Mechanisms underlying the functional cooperation between PPARα and GRα to attenuate inflammatory responses

Glucocorticoids (GCs) act via the glucocorticoid receptor (NR3C1, GR alpha) to combat overshooting responses to infectious stimuli, including lipopolysaccharide (LPS). As such, GCs inhibit the activity of downstream effector cytokines, such as tumor necrosis factor (TNF). PPAR alpha (NR1C1) is a nuclear receptor described to function on the crossroad between lipid metabolism and control of inflammation. In the current work, we have investigated the molecular mechanism by which GCs and PPAR alpha agonists cooperate to jointly inhibit NF-kappa B-driven expression in A549 cells. We discovered a nuclear mechanism that predominantly targets Mitogen- and Stress-activated protein Kinase-1 activation upon co-triggering GR alpha and PPAR alpha. In vitro GST-pull down data further support that the anti-inflammatory mechanism may additionally involve a non-competitive physical interaction between the p65 subunit of NF-kappa B, GR alpha, and PPAR alpha. Finally, to study metabolic effector target cells common to both receptors, we overlaid the effect of GR alpha and PPAR alpha crosstalk in mouse primary hepatocytes under LPS-induced inflammatory conditions on a genome-wide level. RNA-seq results revealed lipid metabolism genes that were upregulated and inflammatory genes that were additively downregulated. Validation at the cytokine protein level finally supported a consistent additive anti-inflammatory response in hepatocytes

Chromatin recruitment of activated AMPK drives fasting response genes co-controlled by GR and PPARα

Adaptation to fasting involves both Glucocorticoid Receptor (GRα) and Peroxisome Proliferator-Activated Receptor α (PPARα) activation. Given both receptors can physically interact we investigated the possibility of a genome-wide cross-talk between activated GR and PPARα, using ChIP- and RNA-seq in primary hepatocytes. Our data reveal extensive chromatin co-localization of both factors with cooperative induction of genes controlling lipid/glucose metabolism. Key GR/PPAR co-controlled genes switched from transcriptional antagonism to cooperativity when moving from short to prolonged hepatocyte fasting, a phenomenon coinciding with gene promoter recruitment of phosphorylated AMP-activated protein kinase (AMPK) and blocked by its pharmacological inhibition. In vitro interaction studies support trimeric complex formation between GR, PPARα and phospho-AMPK. Long-term fasting in mice showed enhanced phosphorylation of liver AMPK and GRα Ser211. Phospho-AMPK chromatin recruitment at liver target genes, observed upon prolonged fasting in mice, is dampened by refeeding. Taken together, our results identify phospho-AMPK as a molecular switch able to cooperate with nuclear receptors at the chromatin level and reveal a novel adaptation mechanism to prolonged fasting

The autophagy receptor SQSTM1/p62 mediates anti-inflammatory actions of the selective NR3C1/glucocorticoid receptor modulator compound A (CpdA) in macrophages

Glucocorticoids are widely used to treat inflammatory disorders; however, prolonged use of glucocorticoids results in side effects including osteoporosis, diabetes and obesity. Compound A (CpdA), identified as a selective NR3C1/glucocorticoid receptor (nuclear receptor subfamily 3, group C, member 1) modulator, exhibits an inflammation-suppressive effect, largely in the absence of detrimental side effects. To understand the mechanistic differences between the classic glucocorticoid dexamethasone (DEX) and CpdA, we looked for proteins oppositely regulated in bone marrow-derived macrophages using an unbiased proteomics approach. We found that the autophagy receptor SQSTM1 but not NR3C1 mediates the anti-inflammatory action of CpdA. CpdA drives SQSTM1 upregulation by recruiting the NFE2L2 transcription factor to its promoter. In contrast, the classic NR3C1 ligand dexamethasone recruits NR3C1 to the Sqstm1 promoter and other NFE2L2-controlled gene promoters, resulting in gene downregulation. Both DEX and CpdA induce autophagy, with marked different autophagy characteristics and morphology. Suppression of LPS-induced Il6 and Ccl2 genes by CpdA in macrophages is hampered upon Sqstm1 silencing, confirming that SQSTM1 is essential for the anti-inflammatory capacity of CpdA, at least in this cell type. Together, these results demonstrate how off-target mechanisms of selective NR3C1 ligands may contribute to a more efficient anti-inflammatory therapy

Co-activation of glucocorticoid receptor and peroxisome proliferator-activated receptor-γ in murine skin prevents worsening of atopic march

Children with atopic dermatitis show an increased risk to develop asthma later in life, a phenomenon referred to as "atopic march," which emphasizes the need for secondary prevention therapies. This study aimed to investigate whether relief of skin inflammation by glucocorticoids and peroxisome proliferator-activated receptor agonists might influence the subsequent development of asthma in a murine model for the atopic march in which mice were repeatedly exposed to house dust mite via the skin, followed by exposure to house dust mite in lungs. To abrogate atopic dermatitis, mice received topical treatment with glucocorticoid receptor/peroxisome proliferator-activated receptor-gamma agonists. Nuclear receptor ligand effects were assessed on primary keratinocytes and dendritic cells, as central players in skin inflammation. Prior house dust mite-induced skin inflammation aggravates allergic airway inflammation and induces a mixed T helper type 2/T helper type 17 response in the lungs. Cutaneous combined activation of glucocorticoid receptor/peroxisome proliferator-activated receptor-gamma reduced skin inflammation to a higher extent compared to single activation. Additive anti-inflammatory effects were more prominent in dendritic cells, as compared to keratinocytes. Alleviation of allergic skin inflammation by activation of glucocorticoid receptor/peroxisome proliferator-activated receptor-gamma appeared insufficient to avoid the allergic immune response in the lungs, but efficiently reduced asthma severity by counteracting the Th17 response. Glucocorticoid receptor/peroxisome proliferator-activated receptor-gamma co-activation represents a potent remedy against allergic skin inflammation and worsening of atopic march

Comparative analysis of antibody- and lipid-based multiplexing methods for single-cell RNA-seq

Background Multiplexing of samples in single-cell RNA-seq studies allows a significant reduction of the experimental costs, straightforward identification of doublets, increased cell throughput, and reduction of sample-specific batch effects. Recently published multiplexing techniques using oligo-conjugated antibodies or -lipids allow barcoding sample-specific cells, a process called "hashing." Results Here, we compare the hashing performance of TotalSeq-A and -C antibodies, custom synthesized lipids and MULTI-seq lipid hashes in four cell lines, both for single-cell RNA-seq and single-nucleus RNA-seq. We also compare TotalSeq-B antibodies with CellPlex reagents (10x Genomics) on human PBMCs and TotalSeq-B with different lipids on primary mouse tissues. Hashing efficiency was evaluated using the intrinsic genetic variation of the cell lines and mouse strains. Antibody hashing was further evaluated on clinical samples using PBMCs from healthy and SARS-CoV-2 infected patients, where we demonstrate a more affordable approach for large single-cell sequencing clinical studies, while simultaneously reducing batch effects. Conclusions Benchmarking of different hashing strategies and computational pipelines indicates that correct demultiplexing can be achieved with both lipid- and antibody-hashed human cells and nuclei, with MULTISeqDemux as the preferred demultiplexing function and antibody-based hashing as the most efficient protocol on cells. On nuclei datasets, lipid hashing delivers the best results. Lipid hashing also outperforms antibodies on cells isolated from mouse brain. However, antibodies demonstrate better results on tissues like spleen or lung