After performing a 45-plex cytokine array on plasma samples from 108 patients at hospital admission, five cytokines displayed statistically significant differences according to degree of severity in COVID-19. Indeed, high levels of HGF and IL-1α coupled with low levels of IL-27 at admission can predict bad clinical outcome compared to the patient subset with better prognosis; these cytokines are therefore particularly important as predictors of admission to intensive care. Moreover, this multivariate model was especially sensitive in order to identify those patients who end up as critical (AUC = 0.794; specificity = 69.74; sensitivity = 81.25) following hospital admission. Lastly, we have also described how the combination of high levels of HGF and IL-1α at admission can predict mortality, showing significant results in the survival analysis (p = 0.033 and p = 0.011, respectively).
During recent months, several studies have tried to understand the cytokine profile in patients with COVID-19. Most of them relate the severity of lung disease to high levels of multiple cytokines in blood according to what has been defined as a cytokine storm. Indeed, some authors even describe three different clinical phenotypes of COVID-19 based on cytokine levels.22 In this regard, Huang et al. suggest that the cytokine storm is associated with severity after analysing 27 cytokines in 41 patients: those in the intensive care unit (ICU) had higher plasma levels of IL-2, IL-7, IL-10, GSCF, IP-10, MCP-1, MIP-1A and TNF-α.17 In a similar manner, Liu et al. studied 40 patients, 13 of them severe, and found increased plasma levels of IL-6, IL-10, IL-2 and IFN-γ in severe compared to mild cases.23 Zhao et al. studied 71 patients (53 mild and 18 severe) and 18 healthy volunteers, describing that IL-1RA and IL-10 correlated with disease severity24, and Zhang et al. analysed 326 patients, finding higher levels of IL-6 and IL-8 in severe or critical patients.25 Nevertheless, these studies display several limitations, such as small sample size, the study of low numbers of cytokines and the lack of a well-defined degree of severity. Moreover, patients who required mechanical ventilation were not usually differentiated from patients with severe disease, despite this aggressive intervention increasing cytokine levels. Lastly, these studies usually applied basic statistical approaches. Therefore, and in order to overcome these limitations, he have analysed in duplicate the plasma levels of 45 cytokines from an extremely well categorized cohort of 108 COVID-19 patients, who were classified into severity groups based on their clinical evolution defined by objective criteria, at which time we also performed an exhaustive statistical analysis. Hence, we have considered all confounders by using both univariate and multivariate regression analysis, showing at least an internal validation.
Other studies have performed a similar approach to the one described here, such as that by Han et al., which classified 102 patients into moderate, severe and critical groups according to their symptoms and also presented a control group of healthy volunteers. However, that study showed higher serum levels of TNF-α, IFN-γ, IL-2, IL-4, IL-6, IL-10 and CRP compared to the controls; using a logistic regression analysis, IL-6 and IL-10 were found to predict disease severity and internal validation could further confirm this result.26 However, they only analysed six cytokines and a duplicate analysis was not performed on each sample. In a similar manner, Meizlish et al. analysed a cohort with 49 adult patients (40 in the ICU and 9 in other units), as well as 13 healthy volunteers. They analysed 78 circulating proteins with immunological functions. Their study identified a neutrophil activation signature composed of neutrophil activators (G-CSF, IL-8) and effectors (resistin, lipocalin-2 and HGF), which had greater power to identify critically ill patients.27 As a default, the small number of patients and the different degrees of pulmonary severity do not differ in the non-critical patients.
Based on the results displayed by these two studies, and in agreement with ours, we can conclude that there is no specific cytokine pattern correlating with disease severity. On the one hand, high levels of HGF were associated with a risk of up to 3.5 times higher of being critical with mechanical ventilation. This growth factor, which has already been related to severity in other studies, primarily elicits its effects on epithelial cells. In a similar manner, IL-1α, which is a pro-inflammatory cytokine from the innate immune system mainly produced by macrophages but also epithelial cells, can also predict a bad prognosis and disease outcome. Hence, both cytokines could be reflecting the tissue damage elicited by the macrophage infiltration to the lungs.28,29 Nonetheless, low levels of IL-27, which belongs to the IL-12 family and is therefore involved in Th1 differentiation, is a good prognosis biomarker in COVID-19 patients. Together, these results suggest that, although in our hands the cytokine storm may not be the trigger of the bilateral pneumonia, there is certainly a mixed and altered cytokine profile that drives disease progression and inflammation, as highlighted by the fact that high HGF levels combined with low IL-27 levels are revealed as early mortality markers.
Since the beginning of this health crisis, treatment strategies in the most severe cases were aimed at blocking interleukins such as IL-6 (tocilizumab), IL-1 (anakinra) and TNF-α (infliximab, adalimumab, etc.). Nevertheless, there is no clear evidence about their utility. Indeed, tocilizumab has been reported to be ineffective in preventing intubation or death in hospitalized patients with moderate COVID-19 infection.30 Only the use of dexamethasone (two drops daily for 10 days) decreased mortality at Day 28 in patients who were receiving invasive mechanical ventilation.31 However, even with a corticosteroid, this approach did not identify the key immune components involved in this process. Accordingly, and with the results reported here, it is to be expected that these strategies do not cause a reduction in mortality because increased levels of IL-6, IL-1 or TNFα are not directly responsible for driving disease severity in these patients. Therefore, even though increased levels of plasma cytokines in COVID-19 patients have been largely reported, the identification of disease progression and severity biomarkers remains an urgent need. In this regard, we hereby report that HGF, IL-1α and IL-27 contribute to the deterioration of the disease and the adverse outcome of COVID-19, revealing these three compounds as novel biomarkers and as future therapeutic targets in COVID-19.