Shorter telomere lengths in patients with severe COVID-19 disease

The incidence of severe manifestations of COVID-19 increases with age with older patients showing the highest mortality, suggesting that molecular pathways underlying aging contribute to the severity of COVID-19. One mechanism of aging is the progressive shortening of telomeres, which are protective structures at chromosome ends. Critically short telomeres impair the regenerative capacity of tissues and trigger loss of tissue homeostasis and disease. The SARS-CoV-2 virus infects many different cell types, forcing cell turn-over and regeneration to maintain tissue homeostasis. We hypothesize that presence of short telomeres in older patients limits the tissue response to SARS-CoV-2 infection. We measure telomere length in peripheral blood lymphocytes COVID-19 patients with ages between 29 and 85 years-old. We find that shorter telomeres are associated to increased severity of the disease. Individuals within the lower percentiles of telomere length and higher percentiles of short telomeres have higher risk of developing severe COVID-19 pathologies.


INTRODUCTION
The current COVID-19 pandemic (https://www.who.int/) is produced by the SARS-CoV-2 virus, a novel zoonotic Coronavirus of the betacoronavirus genus that most likely crossed species from bats to humans leading to a pneumonia outbreak initially reported in Wuhan, China and now affecting the majority of countries. SARS-CoV-2 causes from mild flu-like symptoms in approximately 80% of the cases to a severe lung and multi-organic failure which can result in death of a significant percentage of patients. Pathologies associated with SARS-CoV-2 include severe lung failure, diarrhea, heart infarct, and brain pathologies among others [1][2][3]. This wide viral tropism is mediated by expression of the Angiotensin-converting enzyme 2 (ACE2), which acts as the receptor protein for the virus to enter the host cells. In particular, the SARS-CoV-2 spike protein directly binds the ACE2 human protein [4][5][6][7]. The human ACE protein is expressed in alveolar type II (ATII) cells in the lung [8], as well as in the kidney, the heart and the gut [9][10][11][12][13][14]. This expression pattern of the ACE protein explains that a preferential site for SARS-CoV-2 infection is the lung [4,15,16], although the virus can also infect kidney, intestine, and heart cells causing severe pathologies in all these tissues [1-3, 11, 17, 18]. In this regard, it caught our attention that a common outcome of SARS-CoV-2 infection seems to be induction of fibrosis-like phenotypes in the lung and kidney, suggesting that the viral infection maybe exhausting the regenerative potential of tissues [11,[16][17][18].
In contrast to influenza infection, that causes a high mortality in infants [19][20][21][22][23][24], SARS-CoV-2 infection causes low mortality in infants or children but results in a progressively increased mortality with increasing age reaching up to 15% mortality in individuals that are ≥80 years old (see https://covid19.isciii.es/ for mortality data in Spain). These findings suggest that molecular mechanisms at the origin of organismal aging maybe

Shorter telomere lengths in patients with severe COVID-19 disease
Raul Sanchez-Vazquez 1,* , Ana Guío-Carrión 1,* , Antonio Zapatero-Gaviria 2 , Paula Martínez 1 , Maria A. Blasco 1 AGING influencing the outcome of SARS-CoV-2 infection by increasing lethality. One of such molecular events underlying aging is the progressive shortening of telomeres throughout life, which can cause exhaustion of the proliferative potential of stem cells and immune cells among others [25][26][27].
Telomeres are specialized structures at the chromosome ends, which are essential for chromosome-end protection and genomic stability [28]. Vertebrate telomeres consist of tandem repeats of the TTAGGG DNA sequence bound by a six-protein complex known as shelterin, which prevents chromosome end-to-end fusions and telomere fragility [29,30]. As cells divide and DNA has to be replicated, telomeres become progressively shorter owing to the so-called "end replication problem" [31,32]. Thus, telomere shortening occurs associated with increasing age in humans [33], mice [34] and other species, and the rate of telomere shortening has been shown to correlate with species lifespan [35]. When telomeres become critically short this results in loss of telomere protection, leading to activation of a persistent DNA damage response [36] and loss of cellular viability by induction of apoptosis and/or senescence [30].
Telomerase is a reverse transcriptase that is able to elongate telomeres de novo by adding TTAGGG repeats to chromosome ends [37]. Telomerase is active in embryonic stem cells, thereby ensuring sufficiently long telomeres with generations in a given species. After birth, however, telomerase expression is silenced in the majority of cell types causing telomeres to shorten with age.
We have shown by using telomerase-deficient mice with critically short telomeres, that short telomeres are sufficient to impair the ability of stem cells to regenerate different tissues, including skin, brain and bone marrow [38][39][40][41]. In humans, mutations in telomerase or telomere-binding proteins can also lead to very short telomeres and appearance of pathologies characterized by loss of the regenerative capacity of tissues and presence of fibrosis in lungs, liver or kidney, as well as by intestinal atrophy and bone marrow aplasia [42].
In particular, we previously demonstrated that short or dysfunctional telomeres are at the origin of pulmonary fibrosis in mouse models of the disease [43]. In particular, induction of telomere dysfunction specifically in alveolar type II (ATII) cells by deletion of an essential telomere protective protein in these cells, TRF1, is sufficient to induce progressive and lethal pulmonary fibrosis phenotypes in mice, which are concomitant with induction of telomeric DNA damage, cell death and senescence [43]. These findings demonstrate that dysfunctional telomeres in lungs ATII cells lead to loss of viability of these cells and induction of fibrosis. Also in support of this notion, we have demonstrated that therapies aimed to elongate telomeres, such as a telomerase gene therapy using adeno-associated vectors (AAV9-TERT) can stop the progression of pulmonary fibrosis associated to short telomeres in mouse models of the disease by increasing telomere length in ATII cells, as well as their proliferative potential [44], thus demonstrating the importance of sufficiently long telomeres to allow tissue regeneration.
Importantly, as SARS-CoV-2 infects different cell types in humans, including ATII cells in the lungs, it is plausible that viral infection could damage these different cell types forcing an increased turn-over of different regenerative cell types. While in young individuals with sufficiently long telomeres, regenerative cell types, such as lung ATII cells could undergo these extra cell divisions and contribute to tissue healing, older individuals with shorter telomeres may fail to allow cell proliferation and regeneration, thus leading to tissue failure. Thus, here we set to assess whether telomere length in COVID-19 patients correlated with development of more severe COVID-19 pathologies.

Pathologies in COVID-19 patient cohort
In order to assess the potential impact of telomere length on pathologies associated to COVID-19 disease, we obtained both DNA and mononuclear cells from peripheral blood samples from patients hospitalized at the IFEMA field hospital in Madrid, which was constructed to treat COVID-19 patients. A total of 61 female and 28 male patients of ages ranging from 29 to 85 years old were included in the study ( Table 1). The patient cohort had different severity of pathologies and received the treatments indicated in Table 1. None of the patients included in this study died as a consequence of the COVID-19 disease.
In order to correlate patient severity with telomere length, we first grouped the patients according the a severity score ranging from 1 to 4, with severity score 1 in the case of patients with low fever and cough but without any radiological features of pneumonia to patients with severity score of 4 in the cases of patients with features of Acute Respiratory Distress Syndrome (ARDS) requiring mechanical ventilation along with presence of multiorgan dysfunction failure, metabolic acidosis and coagulation dysfunction (Materials and Methods).

Determination of telomere length in COVID-19 patients
In order to determine telomere length in our patient cohort, peripheral blood was extracted from the arm from the different patients and used to measure telomere length by three independent techniques (Materials and Methods).
First, we determined telomere length in DNA extracted from peripheral blood by using both the Southern blotting-based Telomere Restriction Analysis (TRF; see [38]) and the quantitative-PCR (qPCR) telomere length analyses [45]. In addition to these two technologies based on DNA samples, we also measured telomere length on fresh peripheral blood mononuclear cells by using the AGING more precise high-throughput quantitative fluorescence in situ hybridization (HT Q-FISH) previously described by us [34,35,46], which allows determination of individual telomere fluorescence signals using tens of thousands of cells from a single patient. The fact that HT Q-FISH can determine individual telomere fluorescence spots in interphasic nuclei, each spot usually formed by several clustered telomeres, allows to the determine the abundance of very short telomeres.
We observed a very significant correlation between the telomere length measurements obtained by the three different techniques ( Figure 1A), thus indicating the robustness of the data on telomere length obtained in our patient cohort. Given the good correlation of the telomere length data obtained with the three technologies, we decided to perform the rest of the analysis with the telomere length data obtained by HT Q-FISH, as it measures telomeres in a single cell manner and it also allows to measure individual telomere spots within single nuclei.
The rate of telomere shortening in the patient cohort was of 77 bp/year ( Figure 1B). This rate of telomere shortening is in the range previously published by us and others [45][46][47][48]. In agreement with telomere shortening with increasing patient age, we also observed an increase in the abundance of short telomeres AGING (ie, telomere fluorescence spots corresponding to less than 3 kb of telomere length) which increased at a rate of 8.8 % per year ( Figure 1B).
When we analyzed the data segregated by gender, linear regression of telomere length data in COVID-19 female patients showed that their telomeres were consistently longer than those of male patient at all age ranges, as well as they showed a lower percentage of short telomeres compared to male patients (Figure 2A-2D), also in agreement with previous findings [46]. Again, when segregated by gender, the rates of telomere shortening were in a range of 70-80 bp/year (Figure 2A). Similarly, the increase in the percentage of short telomeres with age was also similar in both genders (Figure 2A, 2B).
The fact that COVID-19 female patients had longer telomeres than men patients at different age ranges is in line with the fact that female COVID-19 patients show a lower mortality than males (see COVID-19 Sex-Disaggregated Data Tracker available at: http://globalhealth5050.org/covid19).

Age and telomere length correlate with COVID-19 severity
In order to assess whether short telomeres correlated with the severity of COVID-19 disease, we used a Pearson correlation analysis between the mean telomere length or the percentage of short telomeres (<3 kb) as determined by the HT Q-FISH technique, and either age or the severity score of the different COVID-19 patients ranging from 1 (less severe) to 4 (more severe) (see Materials and Methods).
As expected, we observed a significant inverse correlation between mean telomere length (TL) and age of the COVID-19 patients (r=-0.3985; p=0.0001; Figure  3A). We also observed a significant direct correlation between percentage of short telomeres (ie, telomeres < 3Kb) and patient age (r=0.285; p=0.0067; Figure 3B). Thus, these findings confirm a significantly higher incidence of short telomeres with increasing age in the COVID-19 patients. We also observed an inverse correlation between mean telomere length (TL) and the severity score of the COVID-19 patients when using HT Q-FISH (r=-0.1752; p=0.1026; Figure 3A) and a direct correlation between the percentage of short telomeres (ie, telomeres < 3Kb) and the severity score (r=0.1454; p=0.1766; Figure 3B), although these correlations did not reach statistical significance. To further analyze this, we performed similar analysis with the telomere length data obtained by TRF and by PCR ( Figure 3C, 3D). Again, we confirmed a significant inverse correlation between mean telomere length (TL) and age of the COVID-19 patients by TRF (r=-0.4675; p<0.0001; Figure 3C) as well as by PCR (r=-0.405; p=0.0001; Figure 3D) techniques. Importantly, by these two DNAbased techniques, the correlation between telomere length and COVID-19 severity reached statistical significance (TRF: r=-0.3119, p=0.004; Figure 3C; PCR: r= -0.2308, p=0.036; Figure 3D. AGING Finally, we also observed a significant direct correlation between age of the COVID-19 patients and the severity score of the disease (r=0.2299; p=0.0312; Figure 4A, 4B). Furthermore, we observed an inverse correlation between age and mean telomere length (TL) and a direct correlation between age and the percentage of short telomeres when using HT Q-FISH ( Figure 4A, 4B).
Together, these findings suggest significant correlations of age as well as telomere length with COVID-19 severity.

Higher severity of COVID-19 disease in patients in the lower percentiles of telomere length
The findings suggest that COVID-19 patients with shorter telomere length may have a higher risk of more severe pathologies. To further test this, we divided the patients in quartiles according to either their mean telomere length or their percentage of short telomeres using the telomere signal fluorescence data obtained by HT Q-FISH. We observed that patients in the lower quartile of mean telomere length (<25%) had a higher severity score (p=0.06; Figure 5A). Similarly, the  AGING patients in the higher quartile of percentage of short telomeres had significantly higher severity scores of the disease (p=0.049; Figure 5B).

Different rates of telomere shortening in patients with different severity scores
As patients in the lower quartile of telomere length have a significantly higher risk of severe COVID-19 pathologies, we set to investigate whether the rates of telomere shortening in higher severity score patients were significantly higher than in the lower severity score patients. To this end, we pooled together the patients in "mild-moderate" and "severe-acute" severity groups. We found that patients with a "severe-acute" diagnosis showed a significantly faster rate of telomere shortening compared to the "mild-moderate" diagnosis as determined by HT Q-FISH (p=0.024; Figure 6A). Of note, patients with "severe-acute" COVID-19 disease have shorter telomeres along all age groups compared to patients with "mild-moderate" COVID-19 disease. Similarly, we found an increased rate of accumulation AGING of short telomeres in patients with a "severe-acute" diagnosis compared to patients with a "mild-moderate" severity score (p=0.08; Figure 6B).
In order to address whether this association between Covid-19 severity and short telomeres was independent of the age, we grouped the patients in different age groups (below 40 years of age; between 40-49; 50-59; 60-69 and over 70 years of age) and compared the fluorescence of individual telomere foci in patients showing either "mild-moderate" or "severe-acute" COVID-19 severity. We found that for age groups above 60 years of age, telomeres were shorter in the groups with "severe-acute" severity compared to "mildmoderate" severity ( Figure 6C).

DISCUSSION
Data from COVID-19 around the world shows that patients of older age groups show a higher severity of the disease and a higher mortality. Male patients also show a higher mortality than female patients (see COVID-19 Sex-Disaggregated Data Tracker available at: http://globalhealth5050.org/covid19). This suggest that molecular mechanisms of aging maybe aggravating the pathological consequences of infection by the SARS-CoV-2 virus. Telomere shortening and accumulation of DNA damage steaming from short telomeres has been proposed as one of the primary hallmarks of aging [27]. In particular, short telomeres are known to result in chromosomal instability and loss of cell viability by inducing replicative senescence and/or apoptosis [26]. Importantly, by using mouse models that lack telomerase activity, we and others have shown that short telomeres impair the regenerative capacity of tissues leading to loss of tissue homeostasis and degenerative diseases [40]. Similarly, humans with critically short telomeres owing to mutations in telomerase also show an impaired regeneration capacity and are at a higher risk of developing degenerative diseases in both low proliferative (lung, kidney) and high proliferative tissues (bone marrow, skin) [42].
Given that the SARS-CoV-2 virus infects different cell types in the organisms, including regenerative cell types such as alveolar type II (ATII) cells in the lungs [8][9][10][11][12][13]49], here we hypothesize that individuals with short telomeres would have an impaired regenerative response upon SARS-CoV-2 infection, thus leading to more severe and progressive pathologies, such as fibrosis-like pathologies in the lungs, kidney or liver.
To address this, we have measured telomere length in a total of 89 patients diagnosed with COVID-19 ranging from mild to acute disease. As expected we found that telomere length decreases with age, with women having longer telomeres than men at different age groups, which could explain why COVID-19 disease is more severe in males than females. Interestingly, we also found that those patients that have more severe COVID-19 pathologies have shorter telomeres at different ages compared to the patients with milder disease. Indeed, patients which are in the lower percentile of telomere length also have significantly higher severity scores.
These findings demonstrate that molecular hallmarks of aging, such as presence of short telomeres can influence the severity of COVID-19 pathologies. As short telomeres can be elongated by telomerase, and telomerase activation strategies have been shown by us to delay aging and age related pathologies [50], as well as to have therapeutic effects in diseases associated to short telomeres, such as pulmonary fibrosis [44], it is tempting to speculate that such telomerase activation therapies could ameliorate some of the tissue pathologies remaining in COVID-19 patients, such as fibrosis-like pathologies in the lungs [51] after overcoming the viral infection.

Patients
In this study participated a total of 89 patients (61 female and 28 male patients of ages ranging from 29 to 85 years old) from the IFEMA field hospital installed due to the emergency situation in Madrid, Spain. All these samples were donated to CNIO BioBank which allows their use for biomedical analyses under the existing law requirements in Spain.

Blood samples
A total of 8 ml of blood were collected from the arm of each patient in heparin tubes and 4 ml in EDTA tubes and shipped within less than 24h to the DNA National Bank at Salamanca University, where they were immediately processed at a biosafety level (BSL) 3 (BSL-3) biocontainment level. Peripheral blood mononuclear cells (PBMCs) were purified by Ficoll gradient and frozen in 90% FBS (v/v) supplemented with 10% (v/v) DMSO into a number of aliquots ranging from 1 to 3, according to cell number. PBMCs were stored in vapor phase-nitrogen.
Genomic DNA was extracted directly from blood samples using the Real Blood DNA Kit and stored longterm in TE at -20° C.

Q-PCR Assay to measure average telomere length
Telomere length was measured in genomic DNA isolated from blood samples. We used a modified AGING monochrome multiplex quantitative polymerase chain reaction (PCR) method already described [45]. Finally, T/S ratios were converted into kb by external calibration with the K562 (6.5 kb), CCRF-CEM (7.5 kb), Jurkat (11.5 kb) and HeLa1211 (24 kb) cell lines.

Terminal restriction fragment analysis
Mean telomere length by Telomere Restriction Fragment (TRF) was determined using the method already described [38]. Briefly, genomic DNA was digested by MboI and separated by gel electrophoresis in 0.5X TBE maintained at 14° C, using a CHEF DR-II pulsed-field apparatus (BioRad) for 14 h at 5 V/cm at a constant pulse time of 0.5 s. The gel was transferred to a nylon membrane (Hybond-XL, GE Healthcare) and probed with a 32 P-labeled telomeric probe (TTAGGG)n (a gift from T. de Lange). Mean TRF lengths were determined using an ImageQuant TL.
PBMCs were thawed in complete RPMI 1640 growth media supplemented with 10% FBS (v/v) and seeded at a concentration of 100000 cells/ well in triplicate wells per sample. Cells were left to adhere to the plate for 1 h at 37C in incubator with 95% humidity, 5% CO 2 . Plates were then removed from incubator and then fixed at room temperature (RT) by slowly filling up the wells with 200 ul methanol/acetic acid (3/1, vol/vol) and incubated for 10 to 15 min. The solution was removed, and this was repeated 2 more times, leaving the last fixative volume up for a total of 1 h fixation. Plates were then moved to -20 until processed for highthroughput quantitative FISH (HT Q-FISH).
We performed HT Q-FISH as described before [46]. Briefly, the plates were removed from -20, the fixative solution removed, and the plates were dried on a hot plate at 37° C overnight, followed by rehydration with 200 μL of PBS.

High-throughput microscopy
Quantitative image acquisition was performed on an Opera High Content Screening System (PerkinElmer) 40×/0.9 N.A. water-immersion objective. UV and 561 nm excitation wavelengths were used to detect DAPI and Cy3 telomeric signals, respectively and 60 AGING independent images were captured at different positions of each well. Images were analyzed with Acapella Image analysis software (PerkinElmer). Data were analyzed with SPSS (IBM) and Excel (Microsoft).

Criteria for the diagnosis of COVID-19
Depending on the clinical features of COVID-19, patients are generally divided as mild, moderate, severe and acute.
1. Mild COVID-19: low-grade fever, cough, malaise, rhinorrhea, sore throat with or without hemoptysis, nausea, vomiting, diarrhea, but without any radiological features of pneumonia and absence of mental changes.
2. Moderate COVID-19: fever, respiratory symptoms including dry cough and shortness of breath that may emerge along with the radiological features.
4. Acute COVID-19: usually develops after 7 days in patients with mild/moderate/severe COVID-19 with features of Acute respiratory distress syndrome (ARDS) requiring mechanical ventilation along with presence of multiorgan dysfunction failure, metabolic acidosis and coagulation dysfunction.

Data availability statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.

AUTHOR CONTRIBUTIONS
MAB had the original idea and secured funding. MAB and PM supervised research and wrote the paper. RSV, AGC and PM analyzed the data and performed experiments. AZG provided blood samples and clinical information.

CONFLICTS OF INTEREST
MAB is founder and holds shares of Life Length SL, a Biotech company that measures telomere length for biomedical uses.