4. Discussion
In recent years, with the development of techniques, increasing high throughput sequencing was used in MP studies. Lluch-Senar et al performed a comparative ”-omics” analysis and revealed more toxigenic of type 2 strains than type 1 strains of MP due to higher expression levels of CARDS toxin 14. By using RNA sequencing in bronchoalveolar lavage fluid, Gao M et al found 810 differentially expressed genes in MPP group comparing to control group, suggesting NK and CD8+ T cells are over activated and proliferated in MPP children15. Proteomics is now increasingly used to explore biomarkers of infectious diseases, which can provide us a feasible mean for large-scale screening of SMPP-related proteins, thus enhancing our understanding of the pathogenesis of SMPP. However, till now only two studies performed proteomics analysis in MPP16,17, one used serum samples from 5 RMPP children, 5 non-RMPP children, and 5 healthy children, while the other selected plasma from children with MPP, infectious disease controls and healthy controls. There are very few reports on proteomics analysis of SMPP. To our knowledge, ours is the first proteomics study in GMPPs and SMPPs to explore marker for dynamic progress of SMPP.
In the present study, by using label-free quantitative analysis, we initially explored differentially expressed proteins between GMPP and SMPP children and found 130 proteins. Interestingly, the complement and coagulation cascades pathway were enriched in both GMPP and SMPP groups, which might be explained by the varied pathway activation extent. Since the moderate activation of complement and coagulation cascades pathway assisted in cleaning microbial intruders, it is convincing that organism strived to clean pathogens and retain homeostasis in GMPP through activating the pathway. While dysregulation of the acute phase involving cytokine storm can result in severe clinical symptoms like acute lung injury 18, hypercoagulable stat even thrombosis 4, thus the disorder of complement and coagulation cascades pathway might play a vital role in the incidence of SMPP. In addition, collagen-containing extracellular matrix and platelet degranulation were enriched in both GMPP and SMPP groups (Figure 1B、1C), and KEGG analysis indicated an enriched platelet activation in SMPP group (Figure 1D), which might be explained by the varied airway remodeling and platelet activation extent. Typical characteristics of SMPP includes excessive immune response, hypercoagulable stat even thrombosis, and subsequent airway remodeling such as airway stenosis and BO4. It is worth noting that platelets are key mediators of inflammatory responses. Platelets and the coagulation pathway activate innate immune cells19. Active platelets may regulate pulmonary immune defenses and inflammatory injury20, immunothrombosis21, and is necessary in airway wall remodeling22. Therefore, the disorder of platelet activation might play another vital role in SMPP.
Among all the identified differentially expressed proteins including CRP and SAA, FCGBP was one of the most altered protein with highest fold change. FCGBP may play a role in the maintenance of the mucosal structure as a gel-like component of the mucosa. It can covalently bind and cross-link mucus proteins via its autocatalytically cleaved von Willebrand D domain23. Differentially expression of FCGBP has been reported gallbladder, prostate, thyroid, lung 24, colon, and ovarian cancer. Present in serum, the protein can be found in higher levels in patients with autoimmune diseases 25. Of special note, FCGBP has been postulated to trap HIV-1-antibody complexes at mucosal surfaces 26. The expression of FCGBP was assumed to be modulated by HPV infection 27and it was the highest upregulated early defense gene in catfish skin after microbial infection 28. In recent years, FCGBP has been reported to bind trefoil factor family 3 (TFF3), forming heterodimeric complex 29. Interestingly, TFF3 has been reported a lectin activity, enabling binding to bacterial glycans, such as lipopolysaccharide of H. pylori and exhibiting potential antibacterial activities 30,31. Therefore, it has been considered that FCGBP was a component of first line responder in innate immunological defense, critically regulating pathogen attachment and disease progression in mucosal surface, thus playing roles in cell protections and anti-inflammation in tissues.
In our study, in the early stage, patients with SMPP showed a higher FCGBP expression tendency than that in GMPP group, but did not reach a statistic significance, which may be due to the acute stage of the disease or the relatively severe in GMPP patients who had > 1/2 pulmonary lobe consolidation on chest imaging in our hospital. On the late stage, GMPP patients got reviled and did not need more FCGBP, thus the expression level reduced. However, for SMPP patients, even CRP level restored to baseline, the body temperature returned to normal, the airway damage did not relieved, even airway deformation such as BO occurred, which suggested airway epithelial cell and fibroblastic proliferation, and airway remodeling, and more FCGBP was produced to play anti-inflammation, anti-proliferation, anti-remodeling and cell protection roles. To some extent, these results indicated that FCGBP could be a biomarker for progression of MPP or airway remodeling in MPP. Since we only included 23 patients in our ELISA analysis, well designed and large cohort of randomized controlled trial will be needed before clinical application.
FCGBP is a key regulator in the process of transforming growth factor β1 (TGF-β1) induced epithelial–mesenchymal transition (EMT) in gallbladder cancer32. TGF-β is a platelet-granule constituent21 and airway remodeling is consistent with EMT in MP infection 1. Thus, we think FCGBP and platelet activation are very important in airway remodeling of SMPP. Among the identified TFs, TCF7L1 ranks top 3, which is a member of the T cell factor/lymphoid enhancer factor family of TFs and involves in the wnt signaling pathway to allow human stem cell differentiation precisely33,34. And a study based on smallpox vaccine response genome-wide association indicated that TCF7L1 is critical for lymphocyte IFNγ production or cytotoxic T cell function35, showing its important role in regulating inflammatory response and eradicating foreign pathogens. Besides, numerous studies have linked the activation of CEBPB and RELA with immune and inflammatory responses, implicating they might orchestrate with FCGBP for the development of SMPP. RELA is a subunit of NF-kB, which is a major transcriptional regulator of genes involved in survival, proliferation and inflammation. NF-kB activation has been reported in MP infection36. Our results suggest that these TFs might regulate the expression of FCGBP, thus promote the development and increase the severity of SMPP.
Anti-inflammatory treatment mainly glucocorticoid is extremely important in SMPP. However, glucocorticoid resistance has been found in many SMPP patients 8. Comparing to SMPP, steroid binding was enriched in GMPP (Figure 1C), which might predict a good prognosis. In addition, large amounts of SMPP patients miss their best glucocorticoid therapeutic opportunity in the early stage. Besides, glucocorticoid had side effects like osteoporosis and increased risk of secondary infection. As patients developing into SMPP would lead to respiratory failure, massive pleural effusion, atelectasis, necrotizing pneumonia, even subsequent BO, and even life-threatening, finding drugs preventing GMPP from developing into SMPP is of critical importance. In our study, 25 drugs were predicted, which may give us more chance to treat the disease early and effectively. MTOR inhibitor, sirolimus (rapamycin), which was also identified. Vascular endothelial growth factor (VEGF) released by endothelial cells induces angiogenesis and its elevated expression level is observed in some MPP37 and SMPP patients (unpublished data). MTOR inhibitor, an immunosuppressive agent and a macrolide compound, was isolated in 1975 fromStreptomyces hygroscopicus in a soil sample from Easter Island, which may effectively suppress inflammation and angiogenesis including VEGF, and block T-cell activation and proliferation. Sirolimus may be useful for the treatment of acute lung injury38 and decreases airway remodeling caused by TGF -α induced/EGF receptor-mediated signaling 38. The TOR pathway regulates morphogenesis and responses to host cells in the fungal pathogen Candida albicans39, which suggests it may also regulate the responses to host cells in MPP. Low dosages of sirolimus had a good profile of safety and tolerability in pediatric patients 40. Considering the evidence of angiogenesis, excessive inflammatory response, T cell-proliferation and airway remodeling in SMPP, we highly suspect that sirolimus, a macrolide antibiotic, is a promising drug target for the treatment of SMPP. Further investigations about the drug sensitivity test to MP, the mechanisms of MTOR inhibitor in the treatment of SMPP and randomized clinical trial are needed to promote its clinical usage.