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.