Multi-Omic blood analysis reveals differences in innate inflammatory sensitivity between species

Abstract

Vertebrates differ greatly in responses to pro-inflammatory agonists such as bacterial lipopolysaccharide (LPS), complicating use of animal models to study human sepsis or inflammatory disorders. We compared transcriptomes of resting and LPS-exposed blood from six LPS-sensitive species (rabbit, pig, sheep, cow, chimpanzee, human) and four LPS-resilient species (mice, rats, baboon, rhesus), as well as plasma proteomes and lipidomes. Unexpectedly, at baseline, sensitive species already had enhanced expression of LPS-responsive genes relative to resilient species. After LPS stimulation, maximally different genes in resilient species included genes that detoxify LPS, diminish bacterial growth, discriminate sepsis from SIRS, and play roles in autophagy and apoptosis. The findings reveal the molecular landscape of species differences in inflammation, and may inform better selection of species for pre-clinical models.

Fig. 1.. Rationale of study.

(A) Lethal doses of LPS in different species. Values are selected from representative studies (shown on the x-axis) where LPS was injected i.p. or i.v. and survival reported. Wherever possible, studies using multiple doses of E. coli LPS injected as a single bolus are reported. Resilient species, defined here as those that survive a dose of 1 mg/kg body weight (dashed line), are indicated with red background, sensitive species are indicated with a blue background. References are given in Table S1. (B) Outline of study. Whole blood from 4–5 individuals of 4 resilient species (mouse, rat, rhesus, and baboon) and 6 sensitive species (rabbit, pig, cow, sheep, chimp, human) was incubated ex vivo with 0, 1, 10, or 100 ng/mL E. coli LPS for 2, 6, or 24h followed by leukocyte mRNA sequencing or MS/MS analysis of lipids and proteins was performed on plasma without prior incubation.

Fig. 2.. Overview of plasma proteins identified as differentially abundant in comparison of resilient versus sensitive species.

Three different comparisons were performed resulting in 122 total proteins identified as differentially abundant. Quantitative protein level values are based upon scaled LFQ intensities combined from peptide level intensities. Color scale represented as scaled quantitative abundance differences with brown representing higher abundance and purple lower abundance for each individual protein. A) Mapping of 38 proteins at p <0.05, Pearson correlated. B) Mapping of 41 proteins based upon higher fold-change abundance (+/−3.0 in log2 phase, minimum of 4 occurrences), bimodal correlation. C) Mapping of 43 proteins with yes/no abundance based upon a minimum of 3 species observations, bimodal correlation.

Fig. 3.. Leukocyte transcriptomes at baseline.

(A) Principal component analysis showing overall distribution of individual mRNA abundance between individuals. (B) Regulators of gene expression that are consistent with the differences between expression profiles of resilient and sensitive species. (C) Average expression of four example genes in each species. (D) Clustering of the 50 genes with the greatest expression differences between sensitive and resilient species. Color intensity scale: transcript abundance (log₂ TPM).

Fig. 4.. Cytokine and gene expression responses of leukocytes to LPS stimulation.

(A-D) Cytokine release in a subset of species, measured by Luminex. (E-G) Cluster analysis of the 50 genes with the largest difference in response to 10 ng/mL LPS between sensitive and resilient species after 2h, 6h, and 24h, respectively. Color intensity scale: transcript abundance (log₂ TPM).

Fig. 5.. Genes with divergent responses to LPS stimulation.

(A) The ten genes at each timepoint with the greatest significant (FDR < 0.05) separation in responses between resilient and sensitive animals. Negative numbers indicate genes that are induced less, or repressed more, by LPS in sensitive species than resilient species. (B-D) Responses of individual species in example genes from each timepoint. Each point depicts the change in expression (fold change in TPM following incubation with and without 10 ng/mL LPS relative to incubation for the same times in media without LPS) in blood from an individual animal. Bars indicate median fold change for each species. Dashed line indicates no change on stimulation.