MPGES-1-derived PGE2 suppresses CD80 expression on tumor-associated phagocytes to inhibit anti-tumor immune responses in breast cancer.

Prostaglandin E2 (PGE2) favors multiple aspects of tumor development and immune evasion. Therefore, microsomal prostaglandin E synthase (mPGES-1/-2), is a potential target for cancer therapy. We explored whether inhibiting mPGES-1 in human and mouse models of breast cancer affects tumor-associated immunity. A new model of breast tumor spheroid killing by human PBMCs was developed. In this model, tumor killing required CD80 expression by tumor-associated phagocytes to trigger cytotoxic T cell activation. Pharmacological mPGES-1 inhibition increased CD80 expression, whereas addition of PGE2, a prostaglandin E2 receptor 2 (EP2) agonist, or activation of signaling downstream of EP2 reduced CD80 expression. Genetic ablation of mPGES-1 resulted in markedly reduced tumor growth in PyMT mice. Macrophages of mPGES-1(-/-) PyMT mice indeed expressed elevated levels of CD80 compared to their wildtype counterparts. CD80 expression in tumor-spheroid infiltrating mPGES-1(-/-) macrophages translated into antigen-specific cytotoxic T cell activation. In conclusion, mPGES-1 inhibition elevates CD80 expression by tumor-associated phagocytes to restrict tumor growth. We propose that mPGES-1 inhibition in combination with immune cell activation might be part of a therapeutic strategy to overcome the immunosuppressive tumor microenvironment.

Activation of the peroxisome proliferator-activated receptor γ counteracts sepsis-induced T cell cytotoxicity toward alloantigenic target cells.

UNLABELLED: Sepsis still emerges as a major cause of patient death in intensive care units. Therefore, new therapeutic approaches are mandatory. Because during sepsis progression cytotoxic T lymphocytes (CTLs) can be activated in an autoimmune fashion contributing to multiorgan damage, it remains unclear whether CTLs are activated toward alloantigenic cells. This is important for patients receiving an immunosuppressive therapy to permit organ transplantation and, thus, known to be at high risk for developing sepsis. Therefore, we analyzed whether sepsis activates CTL toward alloantigenic target cells and whether this can be inhibited by PPARγ activation, known to block T helper cell responses. To mimic septic conditions, CTLs were isolated from cecal ligation and puncture-operated mice. CTL cytotoxicity was analyzed following a direct alloantigenic activation regime or following classical ex vivo splenocyte-driven activation in a cytotoxicity assay. With this readout, we found that CTL derived from septic mice enhanced cytotoxicity toward alloantigenic target cells, which was lowered by in vivo and ex vivo PPARγ activation. With CTL derived from T cell-specific PPARγ knockout mice, PPARγ activation was ineffective, pointing to a PPARγ-dependent mechanism. In vivo and ex vivo PPARγ activation reduced Fas and granzyme B expression in activated CTL. KEY MESSAGE: In the sepsis CLP mouse model, CTLs are activated toward alloantigenic target cells. Sepsis-mediated alloantigenic CTL activation is blocked in vivo by PPARγ activation. PPARγ deletion or antagonization restored rosiglitazone-dependent inhibition of CTL cytotoxicity. PPARγ inhibits the expression of Fas and granzyme B in CTLs.

Cytokines induce protein kinase A-mediated signalling by a redox-dependent mechanism in rat renal mesangial cells.

Glomerular mesangial cells are smooth muscle cell-like pericytes and are regarded as key players in kidney diseases. In an inflammatory setting, these cells produce high amounts of inflammatory cytokines, chemokines and redox mediators such as reactive oxygen species or nitric oxide (NO). The temporal production of ROS, NO and other redox mediators markedly contributes to the final outcome of inflammatory diseases. Recently, we reported that platelet-derived growth factor forced mesangial cells to activate the regulatory subunit of protein kinase A (PKA RI) by a redox-dependent mechanism but independent from changes in cyclic AMP. This prompted us to further analyze the dimerization of PKA RI and activation of PKA-driven signalling in an inflammatory context. Stimulation of rat mesangial cells with interleukin-1ß and tumour necrosis factor-α [2 nM] induced the formation of PKA RI heterodimers in a time-dependent manner. PKA RI dimerization was accompanied with the formation of ROS, NO and peroxynitrite as well as a depletion of reduced glutathione. Furthermore, dimerization of PKA RI was paralleled by enhanced activity of PKA as shown by the phosphorylation of vasodilator-stimulated phosphoprotein (VASP) at serine 157 that was independent of the formation of cyclic AMP. Remarkably, exogenously administered peroxynitrite potently induced dimerization of PKA RI, whereas pharmacologic inhibition of inducible NO synthase (iNOS) and scavenging of peroxynitrite reduced PKA RI dimerization and VASP phosphorylation to control levels thus clearly indicating a causal role for endogenously formed peroxynitrite on PKA signalling. Consequently, the treatment of inflammatory diseases with anti-oxidants or NOS inhibitors may alter PKA activity.

Autophagy-dependent PELI3 degradation inhibits proinflammatory IL1B expression.

Lipopolysaccharide (LPS)-induced activation of TLR4 (toll-like receptor 4) is followed by a subsequent overwhelming inflammatory response, a hallmark of the first phase of sepsis. Therefore, counteracting excessive innate immunity by autophagy is important to contribute to the termination of inflammation. However, the exact molecular details of this interplay are only poorly understood. Here, we show that PELI3/Pellino3 (pellino E3 ubiquitin protein ligase family member 3), which is an E3 ubiquitin ligase and scaffold protein in TLR4-signaling, is impacted by autophagy in macrophages (MΦ) after LPS stimulation. We noticed an attenuated mRNA expression of proinflammatory Il1b (interleukin 1, ß) in Peli3 knockdown murine MΦ in response to LPS treatment. The autophagy adaptor protein SQSTM1/p62 (sequestosome 1) emerged as a potential PELI3 binding partner in TLR4-signaling. siRNA targeting Sqstm1 and Atg7 (autophagy related 7), pharmacological inhibition of autophagy by wortmannin as well as blocking the lysosomal vacuolar-type H(+)-ATPase by bafilomycin A1 augmented PELI3 protein levels, while inhibition of the proteasome had no effect. Consistently, treatment to induce autophagy by MTOR (mechanistic target of rapamycin (serine/threonine kinase)) inhibition or starvation enhanced PELI3 degradation and reduced proinflammatory Il1b expression. PELI3 was found to be ubiquitinated upon LPS stimulation and point mutation of PELI3-lysine residue 316 (Lys316Arg) attenuated Torin2-dependent degradation of PELI3. Immunofluorescence analysis revealed that PELI3 colocalized with the typical autophagy markers MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 ß) and LAMP2 (lysosomal-associated membrane protein 2). Our observations suggest that autophagy causes PELI3 degradation during TLR4-signaling, thereby impairing the hyperinflammatory phase during sepsis.

SYNCRIP-dependent Nox2 mRNA destabilization impairs ROS formation in M2-polarized macrophages.

AIMS: During sepsis, macrophages are alternatively activated toward an M2-like phenotype on contact with apoptotic cells (ACs) or their secretion products. Simultaneously, NADPH oxidase-dependent reactive oxygen species (ROS) formation is attenuated, thus contributing to immune paralysis. However, the exact mechanism remains elusive. Here, we provide mechanistic insights into diminished mRNA stability of the NADPH oxidase Nox2 on macrophage M2 polarization and therefore reduced ROS formation in sepsis. RESULTS: Murine J774A.1 macrophages were stimulated with conditioned medium (CM) of apoptotic T cells, which reduced Nox2 mRNA and protein expression, consequently decreasing ROS production. An mRNA pulldown approach coupled to mass spectrometry analysis identified the RNA-binding protein SYNCRIP attached to the Nox2 mRNA 3' untranslated region (3'UTR). The binding of SYNCRIP to the 3'UTR of Nox2 mRNA is attenuated after treatment with CM of apoptotic T cells, followed by Nox2 mRNA destabilization. In in vivo models of polymicrobial sepsis such as cecal ligation and puncture, SYNCRIP was strongly downregulated, which was associated with a decreased Nox2 expression in peritoneal macrophages. INNOVATION: Downregulation of SYNCRIP in macrophages after contact to material of ACs destabilized Nox2 mRNA and impaired ROS formation, thereby contributing to an M2 phenotype shift of macrophages in sepsis. CONCLUSION: M2 polarization of macrophages in sepsis results in an attenuated SYNCRIP binding to the 3'UTR of Nox2 mRNA, destabilizing Nox2 mRNA abundance and expression. Consequently, ROS formation needed to fight against recurrent infections is impaired. In conclusion, SYNCRIP-regulated Nox2 mRNA degradation mediates the hypoinflammatory phase of sepsis.