Multiomic Analysis of Neuroinflammation and Occult Infection in Sudden Infant Death Syndrome

Abstract

Importance: Antemortem infection is a risk factor for sudden infant death syndrome (SIDS)-the leading postneonatal cause of infant mortality in the developed world. Manifestations of infection and inflammation are not always apparent in clinical settings or by standard autopsy; thus, enhanced resolution approaches are needed.

Objective: To ascertain whether a subset of SIDS cases is associated with neuroinflammation and occult infection.

Design, setting, and participants: In this case-control study, postmortem fluids from SIDS cases and controls collected between July 2011 and November 2018 were screened for elevated inflammatory markers, specifically cerebrospinal fluid (CSF) neopterin and CSF and serum cytokines. CSF, liver, and brain tissue from SIDS cases with elevated CSF neopterin were subjected to metagenomic next-generation sequencing (mNGS) to probe for infectious pathogens. Brainstem tissue from a subset of these cases was analyzed by single-nucleus RNA sequencing (snRNAseq) to measure cell type-specific gene expression associated with neuroinflammation and infection. All tissue and fluid analyses were performed from April 2019 to January 2023 in a pathology research laboratory. Included was autopsy material from infants dying of SIDS and age-matched controls dying of known causes.

Exposures: There were no interventions or exposures.

Main outcomes and measures: CSF neopterin levels were measured by high-performance liquid chromatography. Cytokines were measured by multiplex fluorometric assay. mNGS was performed on liver, CSF, brain, and brainstem tissue. snRNAseq was performed on brainstem tissue.

Results: A cohort of 71 SIDS cases (mean [SD] age, 55.2 [11.4] postconceptional weeks; 42 male [59.2%]) and 20 controls (mean [SD] age, 63.2 [16.9] postconceptional weeks; 11 male [55.0%]) had CSF and/or serum available. CSF neopterin was screened in 64 SIDS cases and 15 controls, with no exclusions. Tissues from 6 SIDS cases were further analyzed. For CSF neopterin measures, SIDS samples were from infants with mean (SD) age of 54.5 (11.3) postconceptional weeks (38 male [59.4%]) and control samples were from infants with mean (SD) age of 61.5 (17.4) postconceptional weeks (7 male [46.7%]). A total of 6 SIDS cases (9.3%) with high CSF neopterin were identified, suggestive of neuroinflammation. mNGS detected human parechovirus 3 (HPeV3) in tissue and CSF from 1 of these 6 cases. snRNAseq of HPeV3-positive brainstem tissue (medulla) revealed dramatic enrichment of transcripts for genes with predominately inflammatory functions compared with 3 age-matched SIDS cases with normal CSF neopterin levels.

Conclusions and relevance: Next-generation molecular tools in autopsy tissue provide novel insight into pathogens that go unrecognized by normal autopsy methodology, including in infants dying suddenly and unexpectedly.

Figure 1.. Study Design and Cerebrospinal Fluid (CSF) Neopterin

A, The study design flowchart with darker gray boxes representing data obtained as part of a larger study (not reported here) and the remaining boxes representing data reported here. The study flowchart was designed using Biorender software. B, Levels of CSF neopterin in all controls and all sudden infant death syndrome (SIDS) with available CSF. The human parechovirus 3 (HPeV3)–positive SIDS is shown in orange. snRNAseq indicates single-nucleus RNA sequencing.

Figure 2.. Human Parechovirus 3 (HPeV3) Phylogenetic Analysis and Abundance, Inflammatory Mediators, and Neuropathology

A, Phylogenetic analysis of the human parechovirus 3 (HPeV3)–positive SIDS case with reads derived from metagenomic next-generation sequencing (mNGS) from liver tissue where 99% genome coverage was achieved. Sequences were downloaded from the GenBank database. Multiple alignment was performed using Multiple Sequence Comparison by Log-Expectation. The phylogenetic tree was built using Geneious Tree builder. HKY Genetic distance model was used with the UPGMA tree-building method. B, mNGS-positive tissue and abundance of HPeV3 found in each source with biologic and technical replicates performed. The y-axis is represented as log 10 for reads per million at each source. Cerebrospinal fluid (CSF) levels of neopterin (C) and serum levels of macrophage inflammatory protein 1b (MIP-1b) (D) in the HPeV3-positive SIDS case (n = 1, orange dot), compared with controls (n = 15) and the noninflammatory SIDS cases (n = 3). E, Hematoxylin-eosin–stained section of periventricular white matter showing an area of cystic change (arrow; magnification ×200). Reactive astrogliosis is also present (arrowheads). F, Occasional macrophages are highlighted by the CD68 immunostain (magnification ×200). CCL4 indicates C-C motif chemokine ligand 4.

Figure 3.. Single-Nucleus RNA Sequencing (snRNAseq) Reveals Cell-Type–Specific Gene Regulation Associated With Human Parechovirus 3 (HPeV3) Infection in the Medulla

A, Two-dimensional (2-D) uniform manifold approximation and projection (UMAP) embedding showing transcriptomic similarity/dissimilarity relationships between 24 clusters (cell types) identified across integrated samples. B, Same as panel A but with individual nuclei colors corresponding to the sample from which they were obtained. The percentage sample composition for each cluster (from A) can be visualized in the vertical bar plots. C, Number of genes with significantly higher or lower transcript abundance in HPeV3-positive sudden infant death syndrome (SIDS) compared with 3 noninflammatory SIDS for each cluster. D, Heat maps showing expression profiles (expressed as z scores) of the top 20 most significantly differentially expressed genes for various clusters. ACH indicates acetylcholine; CALCA/B+, calcitonin-related polypeptide α and β; CRH+, corticotropin-releasing hormone; FOXP2+, forkhead box P2; GLU, glutamate; GRIK+, glutamate ionotropic receptor kainite type; HTR2C+, 5-hydroxytryptamine receptor 2C; NEUROD1+, neuronal differentiation 1; PHOX2B+, paired like homeobox 2b; PVALB+, parvalbumin; SMC, smooth muscle cells; 5-HT, 5-hydroxytryptamine/serotonin.