Japanese Scientists Discover That SARS-CoV-2 infection Induces Paracrine Senescence That Leads To Sustained Inflammation Causing Long COVID Issues
Researchers from Osaka University-Japan, have in a new study found that SARS-CoV-2 infection triggers paracrine senescence and leads to a sustained senescence-associated inflammatory response that could also be responsible for a variety of conditions seen in Long COVID
Importantly the study found that cells infected by SARS-CoV-2 produce soluble mediators that cause neighboring cells to lose their capacity to divide, even if they are not infected. This leads to an inflammatory response which continues even after the virus is no longer detectable.
Senescence which is also called biological aging, is the breakdown of the physical body and is a cellular stress response triggered by diverse stressors, including oncogene activation, where it serves as a bona-fide tumor suppressor mechanism. Senescence can be transmitted to neighboring cells, known as paracrine secondary senescence.
To date, there has been numerous reports of post-acute COVID-19 syndrome, in which the inflammatory response persists even after SARS-CoV-2 has disappeared, but the underlying mechanisms of post-acute COVID-19 syndrome remain unknown.
The study team in this new research showed that SARS-CoV-2-infected cells trigger senescence-like cell-cycle arrest in neighboring uninfected cells in a paracrine manner via virus-induced cytokine production.
In cultured human cells or bronchial organoids, these SASR-CoV-2 infection-induced senescent cells express high levels of a series of inflammatory factors known as senescence-associated secretory phenotypes (SASPs) in a sustained manner, even after SARS-CoV-2 is no longer detectable.
The study findings also showed that the expression of the senescence marker CDKN2A and various SASP factor genes is increased in the pulmonary cells of patients with severe post-acute COVID-19 syndrome.
The study also found that mice exposed to a mouse-adapted strain of SARS-CoV-2 exhibit prolonged signs of cellular senescence and SASP in the lung at 14 days after infection when the virus was undetectable, which could be substantially reduced by the administration of senolytic drugs. The sustained infection-induced paracrine senescence described here may be involved in the long-term inflammation caused by SARS-CoV-2 infection.
The study findings were published in the peer reviewed journal: Nature Aging. https://www.nature.com/articles/s43587-022-00170-7
As COVID-19 tends to be more severe in older people, some important clues may exist in the relationship between SARS-CoV-2 infection and aging.
Though numerous changes in various biological responses are associated with aging, the accumulation of senescent cells has recently attracted keen attention. Cellular senescence is a state of irreversible cell-cycle arrest that can be induced by a variety of potentially oncogenic stimuli and thus is considered to serve as an important mechanism of tumor suppression. However, unlike apoptotic cells, senescent cells do not die immediately and thereby accumulate throughout the body during the aging process.
Importantly, senescent cells are not merely nondividing, as they also develop a phenomenon called t
he SASP in which they secrete a variety of proinflammatory factors, such as inflammatory cytokines, chemokines, growth factors and extracellular matrix-degrading enzymes, into the extracellular fluid.
Thus, although the induction of cellular senescence acts primarily as a mechanism of tumor suppression, the excessive accumulation of senescent cells in vivo due to aging is thought to have adverse effects via SASP.
Indeed, the removal of senescent cells by genetic or pharmacological approaches reportedly delays the onset of aging-associated inflammatory diseases in aged mice.
These previous findings prompted the study team to examine the relationship between cellular senescence and SARS-CoV-2 infection.
The study team first set up a system to infect senescent cells with SARS-CoV-2 using primary normal human lung diploid fibroblasts (HDFs), which are commonly used in senescence studies.
Interestingly, the expression level of the ACE2 gene, which is required for SARS-CoV-2 to enter the cells, was increased several-fold upon the induction of cellular senescence in HDFs, as well as in other cultured primary normal human cells.
This is fairly consistent with the previous observation that the levels of the ACE2 gene expression are slightly increased in lung tissues with age.
The study team however were unable to detect SARS-CoV-2 infection in these primary normal human cells, regardless of cellular senescence induction.
However, the study team unexpectedly noticed that signs of cellular senescence, such as cell-cycle arrest, induction of expression of p16INK4a, one of the proteins encoded by the CDKN2a gene locus5 and interleukin-1β (IL-1β) (SASP factor) expression were observed on day 9 after SARS-CoV-2 administration.
This is somewhat consistent with previous reports that certain viral infections can directly induced senescence-like features in cultured cells. https://pubmed.ncbi.nlm.nih.gov/33033152/
Most of these senescence-like cells no longer expressed the protein encoded by SARS-CoV-2, and the SARS-CoV-2 subgenomic RNA was hardly detectable by qPCR on day 9 after SARS-CoV-2 administration to ACE2-HDFs, indicating that SARS-CoV-2 either decreased below the detection limit after inducing the senescence-like phenotype or indirectly prompted this phenotype in uninfected cells.
HDFs ectopically expressing EGFP (EGFP-HDFs), which cannot be infected with SARS-CoV-2, were cocultured with the HDFs ectopically expressing ACE2 and administered SARS-CoV-2. Intriguingly, a significant proportion of EGFP-HDFs were positive for p16INK4a, suggesting that SARS-CoV-2 indirectly induces the senescence-like phenotype.
In order to determine how SARS-CoV-2 indirectly induces the senescence-like phenotype, the study team compared the time course of SARS-CoV-2 infection and senescence-like phenotype induction in ACE2-HDFs. Notably, SARS-CoV-2-infected cells became detectable as early as day 4 after SARS-CoV-2 administration and reached a peak on day 6, but the signs of cellular senescence appeared later, reaching a peak on day 9 when most of the SARS-CoV-2-infected cells had died.
These results are consistent with the observation that the expression levels of virus-induced cytokines, such as IFNβ, IL6 and TNFα, peak on day 6 after infection with SARS-CoV-2 and then decrease markedly, whereas those of other cytokines belonging to the SASP factor, such as IL1β and IL8, persist even after SARS-CoV-2 is no longer detected. Some of these virus-induced cytokines reportedly promote cellular senescence, depending on the biological context.
The study team next tested whether SARS-CoV-2 infection causes the senescence-like phenotype indirectly through these virus-induced cytokines. The incubation of HDFs incapable of SARS-CoV-2 infection with culture supernatants of ACE2-HDFs 6 days after SARS-CoV-2 infection did indeed elicit senescence-like phenotypes, such as irreversible cell-cycle arrest and expression of p16INK4a and SASP. Notably, this effect was greatly attenuated in the presence of the anti-tumor necrosis factor (anti-TNF) agent, but not the anti-type I interferon (IFN) agent or anti-IL-6 agent. Furthermore, similar results were obtained by depleting TNF-α in SARS-CoV-2-infected cells with short interfering RNA before collecting the culture supernatant.
These results suggest that TNF-α plays an important role, at least partially, in the induction of the senescence-like phenotype of HDFs by SARS-CoV-2.
The study also found that TNF-α causes phosphorylation and activation of p38 and activated p38 induces p16INK4a expression and SASP in a DNA damage response-independent manner, strongly suggest that SARS-CoV-2 provokes a senescence-like phenotype at least partly through TNF-α/p38 pathway activation.
SARS-CoV-2 provoked a senescence-like phenotype in hBOs and patients with post-acute COVID-19.
a, hBOs infected with SARS-CoV-2 at m.o.i. 0.1 were cultured for 6 days and then subjected to immunofluorescence staining for SARS-CoV-2 N protein (CoV2-NP (red), p16INK4a (green) and DAPI (blue)). Representative images of days 1, 3 and 6 are shown. The histograms indicate the percentages of cells expressing COV2-NP (top) or p16INK4a (bottom). b,c, hBOs infected with SARS-CoV-2 at m.o.i. 0.1 were treated with a TNF-α inhibitor (TNFi) (500 μg ml−1 etanercept) from day 3 after infection. Immunofluorescence staining for p16INK4a (green) and DAPI (blue) (b) or phospho-p38 (red) and DAPI [blue] (c) is shown. The histograms indicate the intensity of p16INK4a signals or phospho-p38 signals normalized by DAPI count. d, Single-cell RNA transcriptomic analysis of lung tissue from patients with severe COVID-19 (ref. 28). Epithelial cells population of uniform manifold approximation and projection (UMAP) plots were divided into healthy controls (25 and 52 years old) and patients with COVID-19 (28, 54 and 57 years old), and the distribution of cells expressing senescent cell marker (CDKN2A) and SASP factor genes is shown. Arrowheads indicate a basal cell population. For all graphs, error bars indicate mean ± s.d. of biological triplicate measurements. Scale bars, 50 μm (a–c). Statistical significance was determined by two-way ANOVA followed by Sidak’s multiple comparison test (a) or one-way ANOVA followed by Dunnett’s multiple comparison test (b,c). a.u., arbitrary units.
A substantial proportion of SARS-CoV-2-infected hBOs expressed activated caspase-3, indicating that SARS-CoV-2-infected cells are more susceptible to death, as seen in ACE2-HDFs. This may explain why most of the senescent cells that emerge after SARS-CoV-2 becomes undetectable are SARS-CoV-2 uninfected.
Interestingly, individuals who have developed post-acute sequelae of SARS-CoV-2 infection reportedly have higher levels of cytokines such as TNF-α in the early stages of recovery, consistent with the experimental data. https://pubmed.ncbi.nlm.nih.gov/34677601/
These results indicate that, at least in certain biological contexts, SARS-CoV-2 provokes paracrine senescence via cytokines secreted by infected cells. It should also be noted that the more virulent SARS-CoV-2 variant (B.1.1.7), first observed in the United Kingdom, also induced a senescence-like phenotype in ACE2-HDFs with slightly faster kinetics than the original SARS-CoV-2, suggesting that the induction of the senescence-like phenotype is common in normal human cells infected with SARS-CoV-2.
Considering that SASP sustains inflammatory responses and that the persistence of inflammation is likely to be one of the causes of post-acute COVID-19 syndrome, the study team next explored the possibility that the senescence-like phenotype contributed to the sustained inflammatory response in post-acute COVID-19 syndrome.
The team reanalyzed data from a recently published single-cell transcriptomic analysis of lung tissue from patients with severe COVID-19. In that study, SARS-CoV-2 was no longer detected in the patients with prolonged COVID-19 disease, but the pathology showed extensive evidence of damage and fibrosis resembling end-stage pulmonary fibrosis. https://pubmed.ncbi.nlm.nih.gov/33257409/
The expression levels of p16INK4a (CDKN2A) and several SASP factor genes, such as IL32, CXCL14 and MMP1029,30,31, were increased in the lung cells of patients with severe COVID-19 as compared with those of healthy individuals, especially in basal cells and, to a lesser extent, in ciliated cells. This is consistent with the observation that a significant proportion of basal cells and a slightly smaller proportion of ciliated cells express p16INK4a in SARS-CoV-2-infected hBOs after SARS-CoV-2 is no longer detectable. Thus, it is tempting to speculate that the senescence-like phenotype induced by SARS-CoV-2 infection may be involved in the development of post-acute COVID-19 syndrome.
To further explore this possibility, the study team next examined whether SARS-CoV-2 infection induced p16INK4a expression in vivo using Syrian hamsters. Unlike mice, hamsters can be infected with SARS-CoV-2, and the expression level of p16INK4a was significantly increased from day 7 after infection in the lung, when SARS-CoV-2 became hardly detectable in hamsters. This is consistent with the observation that a substantial proportion of SARS-CoV-2-infected cells have positive TdT-mediated dUTP nick end labelling (TUNEL) staining, a marker of apoptosis, at day 5 after infection. Moreover, the expression levels of p16INK4a and SASP factors remained high even at days 14 and 45, when SARS-CoV-2 was almost undetectable, indicating that the senescence-associated inflammatory response may persist to some extent even after SARS-CoV-2 decreased below the detection limit in Syrian hamsters.
However, this phenomenon was not observed when Syrian hamsters were infected with the influenza A (H1N1) virus, a respiratory virus that does not cause long-term symptoms after recovery, suggesting that this phenomenon is likely to be unique to SARS-CoV-2 infection.
In order to further prove that SARS-CoV-2 provokes and sustains a senescence-associated inflammatory response, the study team attempted to eliminate senescent cells in Syrian hamsters by using senolytic drugs that can selectively kill senescent cells. Among the reported senolytic drugs the team tested, ABT-263 and ARV-825 specifically reduced the number of senescent human fibroblasts at the reported optimal concentrations. https://pubmed.ncbi.nlm.nih.gov/33328614/
In hamster fibroblasts however, only ABT-263 showed slight senolytic activity. Moreover, the administration of ABT-263 failed to decrease the expression levels of p16INK4a and SASP factors in Syrian hamsters infected with SARS-CoV-2, suggesting that some differences may exist in the pathways that regulate the survival of senescent cells between humans and hamsters.
To circumvent this problem, the study team used mice that are known to be sensitive to senolytic drugs. To this end, the team used a mouse-adapted strain of SARS-CoV-2 (MA10), which shows dose- and age-related increases in pathogenesis in standard laboratory mice and recapitulates key features of COVID-19 in humans. Similar to the Syrian hamsters, signs of cellular senescence, including expression of p16INK4a and p19ARF (critical inducers of cellular senescence encoded by the CDKN2A gene) and SASP factor genes, were increased at day 14 after infection in the lung, when SARS-CoV-2 became hardly detectable in BALB/c mice. Intriguingly, the administration of ABT-263 substantially decreased the expression levels of these senescence-associated genes in BALB/c mice infected with MA10, further supporting the idea that SARS-CoV-2 infection provokes and sustains senescence-associated inflammatory phenotypes even after SARS-CoV-2 is no longer detectable in vivo.
A previous study reported that the elimination of senescent cells, termed senolysis, reduced mortality in aged mice infected with mouse hepatitis virus (MHV), a virus in the same family as SARS-CoV-1 and 2. https://pubmed.ncbi.nlm.nih.gov/34103349/
However, the mechanisms involved in viral entry into cells and the pathogenesis are very different between MHV and SARS-CoV-2, and thus, the extent to which the data obtained from MHV can be applied to SARS-CoV-2 remains unclear.
Another recent study reported that SARS-CoV-2 rapidly causes senescent phenotypes in infected cells within 5 days of infection in both cultured human cells ectopically expressing ACE2 and Syrian hamsters. https://pubmed.ncbi.nlm.nih.gov/34517409/
They were also able to eliminate senescent cells by administering senolytic drugs such as ABT-263 or dasatinib plus quercetin (D + Q) to SARS-CoV-2-infected hamsters using a protocol similar to the present study.
These studies showed that the administration of senolytic drugs to SARS-CoV-2-infected rodents reduced the levels of inflammatory factors classified as SASP, implying that senolysis may be effective in alleviating post-acute COVID-19 syndrome.
However, SASP has both harmful and beneficial effects and it has recently been reported that the removal of accumulated senescent cells in mice resulted in severe liver dysfunction. https://www.sciencedirect.com/science/article/pii/S1550413120302412
In this regard, it is interesting to note that hamsters infected with SARS-CoV-2 showed resistance to superinfection with influenza virus A H1N1. Thus, it is also tempting to speculate that SARS-CoV-2-induced senescent cells may have some beneficial effects, depending on the biological context. Accordingly, a more rigorous analysis is needed to determine whether senolysis can serve as a preventive measure against post-acute COVID-19 syndrome. Nevertheless, the study findings provide valuable new insights into the mechanism of post-acute COVID-19 development and suggest new possibilities for its control.
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