Eva Verjans

Dual role of macrophage migration inhibitory factor (MIF) in human breast cancer

BackgroundMacrophage migration inhibitory factor (MIF) is a pleiotropic cytokine and mediator of acute and chronic inflammatory diseases. MIF is overexpressed in various tumours and has been suggested as a molecular link between chronic inflammation and cancer. MIF overexpression is observed in breast cancer but its causal role in the development of this tumour entity is unclear.MethodsMIF levels in breast cancer cell lines were determined by ELISA and Western blot. CD74 was measured by Western blot, fluorescence microscopy and flow cytometry. Cell proliferation was studied by BrdU incorporation, cell adhesion by Matrigel adhesion assay, and cell invasion by migration assay through Matrigel-coated filters using the Transwell system. MIF expression in primary human breast cancers was measured by tissue microarray and a semi-quantitative immunoreactivity score (IRS) and comparison with histopathological parameters and patient outcome data.ResultsMIF was abundantly expressed in the non-invasive breast cancer cell lines MDA-MB-468 and ZR-75-1, but not in invasive MDA-MB-231 cells, which in turn expressed higher levels of the MIF-receptor CD74. Stimulation with exogenous MIF led to a dramatic upregulation of MIF secretion (50-fold) in MDA-MB-231 cells. Autocrine MIF promoted tumour cell proliferation, as indicated by blockade of MIF or CD74 in MDA-MB-231 and MDA-MB-468, and MDA-MB-231 invasiveness was enhanced by exogenous MIF. We correlated the expression of MIF with histopathological parameters and patient outcome data, using a tissue microarray of 175 primary invasive breast cancers and 35 normal control tissues. MIF was upregulated in breast cancer versus normal tissue (median IRS = 8 versus 6). MIF expression showed positive correlations with progesterone (p = 0.006) and estrogen (p = 0.028) receptor expression, markers of a favourable prognosis and a negative correlation to tumour size (p = 0.007). In line with these data, disease-specific overall (OS) as well as recurrence-free (RFS) survival was significantly improved in breast cancer patients with abundant cytosolic MIF expression compared to MIF low expressers (5-year OS = 67% versus 50%, p = 0.0019; 5-year RFS = 52% versus 36%, p = 0.0327).ConclusionWe conclude that intracellular expression of MIF in breast cancer cells is beneficial, whereas extracellular MIF may play a pro-oncogenic role in promoting breast cancer cell-stroma interactions.

Milrinone Relaxes Pulmonary Veins in Guinea Pigs and Humans

Introduction The phosphodiesterase-III inhibitor milrinone improves ventricular contractility, relaxes pulmonary arteries and reduces right ventricular afterload. Thus, it is used to treat heart failure and pulmonary hypertension (PH). However, its action on pulmonary veins (PVs) is not defined, although particularly PH due to left heart disease primarily affects the pulmonary venous bed. We examined milrinone-induced relaxation in PVs from guinea pigs (GPs) and humans. Material and Methods Precision-cut lung slices (PCLS) were prepared from GPs or from patients undergoing lobectomy. Milrinone-induced relaxation was studied by videomicroscopy in naïve PVs and in PVs pre-constricted with the ETA-receptor agonist BP0104. Baseline luminal area was defined as 100%. Intracellular cAMP was measured by ELISA and milrinone-induced changes of segmental vascular resistances were studied in the GP isolated perfused lung (IPL). Results In the IPL (GP), milrinone (10 µM) lowered the postcapillary resistance of pre-constricted vessels. In PCLS (GP), milrinone relaxed naïve and pre-constricted PVs (120%) and this relaxation was attenuated by inhibition of protein kinase G (KT 5823), adenyl cyclase (SQ 22536) and protein kinase A (KT 5720), but not by inhibition of NO-synthesis (L-NAME). In addition, milrinone-induced relaxation was dependent on the activation of KATP-, BKCa 2+- and Kv-channels. Human PVs also relaxed to milrinone (121%), however only if pre-constricted. Discussion Milrinone relaxes PVs from GPs and humans. In GPs, milrinone-induced relaxation is based on KATP-, BKCa 2+- and Kv-channel-activation and on cAMP/PKA/PKG. The relaxant properties of milrinone on PVs lead to reduced postcapillary resistance and hydrostatic pressures. Hence they alleviate pulmonary edema and suggest beneficial effects of milrinone in PH due to left heart disease.

Levosimendan Relaxes Pulmonary Arteries and Veins in Precision-Cut Lung Slices - The Role of KATP-Channels, cAMP and cGMP.

Introduction Levosimendan is approved for left heart failure and is also used in right heart failure to reduce right ventricular afterload. Despite the fact that pulmonary arteries (PAs) and pulmonary veins (PVs) contribute to cardiac load, their responses to levosimendan are largely unknown. Materials and Methods Levosimendan-induced vasorelaxation of PAs and PVs was studied in precision-cut lung slices from guinea pigs by videomicroscopy; baseline luminal area was defined as 100%. Intracellular cAMP- and cGMP-levels were measured by ELISA and NO end products were determined by the Griess reaction. Results Levosimendan relaxed control PVs (116%) and those pre-constricted with an endothelinA-receptor agonist (119%). PAs were only relaxed if pre-constricted (115%). Inhibition of KATP-channels (glibenclamide), adenyl cyclase (SQ 22536) and protein kinase G (KT 5823) largely attenuated the levosimendan-induced relaxation in control PVs, as well as in pre-constricted PAs and PVs. Inhibition of BKCa 2+-channels (iberiotoxin) and Kv-channels (4-aminopyridine) only contributed to the relaxant effect of levosimendan in pre-constricted PAs. In both PAs and PVs, levosimendan increased intracellular cAMP- and cGMP-levels, whereas NO end products remained unchanged. Notably, basal NO-levels were higher in PVs. The KATP-channel activator levcromakalim relaxed PAs dependent on cAMP/PKA/PKG and increased cAMP-levels in PAs. Discussion Levosimendan initiates complex and divergent signaling pathways in PAs and PVs. Levosimendan relaxes PAs and PVs primarily via KATP-channels and cAMP/cGMP; in PAs, BKCa 2+- and Kv-channels are also involved. Our findings with levcromakalim do further suggest that in PAs the activation of KATP-channels leads to the production of cAMP/PKA/PKG. In conclusion, these results suggest that levosimendan might reduce right ventricular afterload by relaxation of PAs as well as pulmonary hydrostatic pressure and pulmonary edema by relaxation of PVs.

Overexpression of CREMα in T Cells Aggravates Lipopolysaccharide-Induced Acute Lung Injury

Transcription factor cAMP response element modulator (CREM)α contributes to various cellular and molecular abnormalities in T cells, including increased IL-17 and decreased IL-2 expression. For development of acute lung injury (ALI), the invasion and regulation of immune cells are highly important, but the role of T cells remains unclear. In this study, we show that CREMα is upregulated in LPS-induced ALI. During the early phase of ALI (day 1), T cell–specific CREMα overexpression enhances the numbers of T cells and expression of TNF-α in bronchoalveolar lavage fluid and deteriorates lung functions. On day 3 of ALI, CREMα transgenic mice present a stronger inflammatory response with higher levels of TNF-α, IL-6, and IL-17 correlating with increased numbers of T cells and neutrophils in bronchoalveolar lavage fluid, whereas expression of Foxp3 and IL-2 and numbers of regulatory T cells are decreased. These changes result in restricted lung function in CREMα transgenic mice. Finally, an adoptive transfer of CREM−/− CD4+ T cells, but not of wild-type T cells into RAG-1−/− mice results in ameliorated disease levels. Thus, levels of CREM in T cells determine the outcome of ALI, and CREMα transgenic animals represent a model in which proinflammatory T cells aggravate ALI in different phases of the disease. Given the fact that patients with autoimmune diseases like systemic lupus erythematosus show higher levels of CREMα and an increased susceptibility toward infectious complications, our finding is of potential clinical significance and may enable new therapeutic strategies.

The cAMP response element modulator (CREM) regulates T H 2 mediated inflammation

A characteristic feature of allergic diseases is the appearance of a subset of CD4+ cells known as TH2 cells, which is controlled by transcriptional and epigenetic mechanisms. We aimed to analyze the role of CREM, a known transcriptional activator of T cells, with regard to TH2 responses and allergic diseases in men and mice. Here we demonstrate that T cells of asthmatic children and PBMCs of adults with atopy express lower mRNA levels of the transcription factor CREM compared to cells from healthy controls. CREM deficiency in murine T cells results in enhanced TH2 effector cytokines in vitro and in vivo and CREM−/− mice demonstrate stronger airway hyperresponsiveness in an OVA-induced asthma model. Mechanistically, both direct CREM binding to the IL-4 and IL-13 promoter as well as a decreased IL-2 dependent STAT5 activation suppress the TH2 response. Accordingly, mice selectively overexpressing CREMα in T cells display decreased TH2 type cytokines in vivo and in vitro, and are protected in an asthma model. Thus, we provide evidence that CREM is a negative regulator of the TH2 response and determines the outcome of allergic asthma.

CD11c.DTR mice develop a fatal fulminant myocarditis after local or systemic treatment with diphtheria toxin

To assess the role of alveolar macrophages (AMs) during a pulmonary Aspergillus fumigatus infection AMs were depleted by intratracheal application of diphtheria toxin (DTX) to transgenic CD11c.DTR mice prior to fungal infection. Unexpectedly, all CD11c.DTR mice treated with DTX died within 4–5 days, whether being infected with A. fumigatus or not. Despite measurable impact of DTX on lung functional parameters, these constrictions could not explain the high mortality rate. Instead, DTX‐treated CD11c.DTR animals developed fulminant myocarditis (FM) characterized by massive leukocyte infiltration and myocardial cell destruction, including central parts of the hearts stimulus transmission system. In fact, standard limb lead ECG recordings of diseased but not healthy mice showed a “Brugada”‐like pattern with an abnormally high ST segment pointing to enhanced susceptibility for potential lethal arrhythmias. While CD11c.DTR mice are extensively used for the characterization of CD11c+ cells, including dendritic cells, several studies have already mentioned adverse side effects following DTX treatment. Our results demonstrate that this limitation is based on severe myocarditis but not on the expected lung constrictions, and has to be taken into consideration if this animal model is used. Based on these properties, however, the CD11c.DTR mouse might serve as useful animal model for FM.

Nrf2 Is a Central Regulator of Metabolic Reprogramming of Myeloid-Derived Suppressor Cells in Steady State and Sepsis

Arising in inflammatory conditions, myeloid-derived suppressor cells (MDSCs) are constantly confronted with intracellular and extracellular reactive oxygen species molecules and oxidative stress. Generating mice with a constitutive activation of Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) we show a pivotal role of the antioxidant stress defense for development of these immune-modulatory cells. These mice are characterized by a massive increase of splenic CD11b+Gr-1+ cells, which exhibit typical suppressive characteristics of MDSCs. Whole transcriptome analysis revealed Nrf2-dependent activation of cell cycle and metabolic pathways, which resemble pathways in CD11b+Gr-1+ MDSCs expanded by in vivo LPS exposure. Constitutive Nrf2 activation thereby regulates activation and balance between glycolysis and mitochondrial metabolism and hence expansion of highly suppressive MDSCs, which mediate protection in LPS-induced sepsis. Our study establishes Nrf2 as key regulator of MDSCs and acquired tolerance against LPS-induced sepsis.

PReS-FINAL-2350: Overexpression of crem alpha leads to a higher inflammatory response in lps induced acute lung injury (ali) and might therefore trigger infectious complications in patients with autoimmune diseases

Patients with autoimmune diseases are highly susceptible towards infectious complications. In patients with SLE, infections are even one of the most common causes of morbidity, hospitalization and death. CREMα is a transcription factor, which is overexpressed in T cells from patients with systemic lupus erythematosus (SLE). Beyond this, CREMα is also upregulated in a murine model of LPS-induced acute lung injury (ALI).

Overexpression of CREMαlpha accelerates onset and severity of auto-immune mediated disease in a murine model of lupus

The transcription factor cAMP response element modulator (CREM) is a widely expressed transcriptional repressor which is important for the termination of the T cell immune response. CREMα is overexpressed in SLE (Systemic lupus erythematosus) T cells and is supposed to be a key player in orchestrating the transcriptional program of SLE T cells by targeting T cell-relevant genes. To explore the relevance of CREMα in vivo we used a well-established murine lupus model, which is characterized by the introduction of a mutation in the CD95 (Fas) locus. We generated a transgenic mouse with a selective overexpression of CREMα in T cells and introduced a Fas -/- phenotype into the CREMα transgenic mice. CREMα transgenic Fas -/- mice developed a severe lymphadenopathy and splenomegaly as early as 8 weeks of age, while the wildytpe Fas -/- mice did not at this early age. Lymphadenopathy and splenomegaly is paralleled by a massive expansion of pathogenic CD3+CD4-CD8- double negative T cells. Furthermore T cells of CREMα transgenic Fas -/- mice show an enhanced production of IL-21 and IL-17, which are hallmark cytokines of highly inflammatory Th17 cells. Vice versa percentages of regulatory T cells are reduced. The enhanced occurrence of aberrant and inflammatory T cells further leads to increased B cell activation, increased anti-DNA antibody titers and finally shortened life expectation of these mice. Our experiments are the proof of principle for a critical amplifying role of CREMα in autoimmune prone conditions like SLE.