Last week, two back-to-back papers were publish in Cell reporting on the differences in myeloid cells that allows for the discrimination of mild and severe cases of COVID19. We now know that the manifestation of COVID19 is a spectrum – asymptomatic, mild to severe. Most people whom are infected are either asymptomatic or present mild symptoms. However, for some people, COVID19 develops into a severe and fatal illness. The reason behind this observation remains unclear.
These two papers sought to use single-cell technologies to solve this mystery; providing us with new knowledge, at high resolution, on the differences in the intricacies of the immune system between mild and severe COVID19 cases. Here, I will share about the discoveries made by Silvin et al., 2020. Using multi-dimensional single-cell approaches, the group was able to:
1. Observe changes in certain white blood cell types in severe COVID19 patients.
2. Discover potential biomarkers that could be used clinically to stratify COVID19 patients.
3. Uncover potential targets for therapeutic interventions to alleviate severe COVID19.
Necessary background information
What are myeloid cells?
Myeloid cells broadly encompass descendants from common myeloid progenitor cells derived from hematopoietic stem cells in the bon marrow. They include monocytes and granulocytes (figure 1) and they make up a large proportion of white blood cells found in the peripheral blood.
Figure 1 – Types of myeloid cells
Monocyte heterogeneity
Unbeknownst to many, monocytes can actually be classified into three subsets – Non-classical, intermediate and classical (figure 2). Each of the subsets of monocytes are defined by the specific markers2 and have been found to play different roles in our immune system. Functionally, classical monocytes are more phagocytic, intermediate monocytes are seem to be involved in antigen presenting and cytokine secretion3. Non-classical monocytes have been found to play ‘pro-inflammatory’ roles such as promoting neutrophil trans-endothelial migration.
Figure 2 – Types of monocytes
Discovery v.s. validation cohort
A typical biomarker discovery workflow encompasses multiple cohorts i.e. groups of people summarized in figure 3. The process begins with a discovery cohort, where new, unique and biologically significant candidates are discovered. Then, occasionally, there will be a verification cohort to determine if the discovery of those candidates can be reproducible. The clinical outcomes of the patients (e.g. mild and severe) are known during the discovery phase. Lastly, there will be a validation cohort who will be tested only with the candidates to determine if it can reliably distinguish their clinical outcomes.
Figure 3 – Types of cohorts
When blood samples of the discovery cohort were analyzed, the group noted an expansion in the proportion of circulating neutrophils in severe patients. Within these neutrophils, there were drastically more CD10 Low and CD101- immature neutrophils. While the number of monocytes were similar between severe patients and the controls, the monocyte profiles were different. In severe patients, there were significantly lesser numbers of non-classical monocytes. In mild patients, there were significantly higher numbers of intermediate monocytes. However, there were significantly higher numbers of classical monocytes in severe patients (figure 4). Other changes in the immune cell make-up between mild and severe patients were that there are significantly lower numbers of CD4+ and CD8+ T cells in severe patients.
Figure 4 – Monocyte profiles in controls, mild and severe patients
Red- classical, purple - intermediate, blue - non-classical
A closer look at just monocytes revealed that the monocytes of mild patients express a type 1 interferon signature (a type of cytokine i.e. immune signal) that was absent in the control and severe patients. This strong signature, however, disappears after 10 days. This probably is a immune response that was initiated due to the infection and lasted only for 10 days.
Similarly, the neutrophils of mild patients also showed a significant type 1 interferon signature which disappears after 10 days. Neutrophils of severe patients, however, highly expressed genes involved in the production reactive oxygen species, inducible NOS pathway, IL-1 signalling and NFKB pathways, indicative of high levels of inflammation and stress.
Figure 5 – Types of neutrophils
Now, narrowing our focus onto the neutrophils for a second. It was observed that severe patients had more circulating immature neutrophils compared to controls and the mild patients. Specifically, there were more CD10Low CD101- CXCR4+ neutrophils which are ‘pre-neutrophils’ in the severe patients (figure 5). This seems to suggest a dysregulated neutrophil response towards the infection.
In addition, the group also found that severe patients had higher levels of S100A8 and S100A9 genes, which are calgranulin genes that codes for S100A8 and S100A9 alarmins that are released by neutrophils during inflammation. When they are released, they form a stable heterodimer known as calprotectin. Severe patients are found to have dramatically higher levels of calprotectin compared to controls and mild patients. Inflammatory signals CXCL-8, CXCL-12 and IL-6 were also found to be higher in severe patients.
In their validation cohort, they successfully validated their discoveries. While severe patients generally had higher levels of classical monocytes (as in the discovery cohort), they found a strong correlation between numbers of non-classical monocytes with time spent in ICU i.e. the more non-classical monocytes a COVID19 patient has, the more severe the illness and therefore more time spent in ICU (Mean number of days in ICU being 8.83 days).
Collectively, the authors found potential biomarkers of severe COVID19 summarized in Table 1.
Table 1 – Summary of potential biomarkers for mild and severe COVID cases
Most certainly, these biomarkers will require further testing and validation but they have the potential to discriminate and stratify COVID19 patients from a simple blood test. This is important in prioritizing medical attention to the ‘potentially severe’ patients before their illness develops and deteriorates. Another therapeutic avenue has also opened from this study where the authors proposed that targeting the over-production of calprotectin in severe patients may also prevent adverse downstream inflammatory responses caused by calprotectin.
References
Comments