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The current level of knowledge on transcriptome responses triggered by endotoxins and glucocorticoids in immune cells in pigs is limited. Therefore, in the present study, we treated porcine peripheral blood mononuclear cells (PBMCs) with lipopolysaccharide (LPS) and dexamethasone (DEX) separately or combined for 2 hours. The resultant transcriptional responses were examined by mRNA sequencing. We found that the LPS treatment triggered pronounced inflammatory responses as evidenced by upregulation of pro-inflammatory cytokines, chemokines, and related signaling pathways like NF-κB. Concurrently, a series of downregulated pro-inflammatory and upregulated anti-inflammatory molecules were identified. These are involved in the inhibition of TLR, NF-κB, and MAPK cascades and activation of signaling mediated by Tregs and STAT3, respectively. These findings suggested that LPS initiated also an anti-inflammatory process to prevent an overwhelming inflammatory response. The transcriptome responses further revealed substantial crosstalk of immune responses and glucocorticoid receptor (GR) signaling. This was apparent in four aspects: constitutive inhibition of T cell signaling by DEX through a subset of genes showing no response to LPS; inhibition of LPS-induced inflammatory genes by DEX; attenuation of DEX action by LPS paralleled by the regulation of genes implicated in cytokine and calcium signaling; and DEX-induced changes in genes associated with the activation of pro-inflammatory TLR, NF-κB, iNOS, and IL-1 signaling. Consequently, our study provides novel insights into inflammatory and GR signaling in pigs, as well as an understanding of the application of glucocorticoid drugs for the treatment of inflammatory disorders.

The consequences of glucocorticoid receptor (GR) hypersensitivity during infection have so far received little attention. We previously discovered that a natural gain-of-function Ala610Val substitution in the porcine GR aggravates response of pigs to lipopolysaccharide (LPS)-induced endotoxemia, which can be alleviated by dexamethasone (DEX) pretreatment. In this work, we investigated the relevant molecular basis of these phenotypes by transcriptomic profiling of porcine peripheral blood mononuclear cells (PBMCs) carrying different GR genotypes, in unstimulated conditions or in response to DEX and/or LPS in vitro. The Val allele differentially regulated abunda+nt genes in an additive-genetic manner. A subset of more than 200 genes was consistently affected by the substitution across treatments. This was associated with upregulation of genes related i.a. to endo-lysosomal system, lipid and protein catabolism, and immune terms including platelet activation, and antigen presentation, while downregulated genes were mainly involved in cell cycle regulation. Most importantly, the set of genes constitutively upregulated by Val includes members of the TLR4/LPS signaling pathway, such as LY96. Consequently, when exposing PBMCs to LPS treatment, the Val variant upregulated a panel of additional genes related to TLR4 and several other pattern recognition receptors, as well as cell death and lymphocyte signaling, ultimately amplifying the inflammatory responses. In contrast, when stimulated by DEX treatment, the Val allele orchestrated several genes involved in anti-inflammatory responses during infection. This study provides novel insights into the impact of GR hypersensitivity on the fate and function of immune cells, which may be useful for endotoxemia therapy. [ABSTRACT FROM AUTHOR]

The current level of knowledge on transcriptome responses triggered by endotoxins and glucocorticoids in immune cells in pigs is limited. Therefore, in the present study, we treated porcine peripheral blood mononuclear cells (PBMCs) with lipopolysaccharide (LPS) and dexamethasone (DEX) separately or combined for 2 hours. The resultant transcriptional responses were examined by mRNA sequencing. We found that the LPS treatment triggered pronounced inflammatory responses as evidenced by upregulation of pro-inflammatory cytokines, chemokines, and related signaling pathways like NF-κB. Concurrently, a series of downregulated pro-inflammatory and upregulated anti-inflammatory molecules were identified. These are involved in the inhibition of TLR, NF-κB, and MAPK cascades and activation of signaling mediated by Tregs and STAT3, respectively. These findings suggested that LPS initiated also an anti-inflammatory process to prevent an overwhelming inflammatory response. The transcriptome responses further revealed substantial crosstalk of immune responses and glucocorticoid receptor (GR) signaling. This was apparent in four aspects: constitutive inhibition of T cell signaling by DEX through a subset of genes showing no response to LPS; inhibition of LPS-induced inflammatory genes by DEX; attenuation of DEX action by LPS paralleled by the regulation of genes implicated in cytokine and calcium signaling; and DEX-induced changes in genes associated with the activation of pro-inflammatory TLR, NF-κB, iNOS, and IL-1 signaling. Consequently, our study provides novel insights into inflammatory and GR signaling in pigs, as well as an understanding of the application of glucocorticoid drugs for the treatment of inflammatory disorders. [ABSTRACT FROM AUTHOR]

The consequences of glucocorticoid receptor (GR) hypersensitivity during infection have so far received little attention. We previously discovered that a natural gain-of-function Ala610Val substitution in the porcine GR aggravates response of pigs to lipopolysaccharide (LPS)-induced endotoxemia, which can be alleviated by dexamethasone (DEX) pretreatment. In this work, we investigated the relevant molecular basis of these phenotypes by transcriptomic profiling of porcine peripheral blood mononuclear cells (PBMCs) carrying different GR genotypes, in unstimulated conditions or in response to DEX and/or LPS in vitro . The Val allele differentially regulated abunda+nt genes in an additive-genetic manner. A subset of more than 200 genes was consistently affected by the substitution across treatments. This was associated with upregulation of genes related i.a. to endo-lysosomal system, lipid and protein catabolism, and immune terms including platelet activation, and antigen presentation, while downregulated genes were mainly involved in cell cycle regulation. Most importantly, the set of genes constitutively upregulated by Val includes members of the TLR4/LPS signaling pathway, such as LY96 . Consequently, when exposing PBMCs to LPS treatment, the Val variant upregulated a panel of additional genes related to TLR4 and several other pattern recognition receptors, as well as cell death and lymphocyte signaling, ultimately amplifying the inflammatory responses. In contrast, when stimulated by DEX treatment, the Val allele orchestrated several genes involved in anti-inflammatory responses during infection. This study provides novel insights into the impact of GR hypersensitivity on the fate and function of immune cells, which may be useful for endotoxemia therapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Li, Hadlich, Wimmers and Murani.)