To our knowledge, this is the first paper describing an integrated multiomics study in Covid-19 complicated with pulmonary fibrosis patients at the molecular and ionic levels. First, after reviewing the medical records of 85 Covid-19 patients hospitalized in the People's Hospital of Guangxi Zhuang Autonomous Region Yongwu branch, we found that as many as 44.7% of the patients had fibrotic stripe shadows in the early onset, and the incidence rate was close to that of SARS outbreaks in 2003 18. The most common manifestations of chest CT in Covid-19 fibrosis in our 85 cases were summarized as follows: lesions located in bilateral lower lobes, involved in five lobes, and complicated with linear consolidation, bronchiectasis, and pleural incrassation, which partially agreed with a previous report 6. Second, we described the detailed profile of CT images in Covid-19 complicated with pulmonary fibrosis patients, especially focusing on progressive pulmonary fibrosis patients. Third, using proteomics and metabonomics methods, we explored the mechanism of the information pathway related to the occurrence and development of progressive fibrosis, which is expected to predict disease progression and to provide evidence for targeted therapy.
In recent years, proteomics has become an accurate method to screen disease biomarkers 19 and has also been used in the pathogenesis of SARS-CoV-2 infection 16,17. In this study, we found significant differential expression levels of immune system and biological adhesion markers and glycosaminoglycan degradation in pulmonary fibrosis patients. Previous experimental and clinical data demonstrated that the accumulation and deposition of glycosaminoglycan in the extracellular matrix contributed to progressive pulmonary fibrosis 20,21. Moreover, glycosaminoglycan hyaluronan (HA) on AEC2s (stem cells in the adult lung) is critical for restoring renewal capacity and limiting the scope of fibrosis 22. The general properties of cell adhesion molecules (CAMs) involve cell recruitment; they participate in cell-cell interactions and cell-extracellular matrix interactions 23 and then promote the formation of fibrosis by intervention in the microenvironment 24. For example, platelet/endothelial cell adhesion molecule-1 (PECAM-1) is expressed at intercellular junctions on platelets and leukocytes, including NK cells, T cells, monocytes and neutrophils 25, which are involved in chemokine-mediated migration to the inflammatory site 26. Moreover, PECAM-1 participates in the maintenance and restoration of vascular integrity following alveolar-capillary barrier disruption in ARDS 27. In addition, CAMs modulate signal transduction by interacting with extracellular matrix proteins 28. However, the results of this omics analysis are limited. The specific adhesion molecule biomarkers and the occurrence of diseases need further accurate detection.
It has been well documented that the immune system plays an important role in the occurrence and development of fibrosis 29,30 and Covid-19 31. The association of FCGR3B (encodes the lgG receptor FcγRⅢb) copy number variations with susceptibility to idiopathic pulmonary fibrosis (IPF) has been previously reported 32. FcγR binds to serum amyloid P (SAP), inhibits the differentiation of monocytes into fibrocytes 29,30 and affects neutrophil adhesion 29. Moreover, SAP binds to Ca2+-dependent ligands to induce FcγR-mediated phagocytosis in vivo 33. Recently, it was reported that segregated-nucleus-containing atypical monocytes, which were derived from FcεRI+ granulocyte/macrophage progenitors, were involved in fibrosis 34. NK cells lacking FcεRIγ are associated with lower levels of fibrosis 35. Furthermore, samples from 18 patients with IPF showed that the proportion of Breg cells (i.e., Breg cells to total B cells) was decreased, even though this proportion was positively correlated with the diffusion capacity of the lungs 36. Consistent with the results of the above references, our study supported the cross-regulation among pathways of the immune system, calcium signaling and cell adhesion.
As a member of the nuclear receptor and transcription factor family, PPAR is a classical receptor for diabetic therapy 37. However, recent emerging literature suggests a correlation between PPAR and lung fibrosis 38. Activation of the PPARγ pathway provided protection against organ fibrosis in the lung. PPAR agonists are beneficial in treating preclinical models such as bleomycin-induced pulmonary fibrosis 39 and genetically engineered mice 40. Moreover, inhibiting PPARγ promoted fibroproliferative ARDS, which was observed in clinical bronchoalveolar lavage fluid (BAL) samples 41. Our results from both pulmonary fibrosis patients and progressive pulmonary fibrosis patients in metabolomic analysis confirmed the differential expression of the PPAR signaling pathway, which suggested that PPAR signaling is a core pathway in the formation and development of lung fibrosis. TRP channels respond to a series of heterogeneous stimuli and are essential for pathophysiological homeostasis 42. It has been reported that TRP channels participate in lung disease mainly through calcium signaling, recruitment of proinflammatory cells, and neurogenic inflammatory pathways 43. In our study, pathway enrichment revealed that inflammatory mediator regulation of the TRP channel was an important pathway to regulate the occurrence and development of pulmonary fibrosis. Both arginine and ornithine are essential components in the urea cycle. L-arginine is the precursor of nitric oxide 44, which is involved in the vascular system 45. Treatment with L-arginine protects against pulmonary fibrosis progression in a mouse model 46. D-arginine is an inactive form of L-arginine. The results from our study indicated that lung fibrosis was associated with dysfunction of the urea cycle metabolic pathway. D-arginine and D-ornithine could be used as an early warning and a sign of progression of pulmonary fibrosis disease.
This study has several limitations. First, although the 85 Covid-19 cases were all confirmed patients in our hospital, their numbers are relatively few and cannot represent the whole of China or the international situation or even secondary infection. Second, the CV of proteomics was large because the samples were taken from patients' serum. Third, the immune system, PPAR, and TRP pathways screened by proteomics and metabonomics in this study need to be further verified in vivo and in vitro, while the pathways related to nervous system diseases need to be further explored, although it has been reported that Covid-19 patients have nervous system damage 47. Fourth, long-term follow-up of the progression of fibrosis is required.
In conclusion, in this study, proteomics, metabonomics, and correlation analysis were used to screen the related pathways of fibrosis formation and progression in Covid-19 patients, and the results showed that pathways including the immune system (especially the action of FcγR-mediated phagocytosis), PPAR signaling, TRP-inflammatory pathways, and the urea cycle were closely related. Our data provide new ideas for the treatment of Covid-19 and fibrosis patients.