Identification of TLR-specific response and biomarkers in the rhesus macaque
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Toll-Like Receptors (TLRs) are a series of innate immune receptors which detect Pathogen Associated Molecular Patterns (PAMPs) and Damage Associated Molecular Patterns (DAMPs), stimulating pro-survival and pro-inflammatory activity at the site of infection via transcription factors NF-êB, AP-1, and in some cases type 1 interferon. While there are substantial similarities pathways induced by various TLRs, with many inducing NF-êB through adaptor protein myeloid differentiation primary response gene 88 (MyD88), variations exist in the form of differential adaptor protein induction activation and receptor localization. Further, these receptors are subdivided into two major categories, intracellular TLRs which primarily respond to viral agonists and extracellular, which primarily respond to bacterial agonists. In this study, we examined the differences in induced gene expression between agonists of extracellular TLRs, specifically TLR2/6 agonist CBLB612, TLR4 agonist LPS, and TLR5 agonist CBLB502, with the goal of identifying TLR-specific biomarkers and phenotypic differences in response. We compared TLR2, 4, and 5 response in the liver, bladder, duodenum, colon, ileum, and rectum of Rhesus macaques injected with TLR agonists in vivo, with induced mRNA harvested and 30 minutes or 2 hours after treatment and analyzed via microarray analysis. We have found that the liver is strongly responsive to all 3 tested agonists; in contrast, CBLB612 response is relatively weak in the gut relative to CBLB502 or LPS. Within the liver, we have found that much the response to TLR agonists seen at 30 minutes, particularly in response to TLR5 agonist, is transient, likely indicating a biphasic pattern of gene expression whereupon genes induced early on are responsible for gene transcription at later time points rather than simply being the most quickly transcribed. In addition, a significant proportion of the transiently genes induced by CBLB502 contain a binding motif with substantial similarity to that utilized by Tristetraprolin (TTP), gene name ZFP36, a protein known to be involved in mRNA degradation, which may point to its involvement in preventing overexpression of pro-inflammatory response to CBLB502. Finally, we have identified several potential TLR-specific biomarkers, including rapid induction of factors involved in regulation of the circadian rhythm such as BMAL1 by CBLB502, with response to CBLB612 being delayed and LPS response muted in comparison; glycoprotein hormones, alpha chain (GLHA/CGA) and Deiodinase, Iodothyronine, Type II (DIO2), involved in thyroid hormone activation, induced in response to CBLB612; and anti-inflammatory cytokine interleukin 37 (IL37) induced in response to LPS.