Prostaglandin E2 inhibits IL-23 and IL-12 production by human monocytes through down-regulation of their common p40 subunit

The heterodimeric cytokine IL-23 is important for the maintenance of Th17 cells, which are pivotal mediators of autoimmune diseases like rheumatoid arthritis, colitis, and multiple sclerosis. Prostaglandin E2 (PGE2) is a soluble regulator of inflammation that has both pro- and anti-inflammatory properties. PGE2 has been shown to elevate the IL-23 production by dendritic cells (DC). Monocytes are also producers of IL-23 but the effect of PGE2 on IL-23 production by human monocytes has hardly been investigated. We show here that PGE2 blocks the production of IL-23 by LPS-stimulated monocytes in an IL-10 and IL-1 independent manner. This effect was due to the down-regulation of the p40 subunit of IL-23 on mRNA and protein level. The p40 subunit is shared by IL-12 and, consistently, PGE2 also lowered the IL-12 production by monocytes. These effects of PGE2 were cAMP-dependent since the cAMP enhancer forskolin strongly reduced IL-23 and IL-12 production by monocytes. Taken together, PGE2 acts in an anti-inflammatory manner by lowering IL-23 production by monocytes while it has the opposite effect in DC. Our data may help to reconcile controversial point of views on the pro- and anti-inflammatory nature of PGE2 by making a strong case for a cell type-dependent function

mTOR has a developmental stage-specific role in mitochondrial fitness independent of conventional mTORC1 and mTORC2 and the kinase activity.

The mammalian target of rapamycin (mTOR), present in mTOR complex 1 (mTORC1) and mTORC2, is a serine/threonine kinase that integrates nutrients, growth factors, and cellular energy status to control protein synthesis, cell growth, survival and metabolism. However, it remains elusive whether mTOR plays a developmental stage-specific role in tissue development and whether mTOR can function independent of its complexes and kinase activity. In this study, by inducible genetic manipulation approach, we investigated the role of mTOR and its dependence on mTOR complexes and kinase activity in mitochondrial fitness of early, progenitor stage (lineage-negative; Lin-) versus later, lineage-committed stage (lineage-positive; Lin+) of hematopoietic cells. We found that oxidative phosphorylation (OXPHOS), ATP production and mitochondrial DNA synthesis were decreased in mTOR-/- Lin- cells but increased in mTOR-/- Lin+ cells, suggesting that mTOR plays a developmental stage-specific role in OXPHOS, ATP production and mitochondrial DNA synthesis. In contrast to mTOR deletion, simultaneous deletion of Raptor, a key component of mTORC1, and Rictor, a key component of mTORC2, led to increased mitochondrial DNA in Lin- cells and decreased mitochondrial DNA and ATP production in Lin+ cells, suggesting that mTOR regulates mitochondrial DNA synthesis in Lin- and Lin+ cells and ATP production in Lin+ cells independent of mTORC1 and mTORC2. Similar to mTOR deletion, deletion of Raptor alone attenuated glycolysis and increased mitochondrial mass and mitochondrial membrane potential in Lin- cells and increased mitochondrial mass and OXPHOS in Lin+ cells, whereas deletion of Rictor alone had no effect on these mitochondrial parameters in Lin- and Lin+ cells, suggesting that mTOR regulates glycolysis and mitochondrial membrane potential in Lin- cells, OXPHOS in Lin+ cells, and mitochondrial mass in both Lin- and Lin+ cells dependent on mTORC1, but not mTORC2. Either Raptor deficiency or Rictor deficiency recapitulated mTOR deletion in decreasing OXPHOS in Lin- cells and glycolysis in Lin+ cells, suggesting that mTOR regulates OXPHOS in Lin- cells and glycolysis in Lin+ cells dependent on both mTORC1 and mTORC2. Finally, mice harboring a mTOR kinase dead D2338A knock-in mutant showed decreased glycolysis in Lin+ cells, as seen in mTOR-/- Lin+ cells, but no change in glycolysis in Lin- cells, in contrast to the decreased glycolysis in mTOR-/- Lin- cells, suggesting that mTOR regulates glycolysis in Lin+ cells dependent on its kinase activity, whereas mTOR regulates glycolysis in Lin- cells independent of its kinase activity

A selective inhibitor of the immunoproteasome subunit LMP7 blocks cytokine production and attenuates progression of experimental arthritis

The immunoproteasome, a distinct class of proteasome found predominantly in monocytes and lymphocytes, is known to shape the antigenic repertoire presented on class I major histocompatibility complexes (MHC-I). However, a specific role for the immunoproteasome in regulating other facets of immune responses has not been established. We describe here the characterization of PR-957, a selective inhibitor of low-molecular mass polypeptide-7 (LMP7, encoded by Psmb8), the chymotrypsin-like subunit of the immunoproteasome. PR-957 blocked presentation of LMP7-specific, MHC-I-restricted antigens in vitro and in vivo. Selective inhibition of LMP7 by PR-957 blocked production of interleukin-23 (IL-23) by activated monocytes and interferon-gamma and IL-2 by T cells. In mouse models of rheumatoid arthritis, PR-957 treatment reversed signs of disease and resulted in reductions in cellular infiltration, cytokine production and autoantibody levels. These studies reveal a unique role for LMP7 in controlling pathogenic immune responses and provide a therapeutic rationale for targeting LMP7 in autoimmune disorders

Changes in mitochondrial parameters in Lin^- and Lin⁺ cells upon deletion of mTOR.

<p>Changes in mitochondrial parameters in Lin^- and Lin⁺ cells upon deletion of mTOR.</p

mTOR promotes glycolysis in Lin^- cells independent of its kinase activity.

<p>(A) mTOR kinase dead (KD) D2338A mutant knock-in strategy. mTOR KD mutant knock-in was achieved by CRISPR/Cas9 technology. Single guide RNA targeting site (CRISPR target site) is indicated. Amino acid 2338 of mTOR was changed from Asp (D) to Ala (A) (GAC to GCC). In addition, three silent mutations (highlighted in green) were introduced to abolish the restriction enzyme site of Stu I and create the restriction enzyme site of Avr II in the KD allele to facilitate genotyping of the mice (data not shown). The silent mutations also serve to prevent the CRISPR complex from re-cutting the KD allele. (B) Bone marrow cells were harvested from the indicated mice and detected for mTOR and phospho (p)-S6, 4E-BP and Akt by Western blot. Total 4E-BP, S6 and Akt were blotted as loading control. (C-J) Bone marrow cells were harvested from the indicated mice. Lin^- (C, D, G, H) and Lin⁺ (E, F, I, J) cells were fractionated from the bone marrow cells and assayed for oxygen consumption rate (OCR) (C-F) and extracellular acidification rate (ECAR) (G-J). OCR and ECAR profiles are shown in (C, E) and (G, I), respectively. Basal OCR in the absence of oligomycin, FCCP, and antimycin and rotenone from one measurement is shown in (D) and (F). ECAR in the presence of glucose but absence of oligomycin and 2-DG is shown in (H) and (J). Data are representative of three independent experiments. Error bars represent mean ± SD of 5–7 mice. *<i>P</i> < .05, **<i>P</i> < .01 determined by One-way ANOVA followed by Bonferroni test. mTOR^KD: mTOR^loxp/KD;Mx-Cre⁺. NS: no significance.</p

mTOR inhibits OXPHOS in Lin⁺ cells dependent on mTORC1 but not mTORC2.

<p>Bone marrow cells were harvested from the indicated mice. Lin⁺ cells were fractionated from the bone marrow cells and assayed for oxygen consumption rate (OCR) (A) and Atp5I, Nrf1, Cox5a, and Ndufa32 by Quantitative Real-time PCR (B-E). For (B-E), the mRNA expression levels were normalized to one WT mouse. Results are representative of three independent experiments. Error bars represent mean ± SD of 5–8 mice.. **<i>P</i> < .01.</p

mTOR promotes glycolysis in Lin⁺ cells depending on both mTORC1 and mTORC2.

<p>Bone marrow cells were harvested from the indicated mice. Lin⁺ cells were fractionated from the bone marrow cells and assayed for extracellular acidification rate (ECAR) (A) and Slc2a, PDK1, Hif1α, and HK2 by Quantitative Real-time PCR (B-E). For (B-E), the mRNA expression levels were normalized to one WT mouse. Results are representative of three independent experiments. Error bars represent mean ± SD of 5–8 mice. **<i>P</i> < .01.</p

Changes in mitochondrial parameters in Lin^- and Lin⁺ cells upon deletion of mTOR, Raptor or Rictor.

<p>Changes in mitochondrial parameters in Lin^- and Lin⁺ cells upon deletion of mTOR, Raptor or Rictor.</p