Microenvironmental Regulation of Macrophage Transcriptomic and Metabolomic Profiles in Pulmonary Hypertension.

The recruitment and subsequent polarization of inflammatory monocytes/macrophages in the perivascular regions of pulmonary arteries is a key feature of pulmonary hypertension (PH). However, the mechanisms driving macrophage polarization within the adventitial microenvironment during PH progression remain unclear. We previously established that reciprocal interactions between fibroblasts and macrophages are essential in driving the activated phenotype of both cell types although the signals involved in these interactions remain undefined. We sought to test the hypothesis that adventitial fibroblasts produce a complex array of metabolites and proteins that coordinately direct metabolomic and transcriptomic re-programming of naïve macrophages to recapitulate the pathophysiologic phenotype observed in PH. Media conditioned by pulmonary artery adventitial fibroblasts isolated from pulmonary hypertensive (PH-CM) or age-matched control (CO-CM) calves were used to activate bone marrow derived macrophages. RNA-Seq and mass spectrometry-based metabolomics analyses were performed. Fibroblast conditioned medium from patients with idiopathic pulmonary arterial hypertension or controls were used to validate transcriptional findings. The microenvironment was targeted in vitro using a fibroblast-macrophage co-culture system and in vivo in a mouse model of hypoxia-induced PH. Both CO-CM and PH-CM actively, yet distinctly regulated macrophage transcriptomic and metabolomic profiles. Network integration revealed coordinated rewiring of pro-inflammatory and pro-remodeling gene regulation in concert with altered mitochondrial and intermediary metabolism in response to PH-CM. Pro-inflammation and metabolism are key regulators of macrophage phenotype in vitro, and are closely related to in vivo flow sorted lung interstitial/perivascular macrophages from hypoxic mice. Metabolic changes are accompanied by increased free NADH levels and increased expression of a metabolic sensor and transcriptional co-repressor, C-terminal binding protein 1 (CtBP1), a mechanism shared with adventitial PH-fibroblasts. Targeting the microenvironment created by both cell types with the CtBP1 inhibitor MTOB, inhibited macrophage pro-inflammatory and metabolic re-programming both in vitro and in vivo. In conclusion, coordinated transcriptional and metabolic reprogramming is a critical mechanism regulating macrophage polarization in response to the complex adventitial microenvironment in PH. Targeting the adventitial microenvironment can return activated macrophages toward quiescence and attenuate pathological remodeling that drives PH progression.

Human Endogenous Retroviruses: Residues of Ancient Times Are Differentially Expressed in Crohn's Disease.

BACKGROUND: Eight percent of the human genome consists of human endogenous retroviruses (HERV). These genetic elements are remnants of ancient retroviral germ-line infections. Altered HERV expression is associated with several chronic inflammatory diseases. A physiological role of the HERV-derived proteins syncytin-1 and -2 has been described for the integrity of the human placental cell layer in terms of maintaining feto-maternal tolerance. The aim of this project was to investigate HERV expression in Crohn's disease (CD) with a further focus on syncytins in the gut. MATERIAL AND METHODS: Seventy-four ileal and colonic tissue samples of CD patients and healthy controls have been investigated for mRNA expression of major HERV groups by a comprehensive microarray screening. The most prominent differences have been validated by qRT-PCR. Immunohistochemistry (IHC), Western Blot (WB) and qRT-PCR were performed for syncytin-1 and -2. RESULTS: HERV microarray screening revealed a distinct expression profile in ileal and colonic tissue, as well as differential expression in CD compared to healthy controls. qRT-PCR validated differential expression of at least 3 HERV-groups in CD. qRT-PCR, IHC and WB showed a tissue-dependent diminished epithelial expression of syncytins in inflamed CD. CONCLUSION: For the first time, HERV expression has been comprehensively studied in the gut. Between CD and healthy controls we could show a tissue dependent differential HERV expression profile. Notably, we could show that syncytin-1 and -2 are expressed in the epithelial layer in ileal and colonic tissue samples, whereas their diminished tissue-dependent expression in inflamed CD might modulate inflammatory processes at the gut barrier.

Histone deacetylase-mediated regulation of the antimicrobial peptide hBD2 differs in intestinal cell lines and cultured tissue.

Histone deacetylase inhibition (HDACi) has been suggested as a promising approach to bolster TLR-mediated induction of antimicrobial peptides such as human ß-defensin 2 (hBD2). In inflammatory bowel disease (IBD), Crohn's disease (CD) patients display an attenuated expression of hBD2 as compared to ulcerative colitis (UC). Here, we aimed to study if combining HDACi with the therapeutic E. coli Nissle 1917 (EcN), a strong hBD2 inducer, might be a feasible strategy to further modify protective immune responses. Monolayer epithelial cell lines versus cultured human biopsies from healthy controls and CD and UC patients showed diverse effects. In mono-cell systems, we observed a strong NF-kB-dependent enhancement of TLR- but also IL1ß-mediated hBD2 induction after HDACi. In contrast, multicellular colonic biopsy culture showed the opposite result and HDACi was associated with an abolished TLR-mediated hBD2 induction in all tested patient groups. Of note, CD patients showed an attenuated induction of hBD2 by E. coli Nissle as compared to UC. We conclude that the role of HDACs in hBD2 regulation is context-dependent and likely modified by different cell types. Differential induction in different IBD entities suggests different clinical response patterns based on still unknown hBD2-associated mechanisms.

Cannabinoid Receptor-2 Ameliorates Inflammation in Murine Model of Crohn's Disease.

BACKGROUND AND AIMS: Cannabinoid receptor stimulation may have positive symptomatic effects on inflammatory bowel disease [IBD] patients through analgesic and anti-inflammatory effects. The cannabinoid 2 receptor [CB2R] is expressed primarily on immune cells, including CD4+ T cells, and is induced by active inflammation in both humans and mice. We therefore investigated the effect of targeting CB2R in a preclinical IBD model. METHODS: Employing a chronic ileitis model [TNFΔARE/+ mice], we assessed expression of the CB2R receptor in ileal tissue and on CD4+ T cells and evaluated the effect of stimulation with CB2R-selective ligand GP-1a both in vitro and in vivo. Additionally, we compared cannabinoid receptor expression in the ilea and colons of healthy human controls with that of Crohn's disease patients. RESULTS: Ileal expression of CB2R and the endocannabinoid anandamide [AEA] was increased in actively inflamed TNF∆ARE/+ mice compared with controls. CB2R mRNA was preferentially induced on regulatory T cells [Tregs] compared with T effector cells, approximately 2.4-fold in wild-type [WT] and 11-fold in TNF∆ARE/+ mice. Furthermore, GP-1a enhanced Treg suppressive function with a concomitant increase in IL-10 secretion. GP-1a attenuated murine ileitis, as demonstrated by improved histological scoring and decreased inflammatory cytokine expression. Lastly, CB2R is downregulated in both chronically inflamed TNF∆ARE/+ mice and in IBD patients. CONCLUSIONS: In summary, the endocannabinoid system is induced in murine ileitis but is downregulated in chronic murine and human intestinal inflammation, and CB2R activation attenuates murine ileitis, establishing an anti-inflammatory role of the endocannabinoid system.

Intestinal Barrier Function and the Gut Microbiome Are Differentially Affected in Mice Fed a Western-Style Diet or Drinking Water Supplemented with Fructose.

Background: The consumption of a Western-style diet (WSD) and high fructose intake are risk factors for metabolic diseases. The underlying mechanisms are largely unclear.Objective: To unravel the mechanisms by which a WSD and fructose promote metabolic disease, we investigated their effects on the gut microbiome and barrier function.Methods: Adult female C57BL/6J mice were fed a sugar- and fat-rich WSD or control diet (CD) for 12 wk and given access to tap water or fructose-supplemented water. The microbiota was analyzed with the use of 16S rRNA gene sequencing. Barrier function was studied with the use of permeability tests, and endotoxin, mucus thickness, and gene expressions were measured.Results: The WSD increased body weight gain but not endotoxin translocation compared with the CD. In contrast, high fructose intake increased endotoxin translocation 2.6- and 3.8-fold in the groups fed the CD + fructose and WSD + fructose, respectively, compared with the CD group. The WSD + fructose treatment also induced a loss of mucus thickness in the colon (-46%) and reduced defensin expression in the ileum and colon. The lactulose:mannitol ratio in the WSD + fructose mice was 1.8-fold higher than in the CD mice. Microbiota analysis revealed that fructose, but not the WSD, increased the Firmicutes:Bacteroidetes ratio by 88% for CD + fructose and 63% for WSD + fructose compared with the CD group. Bifidobacterium abundance was greater in the WSD mice than in the CD mice (63-fold) and in the WSD + fructose mice than in the CD + fructose mice (330-fold).Conclusions: The consumption of a WSD or high fructose intake differentially affects gut permeability and the microbiome. Whether these differences are related to the distinct clinical outcomes, whereby the WSD primarily promotes weight gain and high fructose intake causes barrier dysfunction, needs to be investigated in future studies.

Intestinal barrier analysis by assessment of mucins, tight junctions, and α-defensins in healthy C57BL/6J and BALB/cJ mice.

The intestinal barrier is gaining increasing attention because it is related to intestinal homeostasis and disease. Different parameters have been used in the past to assess intestinal barrier functions in experimental studies; however most of them are poorly defined in healthy mice. Here, we compared a number of barrier markers in healthy mice, established normal values and correlations. In 48 mice (24 C57BL/6J, 24 BALB/cJ background), we measured mucus thickness, and expression of mucin-2, α-defensin-1 and -4, zonula occludens-1, occludin, junctional adhesion molecule-A, claudin-1, 2 and -5. We also analyzed claudin-3 and fatty acid binding protein-2 in urine and plasma, respectively. A higher expression of mucin-2 protein was found in the colon compared to the ileum. In contrast, the α-defensins-1 and -4 were expressed almost exclusively in the ileum. The protein expression of the tight junction molecules claudin-1, occludin and zonula occludens-1 did not differ between colon and ileum, although some differences occurred at the mRNA level. No age- or gender-related differences were found. Differences between C57BL/6J and BALB/cJ mice were found for α-defensin-1 and -4 mRNA expression, and for urine and plasma marker concentrations. The α-defensin-1 mRNA correlated with claudin-5 mRNA, whereas α-defensin-4 mRNA correlated with claudin-3 concentrations in urine. In conclusion, we identified a number of murine intestinal barrier markers requiring tissue analyses or measurable in urine or plasma. We provide normal values for these markers in mice of different genetic background. Such data might be helpful for future animal studies in which the intestinal barrier is of interest.

Crohn's disease-derived monocytes fail to induce Paneth cell defensins.

Crohn's disease (CD) is associated with a multitude of genetic defects, many of which likely affect Paneth cell function. Paneth cells reside in the small intestine and produce antimicrobial peptides essential for the host barrier, principally human α-defensin 5 (HD5) and HD6. Patients with CD of the ileum are characterized by reduced constitutive expression of these peptides and, accordingly, compromised antimicrobial barrier function. Here, we present a previously unidentified regulatory mechanism of Paneth cell defensins. Using cultures of human ileal tissue, we showed that the secretome of peripheral blood mononuclear cells (PBMCs) from healthy controls restored the attenuated Paneth cell α-defensin expression characteristic of patients with ileal CD. Analysis of the Wnt pathway in both cultured biopsies and intestinal epithelial cells implicated Wnt ligands driving the PBMC effect, whereas various tested cytokines were ineffective. We further detected another defect in patients with ileal CD, because the PBMC secretomes derived from patients with CD were unable to restore the reduced HD5/HD6 expression. Accordingly, analysis of PBMC subtypes showed that monocytes of patients with CD express significantly lower levels of canonical Wnt ligands, including Wnt3, Wnt3a, Wnt1, and wntless Wnt ligand secretion mediator (Evi/Wls). These studies reveal an important cross-talk between bone marrow-derived cells and epithelial secretory Paneth cells. Defective Paneth cell-mediated innate immunity due to inadequate Wnt ligand stimulation by monocytes provides an additional mechanism in CD. Because defects of Paneth cell function stemming from various etiologies are overcome by Wnt ligands, this mechanism is a potential therapeutic target for this disease.

TCF-1-mediated Wnt signaling regulates Paneth cell innate immune defense effectors HD-5 and -6: implications for Crohn's disease.

Wnt signaling regulates small intestinal stem cell maintenance and Paneth cell differentiation. In patients with ileal Crohn's disease (CD), a decrease of Paneth cell α-defensins has been observed that is partially caused by impaired TCF-4 and LRP6 function. Here we show reduced expression of the Wnt signaling effector TCF-1 (also known as TCF-7) in patients with ileal CD. Reporter gene assays and in vitro promoter binding analysis revealed that TCF-1 activates α-defensin HD-5 and HD-6 transcription in cooperation with ß-catenin and that activation is mediated by three distinct TCF binding sites. EMSA analysis showed binding of TCF-1 to the respective motifs. In ileal CD patients, TCF-1 mRNA expression levels were significantly reduced. Moreover, we found specifically reduced expression of active TCF-1 mRNA isoforms. Tcf-1 knockout mice exhibited reduced cryptdin expression in the jejunum, which was not consistently seen at other small intestinal locations. Our data provide evidence that TCF-1-mediated Wnt signaling is disturbed in small intestinal CD, which might contribute to the observed barrier dysfunction in the disease.

Antimicrobial peptides and gut microbiota in homeostasis and pathology.

We survive because we adapted to a world of microorganisms. All our epithelial surfaces participate in keeping up an effective barrier against microbes while not initiating ongoing inflammatory processes and risking collateral damage to the host. Major players in this scenario are antimicrobial peptides (AMPs). Such broad-spectrum innate antibiotics are in part produced by specialized cells but also widely sourced from all epithelia as well as circulating inflammatory cells. AMPs belong to an ancient defense system found in all organisms and participated in a preservative co-evolution with a complex microbiome. Particularly interesting interactions between host barrier and microbiota can be found in the gut. The intestinal cell lining not only has to maintain a tightly regulated homeostasis during its high-throughput regeneration, but also a balanced relationship towards an extreme number of mutualistic or commensal inhabitants. Recent research suggests that advancing our understanding of the circumstances of such balanced and sometimes imbalanced interactions between gut microbiota and host AMPs should have therapeutic implications for different intestinal disorders.

Gastric antimicrobial peptides fail to eradicate Helicobacter pylori infection due to selective induction and resistance.

BACKGROUND: Although antimicrobial peptides protect mucus and mucosa from bacteria, Helicobacter pylori is able to colonize the gastric mucus. To clarify in which extend Helicobacter escapes the antimicrobial defense, we systematically assessed susceptibility and expression levels of different antimicrobial host factors in gastric mucosa with and without H. pylori infection. MATERIALS AND METHODS: We investigated the expression levels of HBD1 (gene name DEFB1), HBD2 (DEFB4A), HBD3 (DEFB103A), HBD4 (DEFB104A), LL37 (CAMP) and elafin (PI3) by real time PCR in gastric biopsy samples in a total of 20 controls versus 12 patients colonized with H. pylori. Immunostaining was performed for HBD2 and HBD3. We assessed antimicrobial susceptibility by flow cytometry, growth on blood agar, radial diffusion assay and electron microscopy. RESULTS: H. pylori infection was associated with increased gastric levels of the inducible defensin HBD2 and of the antiprotease elafin, whereas the expression levels of the constitutive defensin HBD1, inducible HBD3 and LL37 remained unchanged. HBD4 was not expressed in significant levels in gastric mucosa. H. pylori strains were resistant to the defensins HBD1 as well as to elafin, and strain specific minimally susceptible to HBD2, whereas HBD3 and LL37 killed all H. pylori strains effectively. We demonstrated the binding of HBD2 and LL37 on the surface of H. pylori cells. Comparing the antibacterial activity of extracts from H. pylori negative and positive biopsies, we found only a minimal killing against H. pylori that was not increased by the induction of HBD2 in H. pylori positive samples. CONCLUSION: These data support the hypothesis that gastric H. pylori evades the host defense shield to allow colonization.