 and BA.2 neutralizing activity elicited by a comprehensive panel of human vaccines. bioRxiv. https://doi.org/10.1101/2022.03.15.484542
Bruel T, Hadjadj J, Maes P, Planas D, Seve A, Staropoli I, Guivel-Benhassine F, Porrot F, Bolland WH, Nguyen Y, Casadevall M, Charre C, Péré H, Veyer D, Prot M, Baidaliuk A, Cuypers L, Planchais C, Mouquet H, Baele G, Mouthon L, Hocqueloux L, Simon-Loriere E, André E, Terrier B, Prazuck T, Schwartz O (2022) Serum neutralization of SARS-CoV-2 omicron sublineages BA.1 and BA.2 in patients receiving monoclonal antibodies. Nat Med. https://doi.org/10.1038/s41591-022-01792-5
Chen J, Wei GW (2022) Omicron BA.2 (B.1.1.529.2): high potential to becoming the next dominating variant. ArXiv
Zhang L, Narayanan KK, Cooper L, Chan KK, Devlin CA, Aguhob A, Shirley K, Rong L, Rehman J, Malik AB, Procko E (2022) An engineered ACE2 decoy receptor can be administered by inhalation and potently targets the BA.1 and BA.2 omicron variants of SARS-CoV-2. bioRxiv. https://doi.org/10.1101/2022.03.28.486075
Callaway E (2021) Delta coronavirus variant: scientists brace for impact. Nature 595:17–18. https://doi.org/10.1038/d41586-021-01696-3
Maxmen A (2022) Are new omicron subvariants a threat? Here’s how scientists are keeping watch. Nature 604:605–606. https://doi.org/10.1038/d41586-022-01069-4
Gu T, Zhao S, Jin G, Song M, Zhi Y, Zhao R, Ma F, Zheng Y, Wang K, Liu H, Xin M, Han W, Li X, Dong CD, Liu K, Dong Z (2020) Cytokine signature induced by SARS-CoV-2 spike protein in a mouse model. Front Immunol 11:621441. https://doi.org/10.3389/fimmu.2020.621441
Morrison JC, Jia L, Cepurna W, Guo Y, Johnson E (2009) Reliability and sensitivity of the TonoLab rebound tonometer in awake Brown Norway rats. Invest Ophthalmol Vis Sci 50:2802–2808. https://doi.org/10.1167/iovs.08-2465
Kunkel GH, Kunkel CJ, Ozuna H, Miralda I, Tyagi SC (2019) TFAM overexpression reduces pathological cardiac remodeling. Mol Cell Biochem 454:139–152. https://doi.org/10.1007/s11010-018-3459-9
Singh M, George AK, Eyob W, Homme RP, Stansic D, Tyagi SC (2021) High-methionine diet in skeletal muscle remodeling: epigenetic mechanism of homocysteine-mediated growth retardation. Can J Physiol Pharmacol 99:56–63. https://doi.org/10.1139/cjpp-2020-0093
Singh M, Hardin SJ, George AK, Eyob W, Stanisic D, Pushpakumar S, Tyagi SC (2020) Epigenetics, 1-carbon metabolism, and homocysteine during dysbiosis. Front Physiol 11:617953. https://doi.org/10.3389/fphys.2020.617953
Miller A, Mujumdar V, Shek E, Guillot J, Angelo M, Palmer L, Tyagi SC (2000) Hyperhomocyst(e)inemia induces multiorgan damage. Heart Vessels 15:135–143. https://doi.org/10.1007/s003800070030
Stanisic D, George AK, Smolenkova I, Singh M, Tyagi SC (2021) Hyperhomocysteinemia: an instigating factor for periodontal disease. Can J Physiol Pharmacol 99:115–123. https://doi.org/10.1139/cjpp-2020-0224
Jeremic JN, Jakovljevic VL, Zivkovic VI, Srejovic IM, Bradic JV, Bolevich S, Nikolic Turnic TR, Mitrovic SL, Jovicic NU, Tyagi SC, Jeremic NS (2019) The cardioprotective effects of diallyl trisulfide on diabetic rats with ex vivo induced ischemia/reperfusion injury. Mol Cell Biochem 460:151–164. https://doi.org/10.1007/s11010-019-03577-w
Singh M, Kumar V (2003) Transgenic mouse models of hepatitis B virus-associated hepatocellular carcinoma. Rev Med Virol 13:243–253. https://doi.org/10.1002/rmv.392
Bhavani SV, Verhoef PA, Maier CL, Robichaux C, Parker WF, Holder A, Kamaleswaran R, Wang MD, Churpek MM, Coopersmith CM (2022) Coronavirus disease 2019 temperature trajectories correlate with hyperinflammatory and hypercoagulable subphenotypes. Crit Care Med 50:212–223. https://doi.org/10.1097/ccm.0000000000005397
Di Filippo L, De Lorenzo R, D’Amico M, Sofia V, Roveri L, Mele R, Saibene A, Rovere-Querini P, Conte C (2021) COVID-19 is associated with clinically significant weight loss and risk of malnutrition, independent of hospitalisation: a post-hoc analysis of a prospective cohort study. Clin Nutr 40:2420–2426. https://doi.org/10.1016/j.clnu.2020.10.043
Miller DJ, Capodilupo JV, Lastella M, Sargent C, Roach GD, Lee VH, Capodilupo ER (2020) Analyzing changes in respiratory rate to predict the risk of COVID-19 infection. PLoS ONE 15:e0243693. https://doi.org/10.1371/journal.pone.0243693
Akpek M (2021) Does COVID-19 cause hypertension? Angiology. https://doi.org/10.1177/00033197211053903
Tadic M, Cuspidi C, Grassi G, Mancia G (2020) COVID-19 and arterial hypertension: hypothesis or evidence? J Clin Hypertens (Greenwich) 22:1120–1126. https://doi.org/10.1111/jch.13925
Alonso RS, Alonso FOM, Fernandes BF, Ecard VO, Ventura MP (2021) COVID-19-related ocular hypertension secondary to anterior uveitis as part of a multisystemic inflammatory syndrome. J Glaucoma 30:e256–e258. https://doi.org/10.1097/ijg.0000000000001835
Nishiga M, Wang DW, Han Y, Lewis DB, Wu JC (2020) COVID-19 and cardiovascular disease: from basic mechanisms to clinical perspectives. Nat Rev Cardiol 17:543–558. https://doi.org/10.1038/s41569-020-0413-9
Mokhtari T, Hassani F, Ghaffari N, Ebrahimi B, Yarahmadi A, Hassanzadeh G (2020) COVID-19 and multiorgan failure: a narrative review on potential mechanisms. J Mol Histol 51:613–628. https://doi.org/10.1007/s10735-020-09915-3
Wu T, Zuo Z, Kang S, Jiang L, Luo X, Xia Z, Liu J, Xiao X, Ye M, Deng M (2020) Multi-organ dysfunction in patients with COVID-19: a systematic review and meta-analysis. Aging Dis 11:874–894. https://doi.org/10.14336/ad.2020.0520
Kunzmann V, Kretzschmar E, Herrmann T, Wilhelm M (2004) Polyinosinic-polycytidylic acid-mediated stimulation of human gammadelta T cells via CD11c dendritic cell-derived type I interferons. Immunology 112:369–377. https://doi.org/10.1111/j.1365-2567.2004.01908.x
Balfanz P, Hartmann B, Müller-Wieland D, Kleines M, Häckl D, Kossack N, Kersten A, Cornelissen C, Müller T, Daher A, Stöhr R, Bickenbach J, Marx G, Marx N, Dreher M (2021) Early risk markers for severe clinical course and fatal outcome in German patients with COVID-19. PLoS ONE 16:e0246182. https://doi.org/10.1371/journal.pone.0246182
Laguna-Goya R, Utrero-Rico A, Talayero P, Lasa-Lazaro M, Ramirez-Fernandez A, Naranjo L, Segura-Tudela A, Cabrera-Marante O, Rodriguez de Frias E, Garcia-Garcia R, Fernández-Ruiz M, Aguado JM, Martinez-Lopez J, Lopez EA, Catalan M, Serrano A, Paz-Artal E (2020) IL-6-based mortality risk model for hospitalized patients with COVID-19. J Allergy Clin Immunol 146:799-807.e9. https://doi.org/10.1016/j.jaci.2020.07.009
Saji R, Nishii M, Sakai K, Miyakawa K, Yamaoka Y, Ban T, Abe T, Ohyama Y, Nakajima K, Hiromi T, Matsumura R, Suzuki N, Taniguchi H, Otsuka T, Oi Y, Ogawa F, Uchiyama M, Takahashi K, Iwashita M, Kimura Y, Fujii S, Furuya R, Tamura T, Ryo A, Takeuchi I (2021) Combining IL-6 and SARS-CoV-2 RNAaemia-based risk stratification for fatal outcomes of COVID-19. PLoS ONE 16:e0256022. https://doi.org/10.1371/journal.pone.0256022
Santa Cruz A, Mendes-Frias A, Oliveira AI, Dias L, Matos AR, Carvalho A, Capela C, Pedrosa J, Castro AG, Silvestre R (2021) Interleukin-6 is a biomarker for the development of fatal severe acute respiratory syndrome coronavirus 2 pneumonia. Front Immunol 12:613422. https://doi.org/10.3389/fimmu.2021.613422
McLaughlin KM, Bojkova D, Kandler JD, Bechtel M, Reus P, Le T, Rothweiler F, Wagner JUG, Weigert A, Ciesek S, Wass MN, Michaelis M, Cinatl J Jr (2021) A potential role of the CD47/SIRPalpha axis in COVID-19 pathogenesis. Curr Issues Mol Biol 43:1212–1225. https://doi.org/10.3390/cimb43030086
Tal MC, Torrez Dulgeroff LB, Myers L, Cham LB, Mayer-Barber KD, Bohrer AC, Castro E, Yiu YY, Lopez Angel C, Pham E, Carmody AB, Messer RJ, Gars E, Kortmann J, Markovic M, Hasenkrug M, Peterson KE, Winkler CW, Woods TA, Hansen P, Galloway S, Wagh D, Fram BJ, Nguyen T, Corey D, Kalluru RS, Banaei N, Rajadas J, Monack DM, Ahmed A, Sahoo D, Davis MM, Glenn JS, Adomati T, Lang KS, Weissman IL, Hasenkrug KJ (2020) Upregulation of CD47 Is a host checkpoint response to pathogen recognition. MBio. https://doi.org/10.1128/mBio.01293-20
D’Alonzo D, De Fenza M, Pavone V (2020) COVID-19 and pneumonia: a role for the uPA/uPAR system. Drug Discov Today 25:1528–1534. https://doi.org/10.1016/j.drudis.2020.06.013
Cesta MC, Zippoli M, Marsiglia C, Gavioli EM, Mantelli F, Allegretti M, Balk RA (2021) The role of interleukin-8 in lung inflammation and injury: implications for the management of COVID-19 and hyperinflammatory acute respiratory distress syndrome. Front Pharmacol 12:808797. https://doi.org/10.3389/fphar.2021.808797
Li L, Li J, Gao M, Fan H, Wang Y, Xu X, Chen C, Liu J, Kim J, Aliyari R, Zhang J, Jin Y, Li X, Ma F, Shi M, Cheng G, Yang H (2020) Interleukin-8 as a biomarker for disease prognosis of coronavirus disease-2019 patients. Front Immunol 11:602395. https://doi.org/10.3389/fimmu.2020.602395
Baresi G, Giacomelli M, Moratto D, Chiarini M, Conforti IC, Padoan R, Poli P, Timpano S, Caldarale F, Badolato R (2021) Case report: analysis of inflammatory cytokines IL-6, CCL2/MCP1, CCL5/RANTES, CXCL9/MIG, and CXCL10/IP10 in a cystic fibrosis patient cohort during the first wave of the COVID-19 pandemic. Front Pediatr 9:645063. https://doi.org/10.3389/fped.2021.645063
Jiang Y, Rubin L, Peng T, Liu L, Xing X, Lazarovici P, Zheng W (2022) Cytokine storm in COVID-19: from viral infection to immune responses, diagnosis and therapy. Int J Biol Sci 18:459–472. https://doi.org/10.7150/ijbs.59272
Elezkurtaj S, Greuel S, Ihlow J, Michaelis EG, Bischoff P, Kunze CA, Sinn BV, Gerhold M, Hauptmann K, Ingold-Heppner B, Miller F, Herbst H, Corman VM, Martin H, Radbruch H, Heppner FL, Horst D (2021) Causes of death and comorbidities in hospitalized patients with COVID-19. Sci Rep 11:4263. https://doi.org/10.1038/s41598-021-82862-5
Iwasaki M, Saito J, Zhao H, Sakamoto A, Hirota K, Ma D (2021) Inflammation triggered by SARS-CoV-2 and ACE2 augment drives multiple organ failure of severe COVID-19: molecular mechanisms and implications. Inflammation 44:13–34. https://doi.org/10.1007/s10753-020-01337-3
Kaur S, Bansal R, Kollimuttathuillam S, Gowda AM, Singh B, Mehta D, Maroules M (2021) The looming storm: blood and cytokines in COVID-19. Blood Rev 46:100743. https://doi.org/10.1016/j.blre.2020.100743
Zaim S, Chong JH, Sankaranarayanan V, Harky A (2020) COVID-19 and multiorgan response. Curr Probl Cardiol 45:100618. https://doi.org/10.1016/j.cpcardiol.2020.100618
Silva Andrade B, Siqueira S, de Assis Soares WR, de Souza RF, Santos NO, Dos Santos FA, Ribeiro da Silveira P, Tiwari S, Alzahrani KJ, Góes-Neto A, Azevedo V, Ghosh P, Barh D (2021) Long-COVID and post-COVID health complications: an up-to-date review on clinical conditions and their possible molecular mechanisms. Viruses. https://doi.org/10.3390/v13040700
Han H, Ma Q, Li C, Liu R, Zhao L, Wang W, Zhang P, Liu X, Gao G, Liu F, Jiang Y, Cheng X, Zhu C, Xia Y (2020) Profiling serum cytokines in COVID-19 patients reveals IL-6 and IL-10 are disease severity predictors. Emerg Microbes Infect 9:1123–1130. https://doi.org/10.1080/22221751.2020.1770129
Jamal M, Bangash HI, Habiba M, Lei Y, Xie T, Sun J, Wei Z, Hong Z, Shao L, Zhang Q (2021) Immune dysregulation and system pathology in COVID-19. Virulence 12:918–936. https://doi.org/10.1080/21505594.2021.1898790
Wang J, Jiang M, Chen X, Montaner LJ (2020) Cytokine storm and leukocyte changes in mild versus severe SARS-CoV-2 infection: Review of 3939 COVID-19 patients in China and emerging pathogenesis and therapy concepts. J Leukoc Biol 108:17–41. https://doi.org/10.1002/jlb.3covr0520-272r
Buja LM, Wolf DA, Zhao B, Akkanti B, McDonald M, Lelenwa L, Reilly N, Ottaviani G, Elghetany MT, Trujillo DO, Aisenberg GM, Madjid M, Kar B (2020) The emerging spectrum of cardiopulmonary pathology of the coronavirus disease 2019 (COVID-19): Report of 3 autopsies from Houston, Texas, and review of autopsy findings from other United States cities. Cardiovasc Pathol 48:107233. https://doi.org/10.1016/j.carpath.2020.107233
Nugent J, Aklilu A, Yamamoto Y, Simonov M, Li F, Biswas A, Ghazi L, Greenberg H, Mansour G, Moledina G, Wilson FP (2021) Assessment of acute kidney injury and longitudinal kidney function after hospital discharge among patients with and without COVID-19. JAMA Netw Open 4:e211095. https://doi.org/10.1001/jamanetworkopen.2021.1095
Alexander MP, Mangalaparthi KK, Madugundu AK, Moyer AM, Adam BA, Mengel M, Singh S, Herrmann SM, Rule AD, Cheek EH, Herrera Hernandez LP, Graham RP, Aleksandar D, Aubry MC, Roden AC, Hagen CE, Quinton RA, Bois MC, Lin PT, Maleszewski JJ, Cornell LD, Sethi S, Pavelko KD, Charlesworth J, Narasimhan R, Larsen CP, Rizza SA, Nasr SH, Grande JP, McKee TD, Badley AD, Pandey A, Taner T (2021) Acute kidney injury in severe COVID-19 has similarities to sepsis-associated kidney injury: a multi-omics study. Mayo Clin Proc 96:2561–2575. https://doi.org/10.1016/j.mayocp.2021.07.001
Bjornstad EC, Seifert ME, Sanderson K, Feig DI (2021) Kidney implications of SARS-CoV2 infection in children. Pediatr Nephrol. https://doi.org/10.1007/s00467-021-05249-8
Bowe B, Cai M, Xie Y, Gibson AK, Maddukuri G, Al-Aly Z (2020) Acute kidney injury in a National Cohort of Hospitalized US veterans with COVID-19. Clin J Am Soc Nephrol 16:14–25. https://doi.org/10.2215/cjn.09610620
Chen K, Lei Y, He Y, Xiao F, Yu Y, Lai X, Liu Y, Wang J, Dai H (2021) Clinical outcomes of hospitalized COVID-19 patients with renal injury: a multi-hospital observational study from Wuhan. Sci Rep 11:15205. https://doi.org/10.1038/s41598-021-94570-1
de Almeida DC, Franco M, Dos Santos DRP, Santos MC, Maltoni IS, Mascotte F, de Souza AA, Pietrobom PM, Medeiros EA, Ferreira PRA, Machado FR, Goes MA (2021) Acute kidney injury: incidence, risk factors, and outcomes in severe COVID-19 patients. PLoS ONE 16:e0251048. https://doi.org/10.1371/journal.pone.0251048
Ferlicot S, Jamme M, Gaillard F, Oniszczuk J, Couturier A, May O, Grünenwald A, Sannier A, Moktefi A, Le Monnier O, Petit-Hoang C, Maroun N, Brodin-Sartorius A, Michon A, Dobosziewicz H, Andreelli F, Guillet M, Izzedine H, Richard C, Dekeyser M, Arrestier R, Sthelé T, Lefèvre E, Mathian A, Legendre C, Mussini C, Verpont MC, Pallet N, Amoura Z, Essig M, Snanoudj R, Brocheriou-Spelle I, François H, Belenfant X, Geri G, Daugas E, Audard V, Buob D, Massy ZA, Zaidan M (2021) The spectrum of kidney biopsies in hospitalized patients with COVID-19, acute kidney injury, and/or proteinuria. Nephrol Dial Transpl. https://doi.org/10.1093/ndt/gfab042
Flythe JE, Assimon MM, Tugman MJ, Chang EH, Gupta S, Shah J, Sosa MA, Renaghan AD, Melamed ML, Wilson FP, Neyra JA, Rashidi A, Boyle SM, Anand S, Christov M, Thomas LF, Edmonston D, Leaf DE (2021) Characteristics and outcomes of individuals with pre-existing kidney disease and COVID-19 admitted to intensive care units in the United States. Am J Kidney Dis 77:190-203.e1. https://doi.org/10.1053/j.ajkd.2020.09.003
Gok M, Cetinkaya H, Kandemir T, Karahan E, Tuncer İB, Bukrek C, Sahin G (2021) Chronic kidney disease predicts poor outcomes of COVID-19 patients. Int Urol Nephrol 53:1891–1898. https://doi.org/10.1007/s11255-020-02758-7
Gómez-Escobar LG, Hoffman KL, Choi JJ, Borczuk A, Salvatore S, Alvarez-Mulett SL, Galvan MD, Zhao Z, Racine-Brzostek SE, Yang HS, Stout-Delgado HW, Choi ME, Choi AMK, Cho SJ, Schenck EJ (2021) Cytokine signatures of end organ injury in COVID-19. Sci Rep 11:12606. https://doi.org/10.1038/s41598-021-91859-z
Ng JH, Zaidan M, Jhaveri KD, Izzedine H (2021) Acute tubulointerstitial nephritis and COVID-19. Clin Kidney J 14:2151–2157. https://doi.org/10.1093/ckj/sfab107
Punj S, Eng E, Shetty AA (2021) Coronavirus disease 2019 and kidney injury. Curr Opin Nephrol Hypertens 30:444–449. https://doi.org/10.1097/mnh.0000000000000718
Sharma P, Ng JH, Bijol V, Jhaveri KD, Wanchoo R (2021) Pathology of COVID-19-associated acute kidney injury. Clin Kidney J 14:i30–i39. https://doi.org/10.1093/ckj/sfab003
Shetty AA, Tawhari I, Safar-Boueri L, Seif N, Alahmadi A, Gargiulo R, Aggarwal V, Usman I, Kisselev S, Gharavi AG, Kanwar Y, Quaggin SE (2021) COVID-19-associated glomerular disease. J Am Soc Nephrol 32:33–40. https://doi.org/10.1681/asn.2020060804
Chauhan A, Tikoo A, Kapur AK, Singh M (2007) The taming of the cell penetrating domain of the HIV tat: myths and realities. J Control Release 117:148–162. https://doi.org/10.1016/j.jconrel.2006.10.031
Aggarwal R, Bhatia R, Kulshrestha K, Soni KD, Viswanath R, Singh AK, Iyer KV, Khanna P, Bhattacharjee S, Patel N, Aravindan A, Gupta A, Singh Y, Ganesh V, Kumar R, Ayub A, Kumar S, Prakash K, Venkateswaran V, Bhoi D, Soneja M, Mathur P, Malhotra R, Wig N, Guleria R, Trikha A (2021) Clinicoepidemiological features and mortality analysis of deceased patients with COVID-19 in a tertiary care center. Indian J Crit Care Med 25:622–628. https://doi.org/10.5005/jp-journals-10071-23848
Arslan U, Borulu F, Sarac İ, Prof BE (2021) Chronic intracardiac thrombus, a long-term complication of COVID-19: case reports. J Card Surg 36:3939–3943. https://doi.org/10.1111/jocs.15836
Fahmy OH, Daas FM, Salunkhe V, Petrey JL, Cosar EF, Ramirez J, Akca O (2021) Is microthrombosis the main pathology in coronavirus disease 2019 severity?-A systematic review of the postmortem pathologic Findings. Crit Care Explor 3:e0427. https://doi.org/10.1097/cce.0000000000000427
Ghosn L, Chaimani A, Evrenoglou T, Davidson M, Graña C, Schmucker C, Bollig C, Henschke N, Sguassero Y, Nejstgaard CH, Menon S, Nguyen TV, Ferrand G, Kapp P, Riveros C, Ávila C, Devane D, Meerpohl JJ, Rada G, Hróbjartsson A, Grasselli G, Tovey D, Ravaud P, Boutron I (2021) Interleukin-6 blocking agents for treating COVID-19: a living systematic review. Cochrane Database Syst Rev 3:CD013881. https://doi.org/10.1002/14651858.Cd013881
Güven M, Gültekin H (2021) Could serum total cortisol level at admission predict mortality due to coronavirus disease 2019 in the intensive care unit? A prospective study. Sao Paulo Med J 139:398–404. https://doi.org/10.1590/1516-3180.2020.0722.R1.2302021
Haberecker M, Schwarz EI, Steiger P, Frontzek K, Scholkmann F, Zeng X, Höller S, Moch H, Varga Z (2021) Autopsy-based pulmonary and vascular pathology: pulmonary endotheliitis and multi-organ involvement in COVID-19 associated deaths. Respiration. https://doi.org/10.1159/000518914
Liu Z, Liu J, Ye L, Yu K, Luo Z, Liang C, Cao J, Wu X, Li S, Zhu L, Xiang G (2021) Predictors of mortality for hospitalized young adults aged less than 60 years old with severe COVID-19: a retrospective study. J Thorac Dis 13:3628–3642. https://doi.org/10.21037/jtd-21-120
Matsuishi Y, Mathis BJ, Shimojo N, Subrina J, Okubo N, Inoue Y (2021) Severe COVID-19 infection associated with endothelial dysfunction induces multiple organ dysfunction: a review of therapeutic interventions. Biomedicines. https://doi.org/10.3390/biomedicines9030279
Namburu L, Bhogal SS, Ramu VK (2021) COVID-19-induced takotsubo cardiomyopathy with concomitant pulmonary embolism. Cureus 13:e18693. https://doi.org/10.7759/cureus.18693
Navarro Conde P, Alemany Monraval P, Medina Medina C, Jiménez Sánchez A, Andrés Teruel JC, Ferrando Marco J, Puglia Santos V, Mayordomo Aranda E (2020) Autopsy findings from the first known death from severe acute respiratory syndrome SARS-CoV-2 in Spain. Rev Esp Patol 53:188–192. https://doi.org/10.1016/j.patol.2020.04.002
Ng MK, Ngo J, Patel A, Patel D, Ng KK (2020) A case report of rapidly lethal acute respiratory distress syndrome secondary to coronavirus disease 2019 viral pneumonia. Cureus 12:e8228. https://doi.org/10.7759/cureus.8228
Wang Y, Pang SC, Yang Y (2021) A potential association between immunosenescence and high COVID-19 related mortality among elderly patients with cardiovascular diseases. Immun Ageing 18:25. https://doi.org/10.1186/s12979-021-00234-z
Kiseleva AA, Troisi EM, Hensley SE, Kohli RM, Epstein JA (2021) SARS-CoV-2 spike protein binding selectively accelerates substrate-specific catalytic activity of ACE2. J Biochem 170:299–306. https://doi.org/10.1093/jb/mvab041
Acknowledgements
The authors are grateful to all the members in the laboratory for their help, and support.
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The authors express special thanks to the funding sources (NIH: HL-74185, HL-139047, DK116591 and AR-71789).
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MS conceived the idea of the research plan, designed experiments, helped to analyze data, and wrote the initial manuscript’s draft. MS, SPM, and SCT edited, and help finalized the manuscript. NB, YZ, RPH, and SP performed the experiments, and helped write the material, and methods section, and the figure legends for the manuscript.
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Singh, M., Pushpakumar, S., Bard, N. et al. Simulation of COVID-19 symptoms in a genetically engineered mouse model: implications for the long haulers. Mol Cell Biochem 478, 103–119 (2023). https://doi.org/10.1007/s11010-022-04487-0
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DOI: https://doi.org/10.1007/s11010-022-04487-0