BAL in COVID-19: cellularity, immunophenotype, and cytokine profile.
BAL characteristics and cellularity can be extremely useful in clinical practice, helping to identify possible differential diagnosis, and to guide the diagnostic and therapeutic choice of clinicians. BAL and lung cryobiopsy represent unique specimens to investigate the excessive inflammatory pulmonary response to SARS-Cov-2 that represent a major cause of disease severity and death.44,45 Doglioni et al elegantly described the histological and immunohistochemical features observed in the early-phase COVID-19, in cryobiopsies performed in non-intubated patients, with perivascular CD4-T-cell infiltration, capillary and venular changes, florid alveolar type II cells hyperplasia, no hyaline membranes.45 The T-cell perivascular infiltrate was CD 4 positive, but negative for functional activation markers (T-BET, FOXP3, CD25 and CD 30). Few interstitial PD1 + and TCF 1+ T CD8+ lymphocytes were detected. NK cells (CD 56+) and B-cells (CD 20 +) were rare or absent.45 BAL studies can provide precious data on the cellular and molecular component from the distal lung, that nicely integrate histology findings. Compared to lung biopsy BAL is much more easily performed, therefore a considerable number of recent studies have used BAL to evaluate the alveolar cellular profiles that could correlate with clinically meaningful outcomes (e.g. disease severity and mortality) and that could help the understanding of COVID-19 pathogenesis. Dentone et al, described the BAL characteristics and cellularity of 64 COVID-19 patients admitted during March and April 2020 to the Intensive Care Unit (ICU) of Genoa Hospital. 34,4% had coinfections detected by BAL (Candida, Psedumononas, Enterobacter aerogens, Staphylococcus aureus and Klebsiella Pneumoniae).46 BAL samples from individual patients were taken and their total cellularity, subpopulations, and T lymphocytes activation as HLA-DR expression.46 The median cellularity was 68 x 103/ml (IQR 20-145). The majority cells in BAL were neutrophils (70%, IQR 37.5-90.5), followed by macrophages (27% IQR 7-49). Eosinophils were less than 1% (IQR 0.9-3). Lymphocytes were a minority, 1%, with CD3+ 92% (IQR 82-95). Among CD3+ T lymphocytes 52% were CD8+ (IQR 39.5-62.7), with a T CD4+/CD8+ ratio of 0.6 (IQR 0.4-1.2). 20% where HLA-DR+ (IQR 13-32). At multivariate analysis only the percentage of macrophages in the BALF at the time of ICU entry correlated with higher mortality (OR 1.336, 95% CI 1.014-1.759, p = 0.039). The duration of mechanical ventilation was correlated with percentage of TCD8+ in BALF (r = − 0.410, p = 0.008), TCD4+/CD8+ ratio (r = 0.425, p = 0.006) and total lymphocytes TCD3+ (r = 0.359, p = 0.013) in BALF, respectively. The Authors speculate that the lack of lymphocytes in the BALF in patients admitted to the ICU could partly explain a reduced antiviral response. The reason for this depression of lymphocytes could be related to both direct virus damage to the lymphocyte and by cytokine storm induced damage.46 That innate immunity is extensively activated has been confirmed also by Pandolfi et al, that in the BALFs of 33 adults admitted to the ICU reported a marked increase in neutrophils (1.24 X 10^5 ml, 0.85-2.07), reduced numbers of lymphocytes (0.97 X 10^5 ml, 0.024-0.34) and macrophages (0.43 X 10^5 ml, 0.34-1.62) with viral particles inside mononuclear cells (seen by electron transmission microscopy and immunostaining).47 The majority of BAL showed coinfections (26/28). The burden of pro-inflammatory citokines was associated with clinical outcome, IL-6 and IL-8 were significantly higher in ICU patients than in Internal Medicine Ward (IL6 p < 0.01, IL8 p < 0.0001), and also in patients who did not survive (IL6 p < 0.05, IL8 p = 0.05 vs. survivors).47 A recent study by Reynolds and co-workers showed that inflammatory immune dysregulation of the lower airways during severe viral pneumonia (both severe influenza and SARS-Cov-2 were included) is distinct from that of non-severe illness, with an influx of non-classical monocytes, activated T cells and plasmablasts B cells. BAL cytokines were elevated in severe cases, but not in moderate patients. Largest elevation were observed in IL-6, IP-10, MP-1 and IL-8.48 Contrarily to previous reports, Gelarden et al reported in 83 patients intubated for severe COVID-19 a lymphocytosis (i.e. > 15%) in 74.7% of cases (62/83) with a high prevalence of atypical lymphocytes in BAL (72.3%, 60/83).49 BAL lymphocytes, including plasmacytoid and plasmablastic cells, were composed predominantly of T cells with a mixture of CD4+ and CD8+ cells. Both populations had increased expression of T-cell activation markers, suggesting important roles of helper and cytotoxic T-cells in the immune response to SARS-Cov-2 infection in the lung. BAL lymphocytosis was significantly associated with longer hospital stay (p < 0.05) and longer requirement for mechanical ventilation (p < 0.05), whereas the median atypical (activated) lymphocyte count was associated with shorter hospital stay (p < 0.05), shorter time on mechanical ventilation (p < 0.05) and improved survival.49 All these data should be interpreted with great caution because are derived from small, retrospective and monocentric studies with an evident heterogeneity between cohorts in terms of phenotypes, disease severity, duration of intubation, presence of coinfections. Moreover, there is a critical lack of BAL data in non-intubated patients with less severe COVID-19, that limit our ability to understand disease pathogenesis in the early phase of the disease. Besides those evident limits, the current body of evidence suggests that BAL cellular analysis is an invaluable tool to provide useful information for diagnostic and prognostic workup and potentially to expand our understanding of COVID-19 pathogenesis.