Understanding COVID-19 progression with longitudinal peripheral blood mononuclear cell proteomics: Changes in the cellular proteome over time.

The clinical presentation of COVID-19 is highly variable, and understanding the underlying biological processes is crucial. This study utilized a proteomic analysis to investigate dysregulated processes in the peripheral blood mononuclear cells of patients with COVID-19 compared to healthy volunteers. Samples were collected at different stages of the disease, including hospital admission, after 7 days of hospitalization, and 30 days after discharge. Metabolic pathway alterations and increased abundance of neutrophil-related proteins were observed in patients. Patients progressing to critical illness had significantly low-abundance proteins in the pentose phosphate and glycolysis pathways compared with those presenting clinical recovery. Important biological processes, such as fatty acid concentration and glucose metabolism disorder, remained altered even after 30 days of hospital discharge. Temporal proteomic changes revealed distinct pathways in critically ill and non-critically ill patients. Our study emphasizes the significance of longitudinal cellular proteomic studies in identifying disease progression-related pathways and persistent protein changes post-hospitalization.

Glucose metabolism is upregulated in the mononuclear cell proteome during sepsis and supports endotoxin-tolerant cell function.

Metabolic adaptations shape immune cell function. In the acute response, a metabolic switch towards glycolysis is necessary for mounting a proinflammatory response. During the clinical course of sepsis, both suppression and activation of immune responses take place simultaneously. Leukocytes from septic patients present inhibition of cytokine production while other functions such as phagocytosis and production of reactive oxygen species (ROS) are preserved, similarly to the in vitro endotoxin tolerance model, where a first stimulation with lipopolysaccharide (LPS) affects the response to a second stimulus. Here, we sought to investigate how cellular metabolism is related to the modulation of immune responses in sepsis and endotoxin tolerance. Proteomic analysis in peripheral blood mononuclear cells (PBMCs) from septic patients obtained at intensive care unit admission showed an upregulation of proteins related to glycolysis, the pentose phosphate pathway (PPP), production of ROS and nitric oxide, and downregulation of proteins in the tricarboxylic acid cycle and oxidative phosphorylation compared to healthy volunteers. Using the endotoxin-tolerance model in PBMCs from healthy subjects, we observed increased lactate production in control cells upon LPS stimulation, while endotoxin-tolerant cells presented inhibited tumor necrosis factor-α and lactate production along with preserved phagocytic capacity. Inhibition of glycolysis and PPP led to impairment of phagocytosis and cytokine production both in control and in endotoxin-tolerant cells. These data indicate that glucose metabolism supports leukocyte functions even in a condition of endotoxin tolerance.

Combined Transcriptome and Proteome Leukocyte's Profiling Reveals Up-Regulated Module of Genes/Proteins Related to Low Density Neutrophils and Impaired Transcription and Translation Processes in Clinical Sepsis.

Sepsis is a global health emergency, which is caused by various sources of infection that lead to changes in gene expression, protein-coding, and metabolism. Advancements in "omics" technologies have provided valuable tools to unravel the mechanisms involved in the pathogenesis of this disease. In this study, we performed shotgun mass spectrometry in peripheral blood mononuclear cells (PBMC) from septic patients (N=24) and healthy controls (N=9) and combined these results with two public microarray leukocytes datasets. Through combination of transcriptome and proteome profiling, we identified 170 co-differentially expressed genes/proteins. Among these, 122 genes/proteins displayed the same expression trend. Ingenuity Pathway Analysis revealed pathways related to lymphocyte functions with decreased status, and defense processes that were predicted to be strongly increased. Protein-protein interaction network analyses revealed two densely connected regions, which mainly included down-regulated genes/proteins that were related to the transcription of RNA, translation of proteins, and mitochondrial translation. Additionally, we identified one module comprising of up-regulated genes/proteins, which were mainly related to low-density neutrophils (LDNs). LDNs were reported in sepsis and in COVID-19. Changes in gene expression level were validated using quantitative real-time PCR in PBMCs from patients with sepsis. To further support that the source of the upregulated module of genes/proteins found in our results were derived from LDNs, we identified an increase of this population by flow cytometry in PBMC samples obtained from the same cohort of septic patients included in the proteomic analysis. This study provides new insights into a reprioritization of biological functions in response to sepsis that involved a transcriptional and translational shutdown of genes/proteins, with exception of a set of genes/proteins related to LDNs and host-defense system.

Post-treatment with the PPAR-γ agonist pioglitazone inhibits inflammation and bacterial growth during Klebsiella pneumonia.

Agonists of peroxisome proliferator-activated receptor (PPAR)-γ have been suggested as potential adjuvant therapy in bacterial pneumonia because of their capacity to inhibit inflammation and enhance bacterial clearance. Previous studies only assessed the effects of pretreatment with these compounds, thereby bearing less relevance for the clinical scenario. Moreover, PPAR-γ agonists have not been studied in pneumonia caused by Klebsiella pneumoniae, a common human respiratory pathogen of which antibiotic treatment is hampered by increasing antimicrobial resistance. Here we show that administration of the PPAR-γ agonist pioglitazone 6 or 8 h after infection of mice with a highly virulent strain of Klebsiella pneumoniae via the airways results in reduced cytokine and myeloperoxidase levels in the lungs at 24 h after infection, as well as reduced bacterial growth in the lungs and decreased dissemination to distant organs at 42 h post-infection. These results suggest that pioglitazone may be an interesting agent in the treatment of Klebsiella pneumonia.

HIF-1α and Hypoxia Responsive Genes are Differentially Expressed in Leukocytes From Survivors and Non-Survivors Patients During Clinical Sepsis.

ABSTRACT: Hypoxia inducible factor 1 alpha (HIF-1α) is linked to the metabolic and immune alterations in septic patients. Stabilization of HIF-1α by hypoxia or inflammation promotes the expression of several genes related to glycolytic metabolism, angiogenesis, coagulation, cell proliferation, and apoptosis. Here, we analyzed public available blood transcriptome datasets from septic patients and evaluated by PCR array the expression of HIF-1α and other hypoxia responsive genes in peripheral blood mononuclear cells from patients with sepsis secondary to community acquired infections. Samples were collected at intensive care unit admission (D0, n=29) and after 7 days follow-up (D7, n = 18); healthy volunteers (n = 10) were included as controls. Hypoxia and glycolysis were among the top scored molecular signatures in the transcriptome datasets. PCR array showed that 24 out of 78 analyzed genes were modulated in septic patients compared with healthy volunteers; most of them (23/24) were downregulated at admission. This same pattern was observed in surviving patients, while non-survivors presented more upregulated genes. EGLN1, EGLN2, and HIF1AN, inhibitors of HIF-1α activation were downregulated in patients, regardless of the outcome, while HIF-1α and other target genes, such as PDK1 and HMOX1, expression were higher in non-survivors than in survivors, mainly at D7. Non-survivor patients also presented a higher SOFA score and lower PaO2/FiO2 ratio. Our results indicate a differential modulation of hypoxia pathway in leukocytes between septic patients who survived and those who did not survive with an increased intensity at D7, which is possibly influenced by disease severity and may affect the immune response in sepsis.

Lipid metabolism impairment in patients with sepsis secondary to hospital acquired pneumonia, a proteomic analysis.

BACKGROUND: Sepsis is a dysregulated host response to infection and a major cause of death worldwide. Respiratory tract infections account for most sepsis cases and depending on the place of acquisition, i.e., community or hospital acquired infection, differ in etiology, antimicrobial resistance and outcomes. Accordingly, the host response may be different in septic patients secondary to community-acquired pneumonia and hospital acquired pneumonia (HAP). Proteomic analysis is a useful approach to evaluate broad alterations in biological pathways that take place during sepsis. Here we evaluated plasma proteome changes in sepsis secondary to HAP. METHODS: Plasma samples were obtained from patients (n = 27) at admission and after 7 days of follow-up, and were analyzed according to the patients' outcomes. The patients' proteome profiles were compared with healthy volunteers (n = 23). Pooled plasma samples were labeled with isobaric tag for relative and absolute quantitationand analyzed by LC-MS/MS. We used bioinformatics tools to find altered functions and pathways. Results were validated using biochemical estimations and ELISA tests. RESULTS: We identified 159 altered proteins in septic patients; most of them were common when comparing patients' outcomes, both at admission and after 7 days. The top altered biological processes were acute inflammatory response, response to wounding, blood coagulation and homeostasis. Lipid metabolism emerged as the main altered function in patients, with HDL as a central node in the network analysis, interacting with downregulated proteins, such as APOA4, APOB, APOC1, APOL1, SAA4 and PON1. Validation tests showed reduced plasma levels of total cholesterol, HDL-C, LDL-C, non-HDL cholesterol, apolipoproteins ApoA1 and ApoB100, and Paraoxonase 1 in HAP patients. CONCLUSION: Proteomic analysis pointed to impairment of lipid metabolism as a major change in septic patients secondary to HAP, which was further validated by the reduced levels of cholesterol moieties and apolipoproteins in plasma. Our results stress the involvement of lipids in the pathogenesis of sepsis, which is in accordance with previous reports supporting the role of lipid moieties in pathogen toxin clearance and in modulating inflammatory responses.

Trypanosoma cruzi extracellular amastigotes selectively trigger the PI3K/Akt and Erk pathways during HeLa cell invasion.

Cell invasion by Trypanosoma cruzi extracellular amastigotes involves different signaling pathways to induce phagocytosis-like mechanisms. Previous works indicated that PI3K/Akt, Src and Erk might be involved in EA invasion; however, participation of these molecules in this process remains elusive. Here, we observed that EA activated Akt, Erk but not Src. Interference of EA invasion with specific inhibitors corroborated this observation. Our results show that EA is capable of selectively triggering complex signaling pathways. Activation of PI3K/Akt and Erk, kinases related to actin cytoskeleton rearrangement and phagocytosis, reinforces the idea that T. cruzi EA subverts the phagocytic machinery during invasion.

Trypanosoma cruzi: single cell live imaging inside infected tissues.

Although imaging the live Trypanosoma cruzi parasite is a routine technique in most laboratories, identification of the parasite in infected tissues and organs has been hindered by their intrinsic opaque nature. We describe a simple method for in vivo observation of live single-cell Trypanosoma cruzi parasites inside mammalian host tissues. BALB/c or C57BL/6 mice infected with DsRed-CL or GFP-G trypomastigotes had their organs removed and sectioned with surgical blades. Ex vivo organ sections were observed under confocal microscopy. For the first time, this procedure enabled imaging of individual amastigotes, intermediate forms and motile trypomastigotes within infected tissues of mammalian hosts.