SARS-CoV-2 which was first reported in Wuhan, China in Dec, 2019 has so far infected around 6.3% of the total world population with around 1.23% mortality rate as on 18th April, 2022 (https://covid19.who.int/). Furthermore, emerging variants of concern (VoC) from the ancestral SARS-CoV-2 strain has led to sporadic emergence of COVID-19 related morbidity and mortality world-wide. While majority of clinical cases report pulmonary pathologies associated with pneumonia and acute respiratory distress syndrome (ARDS), growing number of evidences suggest cardiovascular, gastrointestinal, renal, neurological, endocrinological manifestations of SARS-CoV-2 infection 1–4. Clinical cases of COVID-19 are also characterized by lymphopenia which is defined by T cell lymphodepletion 5. Previous studies have shown that lymphopenia induced by pathogenic infection could be related to thymic atrophy 6–8.
Thymic atrophy is described as shrinking of the thymus and is related to aging or disease condition and leads to immuno-senescence (loss of T cell repertoire) and inflammaging (self-reactive T cell) (6–11). Moreover, lymphopenia due to pathogenic infection could occur due to direct cellular killing induced by the pathogen or due to changes in the T cell developmental pathway in the thymus. Several mechanisms have been suggested for pathogen induced thymic atrophy which is governed by virus entry and infection. For example thymic atrophy by influenza A virus infection is mediated by IFN-γ cytokine or NK cells cytotoxicity and results in impaired negative selection 7,12. For highly pathogenic avian influenza A virus (HPAIV), thymic atrophy occurs by impaired negative selection for T cells and involvement of glucocorticoids 13,14. For chronic infection caused by hepatitis C virus, HIV infection, etc destruction of thymus usually occurs through CD8 T cells and results in impaired negative selection 15,16. Previous clinical post-mortem studies have suggested changes in the thymic structure which may be lead to changes in the thymic output post COVID-19 6,11,17. However, direct evidence of thymic atrophy and the mechanism involved in it have not been reported mostly due to lack of any animal model study. Golden Syrian hamster and humanized ACE2-transgenic mice are two routinely used models for SARS-CoV-2 infection which mimics upper and lower respiratory tract pathology. Interestingly, several reports have pointed that COVID-19 leads to extra-pulmonary pathologies ranging from gastrointestinal, cardiovascular, neurological, etc in both hamsters and hACE2 mice 18,19. While hACE2 mice infected with SARS-CoV-2 develops acute COVID-19 and ultimately succumbs to infection, hamster represents a mild to moderate model for COVID-19 and have been shown to start recovering post day 6–7 of infection 19–22.
In the current study, we utilized golden Syrian hamster and hACE2 mice model for SARS-CoV-2 infection to investigate the cause of lymphopenia as seen in COVID-19 patients. We observed severe thymic atrophy in hACE2 mice infected with SARS-CoV-2. Immune profile of the thymocytes suggested skewed frequencies of DN and DP population and impaired T cell developmental pathway represented by changes in CD44 vs CD25 frequencies. The loss of T cells was found to be due to apoptosis and was mediated by IFN-γ as neutralization of IFN-γ abrogated thymic atrophy. Rescue of thymic atrophy was also observed with anti-viral drug remdesivir (RDV) which reduces viral load. Interestingly, infected hamsters do not develop severe thymic atrophy. Severe thymic atrophy was also absent from Omicron infection in hACE-Tg mice, however Delta variant infection seems to worsen thymic atrophy. Finally, we showed that P4A2 antibody, which has been recently reported as broadly SARS-CoV-2 neutralizing antibody effect against Delta variant, treated mice were rescued from thymic atrophy and showed no impaired T cell development. Together, we provide the first direct evidence of thymic atrophy induced by SARS-CoV-2 infection and show that the mechanism of thymic atrophy is through IFN-γ. Further, we show this impaired T cell development as a contributing factor for dysregulated peripheral T cell profile. Our results provide mechanism for the induction of lymphopenia and may explain waning T cell immunity post COVID-19 due to loss of TCR repertoire.
SARS-CoV-2 infection in hACE-2 mice causes lymphopenia. hACE2-transgenic (Tg) mice expressing humanized ACE2 receptor driven by epithelial cell cytokeratin-18 (K18) promoter had been shown as a model for acute model of SARS-CoV-2 infection following intranasal challenge with the live virus associated with rapid loss in body mass leading to mortality by day 6–8 post infection 22,23. Consistent with the published reports, we found that hACE2-Tg mice infected with SARS-CoV-2 develops acute COVID-19 pathology characterized by sharp decline in body mass (Fig. 1A & 1B), 80% mortality by day 7 and profound presence of lung inflammation and injury as observed by assessing excised lung and hematoxylin and eosin (H&E) stained lung sections by trained pathologist (Fig. 1C- 1G). In line with this, there was profound localization of N protein in the infected lungs sections as determined by immunohistochemistry (IHC) corresponding to high N gene copy number (by qPCR) (Fig. 1H & 1I). Characteristic of the active infection, mRNA expression of anti-viral genes which are activated post sensing of viral RNA by toll-like receptors (TLRs) or rig-like receptor (RLRs) and activation of stimulator of IFN genes (STING) such as 2’-5’-oligoadenylate synthetase (OAS)-2 and OAS-3, latent RNase (RNaseL), IFN-induced transmembrane (IFITM) protein, adenosine deaminase acting of RNA-1 (ADAR-1) 24 were all significantly increased in infected lung (Fig. 1J). Interestingly, the spleen and lymph node size of the infected mice at 6 dpi (peak of acute infection) showed profound involution with decrease in mass and live cell population (Fig. 1K-1O). Interestingly, we observed significantly reduced frequency as well as cell count of CD45.2+, CD3+, CD4 + and CD8 + lymphocytes in the splenocytes of the infected hACE2-Tg mice at 6 dpi as compared to uninfected mice (UI) (Fig. 1P-1Q). Curiously, there was a 3–4 folds upregulation in the frequency and count of DP cells in the splenocytes, which has been shown to be related to escape of DP cells from the thymus (Fig. 1P-1Q). Together, we found infected hACE2 mice manifests both pulmonary pathology as well as lymphopenia suggestive of dysregulated T cell development in the thymus.
SARS-CoV-2 infection induces thymic atrophy and leads to dysregulated T cell development. In line with the previously published reports, our clinical data showed a significant depletion of peripheral CD3 + and CD8 + T cells of COVID-19 patients as compared to the healthy control while a decreasing trend was observed for CD4 + T cells (Fig. 2A). Changes in the thymus post-acute pathogenic infection have been previously shown to cause lymphopenia as thymus is the site of T cell development and maturation 7,8. Emerging studies now show that lymphopenia is strongly correlated to morbidity and mortality and is associated with more than 50% of the adults and 10% of children infected with SARS-CoV-2 25,26. Lymphopenia is characterized by a substantial decrease in lymphocyte count and reduction in the frequency of peripheral CD4 + T helper and CD8 + T cytotoxic cells 26. In line with this, the thymus of the infected hACE2-Tg mice at 6 dpi showed profound thymic involution (Fig. 2B & 2C) with a 7–8 folds decrease in mass and number of live cells (Fig. 2D). Next in order to understand the influence of SARS-CoV-2 infection on the thymic developmental pathway we looked at the virus entry and localization into the thymus. Our immunofluorescence data for SARS-CoV-2 specific anti-N protein localization indicated prominent presence of N protein in the thymocytes of the infected hACE2-Tg (Fig. 2E). This was a surprising finding since no study has so far reported the expression of humanized ACE2 receptor in the thymus of hACE2-Tg mice. Since cellular injury of pulmonary and extra-pulmonary organs is characteristic pathological manifestation of COVID-19 and is often ascribed to the cellular entry and presence of virus, we evaluated the viral load in different compartments of thymocytes i.e. CD45+, CD45-, CD3+, CD3-, DP, DN cells in the sorted population (Fig. S1A). Our data shows presence of 2-2.5 log10 N gene copy number/ mg mass in all the compartments of thymocytes, suggesting that virus was able to internalize both in CD45- cells (which comprises of thymic epithelial cells) and CD45 + T cells with equal efficiency (Fig. 2F).
Next in order to understand whether virus induced thymic involution could result in dysregulation of T cell development we carried out a detailed immunophenotyping for different developmental stages of T cells in the thymus. We found a substantial increase in the frequency of CD45.2- cells, which was accompanied by expansion of CD45.2 + frequency in the infected thymus, however, there was a significant decrease in the cell count of both CD45.2 + and CD45.2- cells, suggesting that the depletion of CD45.2 + cells were higher than that of CD45.2- cells though there was loss of both CD45.2 + and CD45.2- population due to viral infection (Fig. 2G & S1B). In line with this, we found 2 folds decrease in CD4 + CD8 + double positive (DP) and ~ 5 folds increase in CD4-CD8- double negative (DN) population with ~ 2–3 folds increase in single positive (SP) CD4 + or CD8 + cells indicating a profound dysregulation of T cell developmental pathway in infected thymus (Fig. 2H). Since dysregulated ratio of DP/DN was observed in thymus of infected mice, we made an attempt to understand at which developmental stage of triple negative (TN) population int the thymus is getting affected. Thymic development of TN occurs in 4 distinct stages viz CD44 + CD25- (DN1), CD44 + CD25+ (DN2), CD44-CD25+ (DN3) and CD44-CD25- (DN4) 7,15. We found that thymus of the infected mice showed accumulated frequency for DN1 stage while a decrease in DN2 and DN3 percent frequency was observed for infected samples, suggesting an arrest at the early stage of T cell developmental pathway (Fig. 2I). We found ~ 4–6 folds increase in early and late apoptotic cells CD3 + thymocytes in infected as compared to the uninfected control (Fig. 2J). Similar trends in the induction of early and late apoptosis was found on CD45 + thymocytes, however CD45- thymocytes showed lesser frequency of apoptotic cells suggesting that CD3 + thymocytes are the major depleted population during SARS-CoV-2 infection in mice (Fig S1C & S1D). When time kinetics of infection pathology was studied to understand the early or late phase induction of thymic atrophy, we found that the degree of thymic atrophy was directly proportional to the severity of coronavirus disease-19 (COVID-19) with profound thymic atrophy at day 6 (but not day 3: I3 post challenge) (Fig S1E-S1J).
Several mechanisms have been shown to influence thymic atrophy for pathogenic infection such as NK cells activation, increased levels of glucocorticoids as well as heightened IFN-γ secretion by the thymocytes. Previously studies have shown that pathogenic infections causes thymic atrophy associated with apoptosis of thymocytes which could be mediated by IFN-γ induced tissue injury 7,9,27. Our data shows that SARS-CoV-2 infection results in profound elevation (~ 5 folds) of IFN-γ by CD45 + cells and to lesser extend (~ 2 fold) by CD45- cells (Fig. 2K & 2L). Moreover, SP CD8 + cells were found to be the major contributors of IFN-γ production in the thymus with ~ 6 fold upregulation during infection (Fig. 2M) which could be one of the driving factors for thymic atrophy as has been earlier reported 7. Other pro-inflammatory cytokines such as Granzyme B (GzB), IL-4 and IL-17A was not found to be significantly altered, however interestingly, Perforin-1 (Prf-1) was found to be significantly up-regulated upon infection (Fig. S1K-S1N). Together, we show that SARS-CoV-2 infection in hACE2-Tg mice results in thymic atrophy. Thymic atrophy was characterized by loss of DP population probably due to apoptosis and heightened IFN-γ which could be due to the persistence of virus in the thymus.
Neutralization of IFN-γ alleviates thymic atrophy induced by SARS-CoV-2 infection. IFN-γ, produced by both innate and adaptive arms of the immune system, has been earlier shown to be crucial mediator of inflammation and tissue injury during viral infections. Moreover, IFN-γ is critical in the induction of activation induced cell death (AICD) in T cells 28. Elevated levels of IFN-γ was one of the key component of cytokine release syndrome in human and animal model 29. In fact, our data indicate that a heightened IFN-γ levels in SARS-CoV2-infected hamster and K18-ACE2-Tg mice. In thymus, IFN-γ has been implicated in apoptosis of thymocytes resulting in tissue injury and thymic atrophy 7,10. One previous study have shown that thymic atrophy caused due to influenza A virus could be alleviated by neutralizing IFN-γ 30. Since many aspects of pathogenesis and immunological response of influenza A virus and SARS-CoV-2 bear similarity, we speculated that IFN-γ could be one the key mediators of thymic atrophy for SARS-CoV-2 31, and thus neutralize IFN-γ functions by using anti- IFN-γ neutralizing antibody. Anti-mouse IFN-γ neutralization was done one day prior and one day post SARS-CoV-2 infection in hACE2-Tg mice in order to neutralize the effector functions of IFN-γ induced upon SARS-CoV-2 infection (Fig. 3A). There was marginal protection in the body weight loss (Fig S2A) but no significant decrease in lung viral load in IFN-γ neutralized animals (Fig S2B). However, thymus of the animals receiving anti-IFN-γ antibody neutralization (I + IFN-γ) showed little or no signs of thymic involution in terms of size, mass and number when compared to the thymus from uninfected mice (Fig. 3B-3D). This corroborated with the decreased level of IFN-γ in animals receiving neutralizing antibody (Fig. 3E-3F). In line with the rescue of thymus size, the percentage frequency of CD45.2+/- cells were restored to normal levels as seen in uninfected control thymus (Fig. 3G). Moreover, the percentage frequency of DP and DN cells along with the percentage of DN1-DN4 population was also found to recover to their corresponding uninfected percentage frequency (Fig. 3H-3I). Finally, we showed that neutralization of IFN-γ effectively blunted the apoptosis of thymocytes, thus validating that the important and sufficient mediator of thymic atrophy in SARS-CoV-2 hACE2-Tg mice is IFN-γ (Fig. 3J). Since, SARS-CoV-2 in hACE2-Tg mice resulted in profound IFN-γ response, we asked whether IFN-γ induced tissue injury could also occur in C57BL/6 (WT) mice infected with SARS-CoV-2 without necessitating hACE2 receptor dependent virus entry. Remarkably, SARS-CoV-2 infected WT mice did not show any signs of pulmonary pathology or thymic involution as seen in hACE2 mice (Fig S2C-S2L). Together, our findings show that elevated IFN-γ secretion in SARS-CoV-2 infected hACE2-Tg mice leads to tissue inflammation and injury, thereby causing IFN-γ dependent thymic atrophy.
Use of remdesivir treatment reduces thymic viral load and rescues from thymic atrophy. In the beginning we described that SARS-CoV-2 infection through intranasal route results in virus entry and localization in the thymus. Presence of virus or viral factors have been shown to activate TLR3, TLR7 and TLR8 and RLR which leads to induction of anti-viral genes and cytokines characterized by heightened IFN-γ response 29,32. This would mean that the use of anti-viral drugs which could reduce the viral burden and reduce viral antigens would also effectively reduce the inflammatory IFN-γ production and hence thymic atrophy. To test this, we used remdesivir (RDV, a prototypic anti-viral drug) which has been shown to have significant efficacy against SARS-CoV-2 infection in clinical cases 33,34. Challenged animals receiving remdesivir (I + RDV) treatment significantly reduced lung viral loads and rescued mice from SARS-CoV2 induced pathologies (Fig. 4A, S2M-S2N). In line with this, RDV treatment in SARS-CoV2 infected mice significantly reduced thymic involution with size, mass and number of live thymocytes restored (approx.) to that of the uninfected animals (Fig. 4B-4D). Corresponding to this protection, there was profound reduction in the viral load throughout the thymus as assessed by immuno-fluorescence microscopy and qPCR (Fig. 4E-4F). In addition, animals receiving RDV treatment resulted in alleviation of thymic atrophy immune profile and rescued the normal developmental pathway of thymocytes with restored levels of CD45+/-, DP, DN cells (Fig. 4G-4H). In line with this, the developmental stages of DN population from DN1-DN4 was restored back to their corresponding uninfected profiles (Fig. 4I). Next, we also evaluated the levels of IFN-γ and thymocytes apoptosis as it was important to understand whether RDV mediated rescue of thymic atrophy was operational through clearance of viral load from the thymus or it also resulted in blunted the inflammatory response and related thymic injury. IFN-γ response of CD45 + cells were reduced following RDV treatment and it also resulted in reduced percent frequency of thymocytes entering apoptosis which was expected as viral load and inflammation were earlier shown to be the driving factors for apoptosis of thymocytes (Fig. 4J-4L). Together, we show that reminiscent of IFN-γ, use of RDV (anti-viral drug) could effectively reduce the viral burden could rescue the thymic atrophy induced by SARS-CoV-2 infection.
In contrast to hACE2-Tg mice, golden Syrian hamster model mimics mild to moderate coronavirus disease-19 (COVID-19) as observed in majority of clinical cases 20. In addition, we had earlier shown that the pulmonary pathology of coronavirus disease-19 (COVID-19) following SARS-CoV-2 infection in hamster peaks at 2–4 days post infection (dpi) with highest lung viral load, and starts to decline by 5–6 dpi with comparatively lower viral load and pulmonary pathology on 7 dpi 19. Induction of thymic atrophy was evaluated at early and late phase in infected hamsters i.e. 4 and 7 dpi (Fig S3A). Though there was no significant difference in the size and mass of the excised thymus at 4 and 7 dpi as compared to that of 0 dpi thymus (Fig S3B & S3C), the number of live thymocytes as measured by trypan blue exclusion dye was significantly lower at 4 dpi (peak of infection) as compared to 0 dpi (uninfected) or 7 dpi (recovery phase of infection), suggesting signs of thymic injury at the peak of infection (Fig S3D). Thymus of the 4 dpi hamsters showed 100–500 copy number of viral gene N/ mg of thymus indicating thymic entry of SARS-CoV-2 (Fig S3E). We used anti-mouse CD4 (GK 1.5, cross reactive to hamster CD4) and anti-rat CD8 (cross reactive to hamster CD8) both having cross-reactivity with respective CD markers of hamster to study the development of T cells in thymus. Remarkably, we found 12–15% decrease in the DP cells at 4 dpi and 3–5% decrease in DN at 7 dpi as compared to the 0 dpi thymus DP cells. Similar dysregulated ratios were observed for SP CD4 and CD8 cells at 4 dpi. There was a sharp increase in DN cells at 4 dpi with approx. 2-fold increase as compared to 0 dpi thymus suggestive of dysregulated T cell development (Fig S3F). In line with this, we found 3–4 folds increase in early and late apoptotic cells at 4 dpi thymus accompanied with approx. 3 folds increase in thymic IFN-γ levels as compared to uninfected control (Fig S3G & S3H). Together, our findings show that acute COVID-19, but not moderate COVID-19 leads to severe thymic atrophy.
Delta variant, but not Omicron variant of SARS-CoV-2 induces severe thymic atrophy in hACE2 mice. The original Wuhan strain of SARS-CoV-2 (2019-nCoV) which caused first wave of pandemic in 2020 acquired continuous and considerable mutations in genetic material leading to antigenically different mutations which have been so far characterized into variants of concern (VoC), these VoCs lead to subsequent waves of pandemics across the globe or at different regions of the globe at different time points 35. One such major VoC reported for SARS-CoV-2 was beta variant (B.1.351) that was first detected in South Africa with a large number of mutations in the spike region. The two other notable VoC appeared were Delta variant (B.1.617.2) first detected in India in late 2020 and recently reported omicron variant (B.1.1.529) 36,37. While both B.1.617.2 and B.1.1.529 have been shown to accumulate large amount of mutations in spike and receptor binding domain (RBD) and evade immune response, but only B.1.617.2 but not B.1.1.529 have been shown to cause severe COVID19 in hACE2-Tg animal model 38. Since mutations reported in the VoC have been shown to evade immune response we became interested to investigate the effect of VoC challenge B.1.351, B.1.617.2 and B.1.1.529 on the induction of thymic atrophy (Fig. 5A). Consistent with the previously published reports, mice infected with variants except omicron showed rapid decrease in body mass and with presence of high N-gene copy number (Fig S4A-S4B). Our data demonstrate a most profound thymic atrophy in animals challenged with B.1.617.2 variant which closely mimicked the degree of thymic dysregulation of 2019-nCoV both in terms of involution of size, mass and thymocytes number. Interestingly, animals challenged with B.1.1.529 showed marginal thymic atrophy when compared to the original 2019-nCoV strain in line with the lower lung viral load (Fig. 5B-5D). Consistent with the thymic atrophy we found significant VoC viral load in all the cellular compartment of thymus which was relatively lower for B.1.1.529 variant (Fig. 5E). The profile of developing thymocytes as well as levels of IFN-γ secretion followed the pattern of infectivity with B.1.617.2 showing the highest while B.1.1.529 variant showing lowest dysregulation respectively when compared uninfected control (Fig. 5F-5J & S4C-S4G). Among the other pro-inflammatory cytokines only Prf-1 was found to be significantly elevated across VoC in CD4/CD8 thymocytes sub-sets (Fig S4H-S4K). Together, our data shows that among the 3 VoCs studies ie B.1.351, B.1.617.2 and B.1.1.529, B.1.617.2 (Delta) variants showed most profound thymic atrophy which was higher than that seen in the ancestral strain. While, B.1.1.529 showed milder induction of thymic atrophy.
P4A2 broadly neutralizing monoclonal antibody effectively reduced viral load and mitigated thymic atrophy. We previously demonstrated that the use of anti-viral drug RDV as an effective prophylactic treatment for rescuing thymic atrophy. We further investigated the therapeutic efficacy of P4A2, a murine monoclonal antibody broadly potent against VoC including Omicron by binding to the receptor binding motif (RBM) of SARS-CoV-2 RBD protein 39. Monoclonal antibodies have previously shown to be effective in neutralizing SARS-CoV-2 in clinical settings and have the advantage of being administered as therapeutic dose post challenge even to immuno-compromised individuals 40. We found effective neutralization of VoC and alleviation of thymic involution in mice receiving P4A2 antibody (Fig. 6A-D). Likewise, the profile of the developing thymocytes along with the masking of IFN-γ secretion was also seen with the therapeutic dosing of P4A2 against all the VoC studied (Fig. 6E-6F). Together, we show that the therapeutic dose of P4A2 antibody was effective and sufficient to neutralize VoC challenge and restore the thymic atrophy condition induced by SARS-CoV2 challenge.