Unknown,Transcriptomics,Genomics,Proteomics

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Effect of TLR Ligation on the Differentiation and Function of Human mMDSC

ABSTRACT: Immunosuppressive microenvironments block the activity of tumoricidal T and NK cells, allowing cancers to avoid immune elimination. Monocytic myeloid-derived suppressor cells (mMDSC) are an important component of these immunosuppressive milieus. Human mMDSC respond to stimulation via their Toll-like receptors by differentiating into macrophage. Agonists targeting TLRs 1/2 (such as PAM3) induce mMDSC to mature into immumosuppressive M2-like macrophage through a process that involves TNF and IL6. Agonists targeting TLRs 7/8 (such as R848) cause the same precursors to mature into tumoricidal M1-like macrophages, a process in which IL12 plays a central role. The immune suppression mediated by mMDSC is reversed by exposure to TLR 7/8 agonists which thus might be useful adjuncts for tumor immunotherapy. In this study we looked at several time points. hMDSC from four different donors were sorted from PBMC then cultured in the presence of R848, Pam 3 or left unstimulated for 30, 75 and 225 minutes. Late time points included samples from seven donors after 3 days in culture with the same TLR ligands. For microarray experiments total RNA was extracted, amplified into aRNA and coupled to Cy5. Similarly amplified RNA from universal RNA was coupled to Cy3 and used as a reference for each array.

ORGANISM(S): Homo sapiens

SUBMITTER: Dennis Klinman

PROVIDER: E-GEOD-57032 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Among both healthy and immunocompromised patient populations, pneumonia is a leading cause of death worldwide. Yet, despite structural vulnerability resulting in recurrent exposure to pathogens, the lungs’ mucosal immunity successfully suppresses most infections. We recently reported that these innate defenses can be substantially augmented by inhalational exposure to a crude bacterial lysate, protecting broadly against respiratory pathogens, including lethal pneumonia caused by bacteria, fungi or viruses. The phenomenon of inducible resistance is associated with rapid pathogen killing in the lungs and persists in the absence of the typical leukocytes of innate immunity. Rather, the respiratory epithelium appears to be the predominant effector. Toll-like receptors (TLRs) are highly conserved pattern recognition receptors crucial to host defense through the sensing of pathogen associated molecular patterns. Given the importance of TLRs to mucosal immunity, the presence of numerous pathogen associated molecular patterns in the bacterial lysate, and the induction of many TLR-dependent genes following lysate treatment, we hypothesized that induced resistance follows simultaneous stimulation of multiple TLRs. To test this, we challenged mice deficient in TLR/IL1R adaptor proteins and found that resistance could not be induced in mice lacking MyD88. Having identified this phenomenon to be MyD88-dependent, we sought to determine whether the protective phenomenon could be recapitulated by treatment with synthetic TLR agonists. Mice were treated with aerosolized TLR ligands, alone and in combination, prior to infection with virulent pathogens. While limited protection against pneumonia was afforded by the individual TLR ligands, we discovered that the synergistic combination of diacylated lipopetide TLR2/6 agonist Pam2CSK4 and CpG oligodeoxynucleotide TLR9 agonist ODN2395 induced profound resistance against all tested pathogens. This combination also induced greater than additive pathogen killing in the lungs of challenged mice, and we found that the combination could effectively induce pathogen killing by respiratory epithelial cells in vitro. In order to better understand the mechanisms underlying the inducible pathogen killing by this unique combination of TLR agonists, we performed microarray analysis of murine MLE-15 respiratory epithelial cells following 4 h treatment with PBS (sham treatment), Pam2CSK4 alone, ODN2395 alone, or the combination of both agonists, using Illumina Sentrix MouseRef-8 v2 BeadChips. This allows for assessment of differential gene expression, not only between treated and untreated, but between single and combination treated. The intent of the experiment is to gain insight into the transcriptionally-regulated means by which TLR2/6 and TLR9 signaling pathways synergistically interact. Keywords: Differential expression, epithelium, in vitro

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Effect of TLR Ligation on the Differentiation and Function of Human mMDSC

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