Serum-integrated omics reveal the host response landscape for severe pediatric community-acquired pneumonia

Community-acquired pneumonia (CAP) is the leading cause of death among children under five years of age globally, with 16.4 million hospitalizations every year [1, 2]. In China, a total of 1.42 million cases were reported as having one or more episodes of CAP, resulting in 1.48 million CAP episodes [3]. Approximately 8–20% of children hospitalized with CAP progress to severe disease, and many of these, especially infants, require admission to the pediatric intensive care unit (PICU) [1]. These severe cases require advanced interventions, such as invasive and non-invasive mechanical support to reduce the mortality rate of severe cases.

Diagnosis of pediatric CAP is often difficult due to the poor-quality evidence in clinical data, such as atypical imaging findings, complex clinical indicators, and poor prognostic signs [4, 5]. Failure to provide timely diagnosis and treatment may lead to acid-base balance disorders causing multiple organ failure and even septic shock in critically ill children. Thus, it is essential to develop new methods for early assessment of which cases are likely to become clinically severe. In addition, disease progression of CAP is a complex, multi-system process, and its underlying molecular mechanisms remain unclear. Changes in systemic responses may be caused by a complex set of factors including pathogens, genetic predisposition, and immune response. As a result, these factors may alter proteins and the downstream metabolites involved in disease progression [6]. Therefore, it is important to determine if host-derived proteins and metabolites in the circulation system are connected to the pathogenesis and progression of severe CAP.

Recent multi-omics studies have aimed to identify biomarkers and understand complex systemic changes which contribute to pathogenesis. Serum is the major container for small molecules whose relative amounts can provide valuable insights into disease pathogenesis [7, 8]. Previous studies have used serum proteins and/or metabolites to distinguish infectious disease from healthy controls. For example, one study identified a set of proteins able to accurately distinguish and predict COVID-19 outcomes [9], while in another study, metabolomics was combined with a random forest-based classification model and identified potential biomarkers for diagnosis of Mycoplasma pneumoniae pneumonia [10]. For CAP, metabolomics has been used to distinguish CAP from healthy individuals and identify metabolite signatures which correlate with disease severity [11]. Moreover, plasma lipidomics was also found to be useful in predicting the 90-day mortality prognosis in bacterial CAP [12]. Currently, in CAP, it is unclear which protein or metabolic pathways are involved in disease progression or what their combined roles are, especially in children. Thus, an integrated analysis of the proteome and metabolome may provide new avenues for understanding severe CAP.

Here, we used proteomics and metabolomics to profile the host response in CAP serum samples in a training cohort containing severe CAPs (S-CAPs), non-severe CAPs (NS-CAPs) and healthy controls (CONs). Our study uncovered several host proteins and metabolites that were altered in CAP. To identify potential biomarkers, we developed a machine learning-based pipeline that identified a combination of biomarkers that could accurately distinguish S-CAPs from controls. These selected biomarkers and combinations were then validated using enzyme-linked immunosorbent assay (ELISA) and metabolomics in a second validation cohort. Finally, the proteomics and metabolomics data generated in this study provided a global overview of the molecular changes, which may provide useful insight into the development of new therapeutics for treatment of CAP.

Severe pediatric CAP is a critical public health threat to children’s health. Although bacterial and viral infections may lead to different results, both present with symptoms of pneumonia. Healthcare associated pneumonia is no longer recognized as a distinct entity, but as a form of CAP, and there is increasing evidence of bacterial and virus as etiological agents of CAP. Due to the complexity and heterogeneity of the disease, diagnosis of CAP, especially for severe CAP, remains a clinical challenge. Therefore, it is important to identify early biomarkers that can detect the severity of CAP. For this purpose, we applied proteomics and metabolomics to test the serum protein and metabolite changes associated with severe CAP. To our knowledge, this is the first study to combine proteomic and metabolomic data obtained from children with CAP and different disease severity. Our study identified 2 proteins (CRP, LBP) and 3 metabolites [Fasciculol C, PE (14:0/16:1(19Z)), PS (20:0/22:6(4Z, 7Z, 10Z, 13Z, 16Z, 19Z))], which are good candidates to identify severe CAP cases from non-severe CAP cases and controls. These candidates were further validated in an independent cohort.

In this study, the proteomics and metabolomics data generated also enabled a systematic analysis of the molecular pathology in CAP. The development of children's lung function is not perfect, so age is likely to be an important factor affecting metabolism and morbidity. Therefore, we age-matched the cases and controls to minimize the influence of age on protein and metabolite abundance in each group. Significantly DAPs were identified to be involved in essential biological processes such as cell death, the complement system, coagulation cascades, platelet function and metabolic dysregulation. Our results are consistent with previous findings that severe CAP cases are frequently associated with acute respiratory distress syndrome, sepsis, and multi-organ injury [34], which were pathophysiologically associated with cell death activation pathway, intravascular coagulation and microthrombosis [34]. Our data revealed the molecular changes in CAP sera, which could potentially reflect the occurrence of cell damage in CAP. Here, we observed that severe CAP patients are often accompanied by tissue damage and inflammation. Higher expression of lysosome-related proteins, cytotoxicity-related proteins and phagosome-related proteins were observed in S-CAPs, suggesting that various cell death pathways contribute to the development of severe pneumonia. Lysosomes which are found in pre-existing endolysosomes or autolysosomes act as an important bridge between autophagy and endocytosis [35]. Thus, as an important regulator of cell death, lysosomes, cytotoxicity proteins and the phagosome may be involved in exacerbating CAP leading to the development of severe disease.

Our data also observed activation of the complement system and inflammation system in CAPs. Here, multiple acute phase proteins such as CRP and complement-related proteins were upregulated in CAPs. It has been reported that CRP assists in activation of the complement system [36]. This induces the production of cytokines and chemokines, potentially resulting in a ‘‘cytokine storm’’ [36]; and also recruits macrophages from the peripheral blood, which may lead to acute lung injury. Since ~ 50% of platelets are produced in the lungs [37], these platelets may help to aggravate lung injury and further induce cytokine storm. For example, C4BPB [18] and F11 [19] which are regulators of complement system were significantly decreased in S-CAP cases. PROC, which interacts with C4BP [20], was also downregulated in S-CAPs. Moreover, CFHR3 [21], CFHR4, CFHR5 [22] and CR2 were also decreased in S-CAP patients compared to CONs. Complement and coagulation, together with platelet dysfunction, act as the linchpin in events leading to thromboinflammation [17]. Declining platelet count has also been associated with poor outcomes in CAP patients [23]. Two of the most intriguing proteins downregulated in severe patients were vasodilator-stimulated phosphoprotein (VASP) and integrin alpha-IIb (ITGA2B). VASP is an actin regulatory protein implicated in platelet adhesion [24] while ITGA2B encodes aIIb and is an important gene associated with COVID-19-related stroke [25]. In addition, the expression of most complement proteins, coagulation cascade proteins and platelet-related proteins were negatively associated with disease severity. Interestingly, the levels of platelet-related proteins, such as collagen alpha-1(I) (COL1A1), ITGA2B, fermitin family homolog 3 (FERMT3), talin-1 (TLN1) and VASP were positively correlated with TT levels, while negatively correlated with FIB levels, which are essential clinical indexes. Additionally, levels of fibrinogen alpha (FGA) and fibrinogen beta (FGB) were positively correlated with FDP levels (clinical index). Recently, increasing evidence indicates a potential cross-talk between complement factors and platelet activation, contributing to the pathophysiology of diseases and subsequent tissue remodeling processes [17]. Therefore, activation of the cell death pathway, the inflammatory response and a dysregulated complement, coagulation cascade and platelet function are predicted to cause tissue damage in children with CAP.

Cross-talk between glucose metabolism and nucleotide metabolism were observed in CAP cases. Nucleotides are the building blocks for DNA and RNA synthesis. Glucose metabolic pathways such as the PPP and TCA cycle promote nucleotide formation by increasing the supply of glutamate and/or PRPP [29, 38]. In this study, the levels of PRPP and glutamine were significantly upregulated in NS-CAPs and S-CAPs. Moreover, the nucleotide (CMP) and most deoxynucleotide (dAMP, dGMP, dCMP, dUMP) were also elevated in NS-CAP and/or S-CAPs. One explanation for this “cross-talk” might reflect increased DNA and RNA synthesis in CAP patients due to proliferation of immune cells as nucleotides are required for replication [29]. Modulating nucleotide metabolism may also increase the host immune response against pathogen attack [29, 39]. Furthermore, increased nucleotides and deoxynucleotides in the serum suggests higher RNA turnover and DNA degradation possibly due to apoptosis of host cells or immune cells. Consistent with previous reports, RNA turnover and DNA degradation are increased in inflammatory diseases [40, 41]. The role of increased (deoxy)nucleotides in the pathogenesis of pneumonia requires further research; however, it is possible that higher levels of nucleotides lead to unbalanced deoxyribonucleotide pools which, in turn, contribute to the progression to severe CAP.

In addition to our findings of altered glucose and nucleotide metabolism in CAPs, we also uncovered dysregulated metabolites for lipid metabolism which are important for regulation of signal transduction and immune activation processes. Previously, Ning et al. [11] suggested that sphingolipid metabolism was significantly affected in CAPs, and that lipid dysfunction was one of the potential pathological mechanisms. In another study on serum metabolites and lipid alterations in CAPs, sphingolipids were strongly correlated with respiratory function, the cardiovascular system and liver function [42]. Similarly, our data also showed that lower sphingolipids were detected in both NS and S-CAP patients. In addition, dysregulated expression of APOM was reported to be associated with virus infection [7]. This finding was consistent with our finding that the levels of apolipoproteins, which are involved in the transport and redistribution of lipids, were significantly dysregulated in both NS-CAP and S-CAP patients. Moreover, it is known that pulmonary surfactant is a protein-lipid mixture secreted by type-II alveolar epithelial cells. Impaired surfactant function in lung is thought to be an essential mechanism for pneumonia after pathogen infection. Thus, altered lipid metabolism in this study might have also been induced by surfactant metabolism dysfunction after pathogen infection. Furthermore, it has been reported that CAPs with pulmonary diffusing capacity affect oxygen transport and mitochondria changes in the β-oxidation pathway in children, especially young children. The previous study also reported that lipid catabolism can been improved by enhanced lipolytic and fatty acid β-oxidation pathways [43]. Thus, we hypothesize that lipid metabolism and anaerobic pathways can be altered by the damage of pulmonary diffusing capacity due to lack of adequate oxygen, as well as beta-oxidation pathways in mitochondria due to CAP. Together, these data collectively indicate that dysregulated lipid metabolism is involved in the pathological mechanism of CAP disease progression.

There are still some limitations to this study which needs to be considered. Although our samples were age-matched, there may still be other genetic, clinical or environmental confounding factors such as pathogen type that may not have been detected or controlled for. Furthermore, although our results were verified using an independent cohort, further verification using larger samples sizes are still needed.

In conclusion, this study provides a systematic proteomic and metabolomic investigation of serum samples taken from severe and mild CAP patients as well as control groups. We demonstrated the potential of a panel of serum proteins and metabolites that can identify CAP cases which may progress into severe pneumonia. Although we successfully validated our serum biomarker panel in an independent testing cohort, the two cohort sizes are small and may require larger samples sizes to confirm our findings. Our data also laid out the molecular profile of serum changes in pediatric CAP, which may provide additional useful diagnostic markers and information for the development of therapeutic interventions in children who develop severe pneumonia.