NanoString nCounter Technology – COVID-19 Immunological and Inflammatory Response

Gaining a deeper understanding of COVID-19 pathology through NanoString technology.

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Marita de Waal

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Marita de Waal

The novel coronavirus, SARS-CoV-2, emerged in Wuhan in December 2019, and has since become a global pandemic of coronavirus disease 2019 (COVID-19) (Huang, et al., 2020). As a clinical research services provider, Synexa Life Sciences has positioned itself to be at the forefront of supporting clinical research and development into combatting and overcoming this pandemic. Of the many platforms Synexa has to offer, NanoString nCounter technology has proven of value already in the elucidation of the immunogenomics behind the disease, the details of which have been discussed in this article.
 
The most common symptoms of COVID-19 are fever, shortness of breath, expelling mucus, fatigue, dry cough, and myalgia (Huang, et al., 2020; Qin, et al., 2020). Peripheral blood examination counts of COVID-19 patients have found that many patients have decreased counts of leucocytes, lymphocytes (T-cells in particular), eosinophils, platelets, and haemoglobin, but have higher neutrophil-lymphocyte ratio (NLR) and monocyte-lymphocyte ratio (MLR) (Qin, et al., 2020; Xu, et al., 2020). The virus is highly contagious, and the severity of infection ranges from asymptomatic or mild upper respiratory tract infection to severe infections (Yang, et al., 2020). Cases of respiratory failure, severe viral pneumonia, or secondary haemophagocytic lymphohistiocytosis (sHLH) leading to organ failure, may be fatal (Mehta, et al., 2020). This range in severity appears to be underpinned by differences in host pathogen interaction, host immune response, and pathogen immune evasion strategy (Debnath, et al., 2020). The inflammatory response, in particular, is thought to underpin COVID- 19 pathogenesis and severity (Merad & Martin, 2020; Tay, et al., 2020) and is associated with increased levels of inflammatory cytokines, an exhaustion of circulating lymphocytes (lymphopenia) (Huang & Pranata, 2020; Zhao, et al., 2020), and mononuclear cell infiltration of organs (Tavakolpour, et al., 2020), including the lungs, heart (Xu, et al., 2020), spleen, lymph nodes and kidneys (Diao, et al., 2020; Feng, et al., 2020).

Host Immune Response

When the SARS-CoV-2 virus enters the cell, it does so via the angiotensin‐converting enzyme‐2 (ACE2) entry receptor, triggering the adaptive immune response which attempts to control the viral infection (Xu, et al., 2020). It has been reported that the entry of the virus through the ACE2 receptor dramatically increases the expression of the ACE2 gene, and that ACE2 expression remains high for over 48 hours post infection, suggesting that the ACE2 receptor plays a role in viral susceptibility, as well as in post-infection regulation (Li, et al., 2020).It has been reported that in infection with SARS-CoV, the ACE2 receptor itself is down regulated (Glowacka, et al., 2010), it is possible that infection with SARS-CoV-2 may have the same effect (Li, et al., 2020). ACE2 is expressed multiple tissue types, including renal, cardiovascular and gastrointestinal tissues (Harmer, et al., 2002; Tipnis, et al., 2000). Particularly relevant to SARS-CoV-2 infection, the ACE2 receptor is present in lung alveolar epithelial cells, small intestine enterocytes, arterial and venous endothelial cells and arterial smooth muscle cells (Hamming, et al., 2004).

After entering via the ACE2 receptor, the virus is sensed by Toll‐like receptor 7(TLR7) which leads to activation of the TLR-mediated pro-inflammatory response, with production of alpha interferon, Tumour Necrosis Factor alpha (TNFα), and the secretion of interleukin 12 (IL-12) and IL‐6 (Fung, et al., 2020). This, in turn, results in the formation of CD8+‐specific cytotoxic T cells, and the formation of antigen‐specific B cells and antibody production through the CD4+ helper T cells(Qin, et al., 2020).

When the body does not produce an adequate adaptive immune response to the infection, it results in persistent innate‐induced inflammation. This inflammation is hypothesised to lead to cytokine storm, acute respiratory distress syndrome(ARDS), and diffuse organ involvement, as has been observed in Severe AcuteRespiratory Syndrome (SARS), caused by the SARS coronavirus (Huang, et al.,2020; Salacup, et al., 2020; Xu, et al., 2020). Reduced T-cell activation can potentiate TLR-mediated inflammation in a positive feedback loop (Ong, et al.,2020). In particular, IL-6 plays a pleiotropic role in immune response. While IL-6is crucial for the formation of follicular helper T cells, Th17 subset deviation, and for the formation of long‐lived plasma cells, it can also block CD8+ cytotoxic T cells by inhibiting the secretion of gamma interferon (Ahmadpoor & Rostaing,2020; Debnath, et al., 2020). IL-6 also induces the suppression of cytokine signalling (SOCS-3) and increases the expression of programmed cell death protein 1 (PD-1), which can incapacitate the cell-mediated antiviral response(Velazquez-Salinas, et al., 2019). Some studies have also found elevated plasma levels of pro-inflammatory cytokines such as interleukin-1 (IL-1), IL-6, IL-8, IL-10(Li, et al., 2020), IL-1β, IL-2, IL-7, granulocyte-colony stimulating factor, interferon-γ inducible protein 10, monocyte chemoattractant protein 1, macrophage inflammatory protein 1-α, and TNFα in severe COVID-19 patients(Huang, et al., 2020; Mehta, et al., 2020).

Several studies have also reported that cases of COVID-19 also exhibit low circulating lymphocyte counts (Chen, et al., 2020; Huang, et al., 2020; Wang, etal., 2020). A retrospective study compared the expression of exhaustion markersPD-1 and Tim-3 on the surface of CD4+ and CD8+ T cells from COVID-19patients and healthy controls (Diao, et al., 2020). This study reported that T cell numbers (including total T cells, CD4+ and CD8+ T cells) were significantly lower in patients with severe or fatal COVID-19 disease than in patients who exhibitmild/moderate COVID-19 (Diao, et al., 2020). As ACE2 is absent on T cells, it is unlikely that the decrease in circulating lymphocytes is due to direct infection of the T-cells (Zhou, et al., 2020). COVID-19 symptoms are frequently compared toSevere Acute Respiratory Syndrome (SARS), also caused by a coronavirus, where cytokine storm and other dysregulated immune and inflammatory responses areassociated with disease progression and severity (Huang, et al., 2020; Read, etal., 2020). The numbers of total T cells, CD4+ T and CD8+ T cells appear to benegatively correlated to levels of TNF-α, IL-6, and IL-10 (Diao, et al., 2020;Huang, et al., 2020), which suggests that increased levels of these cytokinesmay be involved in the low levels of T cells detected in COVID-19. It has been suggested that the cytokine storm may promote the apoptosis or necrosis of T-cells (Diao, et al., 2020). This same study also reported an increase in PD-1 andTim-3 exhaustion markers on T-cells in severe COVID-19 patients and correlatedthis with increased levels of IL-10.

One recent NanoString gene expression study called the widely-held link between COVID-19 mortality and the cytokine storm into question after finding that pro-inflammatory gene expression lagged behind the nadir of respiratory function in some cases, suggesting that the inflammatory and immune responses to COVID-19 are dynamic, and vary over the course of illness, implicating a different set of inflammatory cytokines to previous studies (Ong, et al., 2020).This study found that in one patient only the expression of interleukin-1α (IL-1α)and interleukin-1β (IL-1β) preceded the nadir of respiratory function. While no strong conclusions can be drawn from such a limited sample size, it highlights the need for controlled sampling over disease progression.

Risk Factors

Numerous factors play a role in increased risk of COVID-19 mortality, the greatest of which is age (thought to be due to immunosenescence, leading to a decreased number of naïve T-cells (Weiskopf, et al., 2009)) followed by co-morbidities, including hypertension, diabetes mellitus, obesity, cardiovascular and cerebrovascular disease, and being immunosuppressed (Onder, et al., 2020;Herold, et al., 2020). These at-risk populations may be unable to produce an adequate adaptive response (i.e., virus‐specific CD8+ T cells), thus falling victim to innate immunity-driven inflammation (Ahmadpoor & Rostaing, 2020). This can be particularly devastating in countries where the population demographic is skewed towards an older age distribution (e.g. Italy), or with high rates of pulmonary or immune disease such as tuberculosis or HIV/AIDS (Onder, et al.,2020). Underserved populations, especially hospitalised patients who require mechanical ventilation, vasopressors, or are on continuous renal replacement therapy (CRRT) or haemodialysis (HD) have a much greater risk of inpatient mortality (Salacup, et al., 2020).

ACE2 gene polymorphisms have been suggested to play a role in susceptibility or resistance to SARS-CoV-2 infection, as well as suggesting that some polymorphisms may be more common in certain geographic areas or populations than others, but further population genetics and functional studies are required(Debnath, et al., 2020). It is also noteworthy that ACE2 modulates two of the most common COVID-19 comorbidities – hypertension and diabetes mellitus(Patel, et al., 2012). ACE2 disruption impacts vascular function, the renin‐angiotensin system, and exacerbates cardiovascular complications associated with diabetes (Patel, et al., 2012). ACE2 may also play a role in mediating inflammation post-infection (Reddy Gaddam, et al., 2014), and increased ACE2expression after SARS-CoV-2 infection has been linked to increased secretion of several inflammatory cytokines (Li, et al., 2020). ACE2 expression is also involved in mediating the activation of neutrophils, NK cells, Th17 cells, Th2cells, Th1 cells, dendritic cells and TNFα secreting cells (Li, et al., 2020), all of which are associated with the inflammatory response. Interestingly, while there is some evidence of increased susceptibility to COVID-19 in patients with underlying lung conditions (Pranata, et al., 2020), this may not to be linked to differences in ACE2 expression, as one study reported finding no difference in
ACE2 expression between healthy subjects and subjects with chronic airway disease (Li, et al., 2020).

When the body does not produce an adequate adaptive immune response to the infection, it results in persistent innate‐induced inflammation. This inflammation is hypothesised to lead to cytokine storm, acute respiratory distress syndrome(ARDS), and diffuse organ involvement, as has been observed in Severe AcuteRespiratory Syndrome (SARS), caused by the SARS coronavirus (Huang, et al.,2020; Salacup, et al., 2020; Xu, et al., 2020). Reduced T-cell activation can potentiate TLR-mediated inflammation in a positive feedback loop (Ong, et al.,2020). In particular, IL-6 plays a pleiotropic role in immune response. While IL-6is crucial for the formation of follicular helper T cells, Th17 subset deviation, and for the formation of long‐lived plasma cells, it can also block CD8+ cytotoxic Tcells by inhibiting the secretion of gamma interferon (Ahmadpoor & Rostaing, 2020; Debnath, et al., 2020). IL-6 also induces the suppression of cytokine signalling (SOCS-3) and increases the expression of programmed cell death protein 1 (PD-1), which can incapacitate the cell-mediated antiviral response(Velazquez-Salinas, et al., 2019). Some studies have also found elevated plasma levels of pro-inflammatory cytokines such as interleukin-1 (IL-1), IL-6, IL-8, IL-10(Li, et al., 2020), IL-1β, IL-2, IL-7, granulocyte-colony stimulating factor, interferon-γ inducible protein 10, monocyte chemoattractant protein 1, macrophage inflammatory protein 1-α, and TNFα in severe COVID-19 patients(Huang, et al., 2020; Mehta, et al., 2020).

Genetic polymorphisms in immune function‐related genes such as human leukocyte antigen (HLA) are thought to be a possible predictor of COVID-19severity. In a study on 28 COVID‐19 patients with severe respiratory failure, the expression of HLA‐DR was very low and this was accompanied by profound reduction of CD4 lymphocytes, CD19 lymphocytes, and natural killer (NK) cells, indicating that HLA might play an important immune‐regulatory role in COVID‐19(Giamarellos-Bourboulis, et al., 2020). HLA genes may also affect the phenotypes associated with COVID-19 infection.

C-reactive protein (CRP) is used in the diagnosis of early stages of pneumonia, and patients with severe pneumonia exhibit high levels of CRP (Liu, et al., 2020).One study found a correlation between CRP levels and the diameter of the largest lung lesions in patients with COVID-19, suggesting that CRP may be a possible indicator for disease severity (Wang, 2020). Another study found a strong correlation between CRP levels and inpatient mortality (Xu, et al., 2020).In addition to CRP, IL-6 and procalcitonin (PCT) have been found to have some prognostic value, as patients with all three markers at higher levels were more likely to exhibit severe complications, including the need for mechanical ventilation (Herold, et al., 2020; Liu, et al., 2020).

Treatment

At present, there is no commercially available antiviral treatment for COVID-19, and various host-directed treatment and supportive options, including immunomodulators, are being explored. Some of the treatment avenues that have been explored or suggested include:

  • immunosuppressive drugs such as IL-6 blockers (e.g. tocilizumab) or Januskinases (JAK)-signal transducer and activator of transcription signallinginhibitors (Salacup, et al., 2020; Mehta, et al., 2020; Giamarellos-Bourboulis, et al., 2020)
  • recombinant IFN to foster host antiviral response e.g. Type I IFNs (IFN-I)(Mantloa, et al., 2020) as well as Type III IFNs (Park & Iwasaki, 2020)
  • corticosteroids in cases of severe hyperinflammation, whereimmunosuppression could improve mortality (Xu, et al., 2020; Mehta, etal., 2020)
  • intravenous immunoglobulin (Xie, et al., 2020)
  • IL1 or IL1 receptor antagonists, e.g. canakinumab and anakinra (Ong, etal., 2020)
  • chloroquine and hydroxychloroquine have been used to block viralreplication and viral entry into the cell. Hydroxychloroquine is thought toalso block TLR7 and TLR9 signalling, allowing the continuation of theCD8+ cytotoxic viral response (Gautret, et al., 2020; Devaux, et al., 2020)
  • immunomodulators (e.g. azithromycin) which blocks IL‐6 and TNF‐alpha(Gautret, et al., 2020)
  • antivirals (e.g. remdisivir (Al-Tawfiq, et al., 2020), or combination therapyof remdisivir with the IL-6 blocker tocilizumab (Abbaspour Kasgari, et al.,2020))
  • angiotensin-converting enzyme inhibitors (ACEIs) or angiotensin II receptorblockers (ARBs) (usually used to treat hypertension) have been suggestedas possible treatments for COVID-19. However, a retrospective clinicalstudy found no strong association between ACEI/ARB treatment andCOVID-19 health outcome in patients who were already receiving anti-hypertensive treatment (Xu, et al., 2020).
  • Immunisation with the Bacillus Calmette‐Guérin (BCG) vaccine, which ismore commonly mandated in countries with high rates of tuberculosis, hasalso been found in some epidemiological studies to play a role in resilienceto some respiratory infections, including COVID-19. Countries with highrates of BCG vaccination have been found to experience reducedmorbidity and mortality from COVID-19, whereas countries with low ratesof vaccination are more severely infected (Escobar, et al., 2020). Inparticular, the BCG vaccine seems to offer protection to the vaccinatedelderly (Miller, et al., 2020).

 

The field of COVID-19 research is rapidly expanding, and while we are learning ever more about the coronavirus, we are also learning more than ever before about our own natural viral response. While many are hopeful for a vaccine in the near future, in all probability the solution lies in these treatments that modulates our bodies’ own inflammatory response.

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