CCL28 as a modulator of human acute respiratory distress syndrome

Inflammation during ARDS can lead to irreversible lung damage and fibrosis. Previously, we found increased levels of CCL28 in circulation during COVID-19 ARDS when compared to uninfected controls. CCL28 is produced upon inflammatory and hypoxic stimuli, however, it remains unknown which cells secrete CCL28 and its impact in the lung after ARDS. We investigated these aspects in patients9 lungs after ARDS, comparing with non-ARDS controls. We quantified CCL28+, HT2-280+ (mature AT2 cells9 marker), Fibroblast Specific Protein 1+ and CD68+ cells in paraffin-embedded lung slices of 5 ARDS patients with fibrotic evolution and 6 non-ARDS controls. In parallel, we evaluated the impact of CCL28 on cultured AT2 cells isolated from non-ARDS controls using flow cytometry. CCL28-expressing cells were increased in fibrotic lungs after ARDS, both in relative frequency (5.6±3.2% vs 1.9%±0.7, p=0.024), and by tissue area (164±95/mm² vs 41±22/mm², p=0.013). CCL28 was not mainly secreted by AT2 cells, as there were few CCL28+HT2-280+ cells (1.6±1.3/mm² vs 0.3±0.2/mm², p=0.052). Conversely, fibroblasts secrete CCL28 in both ARDS and non-ARDS controls (26±11/mm² vs 24±18/mm², p=0.832). We also found an increased number of macrophages secreting CCL28 in ARDS vs non-ARDS (12±7/mm² vs 3±2/mm², p=0.047). In vitro, upon CCL28 stimulation, AT2 cells displayed a reduction in the median fluorescence intensity of HT2-280 (56.5%±9.2, fold change to the unstimulated), suggesting the loss of the mature phenotype, and of CD63 (29.5%±0.7), a protein of the lamellar bodies. The expansion of CCL28-secreting cells and its effect on epithelial cells supports a role for this chemokine as a modulator of ARDS outcome that deserves further investigation.