Abstract
Background: Patients developing meningococcal septic shock reveal levels of Neisseria meningitidis (106-108/mL) and endotoxin (101-103 EU/mL) in the circulation and organs, leading to acute cardiovascular, pulmonary and renal failure, coagulopathy and a high case fatality rate within 24 h.Objective: To investigate transcriptional profiles in heart, lungs, kidneys, liver, and spleen and immunostain key inflammatory cells and proteins in post mortem formalin-fixed, paraffin-embedded (FFPE) tissue samples from meningococcal septic shock patients.Patients and Methods: Total RNA was isolated from FFPE and fresh frozen (FF) tissue samples from five patients and two controls (acute non-infectious death). Differential expression of genes was detected using Affymetrix microarray analysis. Lung and heart tissue samples were immunostained for T-and B cells, macrophages, neutrophils and the inflammatory markers PAI-1 and MCP-1. Inflammatory mediators were quantified in lysates from FF tissues.Results: The transcriptional profiles showed a complex pattern of protein-coding and non-coding RNAs with significant regulation of pathways associated with organismal death, cell death and survival, leukocyte migration, cellular movement, proliferation of cells, cell-to-cell signaling, immune cell trafficking, and inflammatory responses in an organ-specific clustering manner. The canonical pathways including acute phase response-, EIF2-, TREM1-, IL-6-, HMBG1-, PPAR signaling, and LXR/RXR activation were associated with acute heart, pulmonary, and renal failure. Fewer genes were regulated in the liver and particularly in the spleen. The main upstream regulators were TNF, IL-1β, IL-6, RICTOR, miR-6739-3p, and CD3. Increased numbers of inflammatory cells (CD68+, MPO+, CD3+, and CD20+) were found in lungs and heart. PAI-1 inhibiting fibrinolysis and MCP-1 attracting leukocyte were found significantly present in the septic tissue samples compared to the controls.Conclusions: FFPE tissue samples can be suitable for gene expression studies as well as immunostaining of specific cells or molecules. The most pronounced gene expression patterns were found in the organs with highest levels of Neisseria meningitidis DNA. Thousands of protein-coding and non-coding RNA transcripts were altered in lungs, heart and kidneys. We identified specific biomarker panels both protein-coding and non-coding RNA transcripts, which differed from organ to organ. Involvement of many genes and pathways add up and the combined effect induce organ failure.
Highlights
Patients developing meningococcal septic shock reveal levels of Neisseria meningitidis (106-108/mL) and endotoxin (101-103 endotoxin units (EU)/mL) in the circulation and organs, leading to acute cardiovascular, pulmonary and renal failure, coagulopathy and a high case fatality rate within 24 h
The concentration of RNA isolated from FFPE tissue samples from controls ranged from 41 to 439 ng/μL (n = 8 and median 62 ng/μL)
Our study demonstrates that organismal death and multiple defense mechanisms, both pro-and anti-inflammatory, were heavily activated in tissue samples from our patients
Summary
Total RNA was isolated from FFPE and fresh frozen (FF) tissue samples from five patients and two controls (acute non-infectious death). Lung and heart tissue samples were immunostained for T-and B cells, macrophages, neutrophils and the inflammatory markers PAI-1 and MCP-1. The study was approved by the Regional Medical Ethical Committee of South East Norway (2011/1413C “Translational research, meningococcal disease” and 2011/753 “Studies of invasive meningococcal and pneumococcal disease”). The patients’ samples were collected after informed consent from patient parents or relatives and according to the Helsinki declaration. Systemic meningococcal disease (SMD) was present if N. meningitidis was cultivated and or confirmed by polymerase chain reaction (PCR) in blood, cerebrospinal fluid (CSF) or by organ tissue examination (Brandtzaeg et al, 1989a; Ovstebo et al, 2004; Brusletto et al, 2017). Severe septic shock was defined as persistent hypotension because of bacterial infection, with a systolic blood pressure
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