Up to now, there has been little information about the deep lung inflammatory status in SARS-CoV-2 patients. In the present study we analyzed the broncho-alveolar inflammatory environment of patients admitted to the ICU and IMW of two hospitals situated in the epicenter of the Italian epidemic outbreak. In particular, we evaluated cell differentials, cell activation by morphological features, and cytokines assessment on retrieved BAL fluids. Among cytokines, we decided to focus our attention on IL6, a well-known inflammatory cytokine that has been used as a marker of macrophages activation in the peripheral blood of COVID-19 affected patients [13]; IL8, a pro-inflammatory CXC chemokine that exerts specific chemotactic and activating functions on neutrophils; and IL10, an anti-inflammatory and immunoregulatory cytokine, produced by several immune effectors, whose role in SARS-CoV-2 infected patients is still in debate [9, 14].
In severe ICU COVID-19 patients, the alveolitis was associated with hyperactivation of innate effectors, macrophages and neutrophils, that showed pseudopodia and cell-to-cell contacts (Fig. 2a and b). Moreover, a large number of cytopathic cells with vacuolization, osmiophilic bodies, loss of integrity of both nuclear and plasma membranes and cell fragmentation were present (Fig.S1, Fig. 2 and Fig.S2). Immunostaining with anti-viral capsid and spike antibodies specifically immunoreacted with BAL cells, mostly cytopathic ones (Fig. S1e,f and Fig. 2c and d).
High percentage of neutrophils in ICU patients BAL in contrast to IMW patients were founded (Fig. 1a and b). The high infiltration of neutrophils inside the alveolar space has already been assessed in severe COVID-19 patients, but only after autopsy study [15]. Herein, we quantify the neutrophil percentage in the alveolar space from mild to severe stages of COVID-19, confirming the literature.
Regarding lymphocytes, we observed a strongly and significantly reduced percentage in ICU patients as compared to those in the IMW (Fig. 1), corroborating findings recently reported by Liao and colleagues who analyzed BAL samples from 3 severe and 3 mild SARS-CoV-2 patients. The authors showed a decreased lymphocyte counts in the three severe patients with respect to the mild ones [10]. Thus, we have added another insight regarding the impairment of adaptive immune response to COVID-19 reported in peripheral blood, suggesting the same behavior also at the lung level [6, 8, 16, 17].
Concerning the three analyzed cytokines, we demonstrated that patients admitted to the ICU are characterized by significant high levels of the two pro-inflammatory cytokines, IL6 and IL8, with respect to IMW patients (Fig. 3a,b). This result is sustained by previous reports obtained for Middle East Respiratory Syndrome (MERS) pneumonia patients, demonstrating a severe down-regulation of Th2, and inadequate Th1 immune response in respiratory specimens together with high expression levels of inflammatory cytokines IL-1α and IL-1β and IL-8 (CXCL8) [18]. Furthermore, a recent study about single-cell RNA profiles of alveolar macrophages from 3 SARS-CoV-2 patients identified a prevalence of macrophages expressing FCN1 and several pro-inflammatory genes (CCL2, CCL3, CCL5, IL-8, CXCL9, CXCL10 e CXCL11), while anti-inflammatory macrophages, and in particular macrophages involved in surfactant turnover, were significantly reduced in severe COVID-19 patients [10].
The study herein also demonstrated that upon analyzing survivors in comparison to non-survivors, we found significantly lower IL6 (Fig. 3c) and IL8 (Fig. 3d) levels in the survivor group with respect to non-survivors. The analysis of treatment effect on BAL inflammation, limited to a small number of ICU patients, showed that in patients submitted to a steroid course IL6 was lower than in those treated with tocilizumab or anti-viral drugs (Fig. 4a). It should be noted, however, that tocilizumab, by acting at a receptor level, does not interfere with IL6 release, but rather exerts its action on the IL6-driven inflammatory cascade. The same trend towards a decrease in IL6 was also observed for IL8, which did not reach statistical significance (Fig. 4b). Thus, taking together all our findings, we can suggest that the burden of pro-inflammatory cytokines IL6 and IL8 in deep lung is not only related to the severity of the disease but might also be associated to the outcome. Steroids seem to be able to limit the release of IL6 and IL8 while not significantly affecting IL10 levels (Fig. 4c). This last result is supported by a clinical trial called “RECOVERY”, which reported that dexamethasone reduced deaths by one-third in ventilated patients [19].
The role of IL10 in COVID-19 infection is a matter of ongoing debate. Increased serum levels of IL10 have been described in severe SARS-CoV-2 patients with respect to those affected by mild disease. Moreover, authors have demonstrated a direct correlation with IL6 serum levels, suggesting that both cytokines could be used as predictors of higher risk of disease deterioration [20]. Thus, despite its well-known anti-inflammatory role, an exaggerated release of IL10, together with other pro-inflammatory factors from activated macrophages is integrated in the context of the cytokine storm and considered a marker of disease severity [21]. In the present study, however, there was no significant variation in IL10 BAL levels either in relation to disease severity or according to survival (Fig. 3). In addition, no significant correlation was found between IL10 and IL6 in BAL, while only a negative and significant correlation with IL8 levels was present (Table S2). Thus, on the basis of the present study, a role of BAL IL10 as a specific disease marker cannot be inferred.
We are aware that this study has some limitations. We performed these preliminary analyses on limited sample size given the high risk of infection of the health care personnel. This limited our possibility to make further analyses, such as cell surface activation markers assessment by flow cytometry on macrophage or lymphocytes. Another limitation is the lack of paired assessment of cytokines in the peripheral blood due to the absence of stored serum samples from acute COVID19 patients in our institutions. Finally, we know that the infectious complications registered (Table 1) might have influenced cell differentials and cytokine levels however, the percentage of bacterial and fungal co-infection was very high in all patients in ICU without a significant difference between survivors and non-survivors.
As a result of our study we wish to highlight the possible crucial role of IL8 in COVID-19 infection at the lung level. This cytokine showed higher values among ICU compared to IMW patients (Fig. 3a and b) and was associated to a negative outcome (Fig. 3d). It is known that IL8 acts as a chemoattractant and activating factor of neutrophils, in fact we assessed a direct correlation between IL8 and neutrophils percentage in BAL (Table S1 and Fig. S4b). Hence, we can infer that the release of IL8 represents a crucial step of SARS-CoV-2 pathogenesis and its pathway might represent a possible target of future intervention.