SARS-CoV-2 Causes Prolonged Change To Airway Immune Landscape With Evidence Of Cell Death Due To Ongoing Activation Of Cytotoxic T Cells

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A new study led by researcher from National Heart and Lung Institute, Imperial College London-UK has alarmingly found that SARS-CoV-2 causes a prolonged change to the airway immune landscape in those with persistent lung disease, with evidence of cell death and tissue repair linked to ongoing activation of cytotoxic T cells.

The study findings were published in the peer reviewed journal: Immunity. 

https://www.cell.com/immunity/fulltext/S1074-7613(22)00046-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) related coronavirus disease (COVID19) manifests as a spectrum of acute illnesses ranging from mild respiratory symptoms to severe, sometimes fatal, respiratory failure (Docherty et al., 2020). While the acute impact of COVID19 on morbidity and mortality is well- documented, we are still in the infancy of understanding the longer-term consequences.

Morbidity from a range of persistent symptoms, including breathlessness, fatigue and memory impairment have been noted in patients recovering after the acute illness and described under the umbrella term of “long COVID” (Nalbandian et al., 2021; Sigfrid et al., 2021). Complex respiratory complications have been found in up to 18.4% of inpatients (Drake et al., 2021), and persistent breathlessness reported in more than 50% of patients recovering from COVID19 (Mandal et al., 2021).

The underlying aetiology for persistent respiratory morbidity is likely to be multifactorial but may be due to persistent parenchymal abnormalities and resultant ineffective gaseous exchange. Persistent radiological abnormalities post-COVID19 are common and may be present even up to 6 months post hospital discharge (Fabbri et al., 2021; Guler et al., 2021; Han et al., 2021; Myall et al., 2021). There is, therefore, a pressing need to understand the molecular and cellular basis of post-COVID19 pulmonary syndromes.

The acute immunological and inflammatory events that occur during human respiratory virus infections, including SARS-CoV-2, are relatively well described (Harker and Lloyd, 2021). In contrast, the immunological landscape of the human respiratory tract after recovery from acute viral infection is poorly understood.

SARS-CoV-2 infection results in formation of long-lasting systemic immunological memory, with virus-specific antibodies and T cell responses still detectable in the majority of those infected at least 8 months post infection and higher titers seen in previously hospitalized individuals (Dan et al., 2021).

Circulating lymphocyte counts and the function and frequency of monocytes are also reduced during acute disease, but they appear to return to normal shortly after resolution of acute disease (Mann et al., 2020; Scott et al., 2020). Likewise, plasma concentrations of inflammatory mediators such as IL-6 and CXCL10, that are highly elevated in acute disease, reduce as individuals recover (Rodriguez et al., 2020).

Together, this suggests that systemic inflammatory and immune responses associated with acute disease severity resolve in line with recovery from the acute symptoms. It therefore remains unclear if the severity of inflammation during acute disease is associated with the persistent respiratory pathology seen in some SARS-CoV-2 infected individuals months after infection, or if there is ongoing inflammation in these individuals.

This study examines the relationship between the immune system and respiratory pathology post-COVID19. The immune cell and proteomic composition of the airways and peripheral blood were analyzed in a group of previously hospitalized COVID19 patients with persistent radiological abnormalities in their lungs more than 3 months post discharge. In comparison to healthy individuals, the post-COVID19 airway showed substantial increases in activated tissue resident memory CD8+ and CD4+ tissue-resident memory (Trm) cells, and an altered monocyte pool.

The airway proteome was also distinct from that observed in healthy individuals, with elevation in proteins associated with ongoing cell death, loss of barrier integrity and immune cell recruitment. None of these airway abnormalities were reflected in the proteome or immune cells of the matched peripheral blood.

The scale of these alterations was not linked to the initial severity of disease while in hospital and were heterogenous; Some individuals displaying heightened T cell responses associated with significant increases in CXCR3 chemokines in the airways linked to prolonged epithelial damage and extracellular matrix (ECM) dysregulation, while other individuals exhibited a return to relative airway homeostasis. Subsequent long-term follow-up also suggested that these changes to the airway landscape progressively return to normal.

Discussion

Recovery from COVID19 may be complicated by long-lasting symptoms including breathlessness. Here we studied patients previously hospitalized with COVID19, revealing a persistent proteomic and immunological abnormalities in the airways, but not peripheral blood, many months after acute infection. While there is substantial heterogeneity between patients, we observed upregulation of proteins associated with ongoing cell death, epithelial damage and tissue repair in post-COVID19 airways.

This correlated with the presence of increased numbers of activated tissue resident CD8 T cells. Preliminary evidence suggests this altered airway landscape does improve over the long term, with reductions in airway immune cell numbers 1 year post discharge.

The acute response to SARS-CoV-2 infection is characterized by widespread upregulation of circulating proteins including IFN pathway proteins, chemokines, cytotoxic proteins, and markers of epithelial damage (Arunachalam et al., 2020; Filbin et al., 2021; Gisby et al., 2021). More severe disease is associated with increased inflammatory proteins (e.g. IL-6, TNF, GM-CSF, IL-1RN and IL-18) (Arunachalam et al., 2020; Filbin et al., 2021; Thwaites et al., 2021).

A similar pattern of upregulated proteins, especially chemokines like CXCL10 and cytokines such as IL-6, is seen in the airways during acute COVID19 (Liao et al., 2020; Saris et al., 2021; Szabo et al., 2021). 3-6 months after SARS-CoV-2 infection however, despite the presence of ongoing respiratory morbidity, the plasma proteins differentially expressed during acute disease appear to have returned to similar concentrations to those seen in healthy controls. Even data dimension reduction approaches such as WGCNA fail to highlight any significant associations between COVID19 infection and the plasma proteome months later.

In contrast, the post-COVID19 airways continue to display an abnormal proteome, with both distinct and shared features to that seen in acute disease. Proteins linked to inflammation feature less prominently than in acute COVID19, whereas upregulation of proteins involved in epithelial damage and repair (e.g. the EGFR ligand AREG and the epithelial marker KRT19) persist. MMP-3, which regulates the extracellular matrix (ECM), was also differentially upregulated in the post-COVID19 airway. MMP3 and AREG are both upregulated after influenza A virus (IAV) infection in vivo in mice, and in vitro in human fibroblasts and epithelial cells (Boyd et al., 2020); and both are linked to epithelial repair and fibrosis in the lungs (Morimoto et al., 2018; Yamashita et al., 2011).

Elevated LDH and albumin in the airways provide further evidence of ongoing cell death and damage to respiratory barrier integrity post-COVID19. This observation is reinforced by the upregulation of a module of correlated proteins in the post-COVID19 BAL whose individual members reflect epithelial damage (EPCAM, KRT19), cell death (CASP3) and epithelial repair (TGFA), but also suggest a connection between these processes and immune cell recruitment and survival (CXCL9-11, IL-7). Increased cell death within the airways correlates with the frequency of T cells, primarily CD8 Trm cells, and with heightened respiratory dysfunction.

In mouse models of severe acute respiratory virus infection, CD8 T cells are known to act as a double-edged sword. Although the cytotoxic molecules and cytokines they release are essential for clearing virus, they can also cause tissue damage and immunopathology (reviewed in (Duan and Thomas, 2016; Schmidt and Varga, 2018)). While pre-existing virus specific CD8 Trm cells in the airways is thought to be protective against a re-encounter with the same virus (Jozwik et al., 2015; Wu et al., 2014) little is known about their role in long- term respiratory virus-related pathology, especially in humans.

This is primarily due to the lack of relevant samples collected during the recovery period. Our post-COVID19 data support the concept that sustained activation of CD8 Trm cells in the airways, long after recovery from acute disease, contributes to ongoing damage to the respiratory epithelium, resulting in airway disease.

The mechanism underlying increased Trm cells in the airways is unclear, although several studies have reported virus specific CD8 T cells in lung tissue up to a year post-infection (Cheon et al., 2021; Grau-Exposito et al., 2021; Poon et al., 2021). While virus specific CD4 and CD8 T cells rapidly expand, and form Trm cells, following SARS-CoV-2 infection (Szabo et al., 2021), these cells rapidly contract after resolution of acute disease, with CD8 Trm cells declining more rapidly than CD4 Trm (Slutter et al., 2017).

The lungs of mice previously experienced IAV infection more robustly maintain CD8 Trm cells compared to uninfected lungs however, showing that severe infection promotes a pro-Trm niche (Slutter et al., 2017). This fits with our observation that CD8 Trm cell numbers vary dependent on the proteins and extent of damage in the airways, and change longitudinally in the same individuals, while CD4 Trm cells remain relatively static.

A number of factors may contribute to the heterogeneity of the CD8 Trm niche in the post-COVID19 airway. Firstly, while all our post-COVID19 samples were taken from patients who tested negative for SARS-CoV-2 by qPCR immediately prior to bronchoscopy, persistent antigen has been observed months after other respiratory infections such as IAV (Kim et al., 2010), and SARS-CoV-2 antigen

depots could drive ongoing cytotoxic activity and maintenance of CD8 Trm cells. Secondly, the persistence of lung resident Trm cells is reliant on the availability of local T cell survival signals such as IL-7 (Szabo et al., 2019) and the CXCR3 ligands (Slutter et al., 2013). Indeed IL-7 and the CXCR3 ligands are part of the protein network that is maintained in the post-COVID19 airway.

Lastly there is some evidence for indicating the development of auto-immunity in some patients recently recovered from COVID19 (Lucas et al., 2020; Wang et al., 2020). It is likely that these different mechanisms collectively act to shape CD8 Trm cell responses, and other immune cells, in the post- COVID19 airway, and the scale and duration of ongoing epithelial damage and respiratory dysfunction observed.

B cell frequencies were more elevated in individuals with more widespread lung abnormalities and reduced gas exchange. During acute infection or after vaccination B cells are critical in the generation of protective virus-specific antibody. Virus-specific B cells can be detected in the lungs up to 6-months post SARS-CoV-2 infection (Poon et al., 2021) but represent a minority of the B cells present in the human lung.

Increased frequencies of airway and lung B cells, similar to those seen in the post-COVID19 airway, are commonly seen in a range of respiratory diseases including COPD and interstitial lung diseases (ILD) (Desai et al., 2018; Polverino et al., 2016). B cell frequencies do not correlate with virus-specific antibody, which is more tightly linked to the T cell responses suggesting a common antigen specific driver that B cells are not dependent on.

Precisely how B cells contribute to ongoing respiratory pathology post- COVID19 is unclear; they can produce both pro-inflammatory and regulatory factors, and disruption of regulatory B cell function has been shown to be associated with fibrotic lung disease (Asai et al., 2019). B cells can also promote tissue repair by inducing activation and migration of fibroblasts (Ali et al., 2021). Thus, in the post- COVID19 airway B cells may be directly promoting aberrant tissue repair.

Functional impairment of monocytes and DCs in the peripheral blood of acutely infected patients (Arunachalam et al., 2020; Laing et al., 2020; Mann et al., 2020), and hyperactivation of airway monocyte populations, are features of acute severe COVID19 (Liao et al., 2020; Szabo et al., 2021).

In our post-COVID19 patients, peripheral blood monocyte had normalized and did not correlate with markers of pulmonary dysfunction, but BAL intermediate monocytes were increased in patients with greater CT abnormalities. In humans, following inflammatory insults, monocytes are recruited to the airways to differentiate into new AMs (Byrne et al., 2020). Severe viral infection can cause rapid depletion of the airway macrophage pool (Pribul et al.,

2008), and different subsets of monocytes contribute differentially to the replenishment of lung macrophages (Evren et al., 2021). Monocyte to macrophage transition is also more pronounced in chronic lung disease, with the newly generated monocyte-derived macrophages acting in a pro-fibrotic fashion (Misharin et al., 2017). Increases in intermediate monocytes may therefore be indicative of heightened monocyte differentiation into airway macrophages, the numbers of which are increased in the post-COVID19 airway compared to healthy controls. Amplification of this process may then contribute to ongoing repair within the lungs.

The progressive resolution of radiological abnormalities in the majority of post- COVID19 patients has been described (Han et al., 2021), and within our study even the 3 patients with persistent respiratory abnormalities show improved CT and reduced airway immune cell infiltration. This fits with the hypothesis that SARS-CoV-2 infection can result in organizing pneumonia, with subsequent changes reflecting ongoing epithelial damage and healing parenchyma rather than established fibrosis (Kory and Kanne, 2020). Moreover, the involvement of the immune response in different aspects of ongoing respiratory disease post-COVID19 suggests this recovery could be accelerated using immunomodulatory treatments.

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