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The gastric bacterium causes a persistent infection that is directly responsible for gastric ulcers and gastric cancer in some patients and protective against allergic and other immunological disorders in others. The two outcomes of the -host interaction can be modeled in mice that are infected as immunocompetent adults and as neonates, respectively. Here, we have investigated the contribution of the immunomodulator VacA to -specific local and systemic immune responses in both models. We found that neonatally infected mice are colonized at higher levels than mice infected as adults and fail to generate effector T-cell responses to the bacteria; rather, T-cell responses in neonatally infected mice are skewed toward Foxp3-positive (Foxp3) regulatory T cells that are neuropilin negative and express RORγt. We found these peripherally induced regulatory T cells (pTregs) to be enriched, in a VacA-dependent manner, not only in the gastric mucosa but also in the lungs of infected mice. Pulmonary pTreg accumulation was observed in mice that have been infected neonatally with wild-type but not in mice that have been infected as adults or mice infected with a VacA null mutant. Finally, we traced VacA to gastric lamina propria myeloid cells and show that it suppressed interleukin-23 (IL-23) expression by dendritic cells and induced IL-10 and TGF-β expression in macrophages. Taken together, the results are consistent with the idea that creates a tolerogenic environment through its immunomodulator VacA, which skews T-cell responses toward Tregs, favors persistence, and affects immunity at distant sites. has coexisted with humans for at least 60.000 years and has evolved persistence strategies that allow it to evade host immunity and colonize its host for life. The VacA protein is expressed by all strains and is required for high-level persistent infection in experimental mouse models. Here, we show that VacA targets myeloid cells in the gastric mucosa to create a tolerogenic environment that facilitates regulatory T-cell differentiation, while suppressing effector T-cell priming and functionality. Tregs that are induced in the periphery during infection can be found not only in the stomach but also in the lungs of infected mice, where they are likely to affect immune responses to allergens.
Copyright © 2019 Altobelli et al.
Background & Aims - Chronic inflammation is a predisposing condition for colorectal cancer. Many studies to date have focused on proinflammatory signaling pathways in the colon. Understanding the mechanisms that suppress inflammation, particularly in epithelial cells, is critical for developing therapeutic interventions. Here, we explored the roles of transforming growth factor β (TGFβ) family signaling through SMAD4 in colonic epithelial cells.
Methods - The gene was deleted specifically in adult murine intestinal epithelium. Colitis was induced by 3 rounds of dextran sodium sulfate in drinking water, after which mice were observed for up to 3 months. Nontransformed mouse colonocyte cell lines and colonoid cultures and human colorectal cancer cell lines were analyzed for responses to TGFβ1 and bone morphogenetic protein 2.
Results - Dextran sodium sulfate treatment was sufficient to drive carcinogenesis in mice lacking colonic expression, with resulting tumors bearing striking resemblance to human colitis-associated carcinoma. Loss of SMAD4 protein was observed in 48% of human colitis-associated carcinoma samples as compared with 19% of sporadic colorectal carcinomas. Loss of increased the expression of inflammatory mediators within nontransformed mouse colon epithelial cells in vivo. In vitro analysis of mouse and human colonic epithelial cell lines and organoids indicated that much of this regulation was cell autonomous. Furthermore, TGFβ signaling inhibited the epithelial inflammatory response to proinflammatory cytokines.
Conclusions - TGFβ suppresses the expression of proinflammatory genes in the colon epithelium, and loss of its downstream mediator, SMAD4, is sufficient to initiate inflammation-driven colon cancer. Transcript profiling: GSE100082.
The tumor-cell microenvironment is recognized as a dynamic place where critical cell interactions occur and play an important role in altering tumorigenesis. While many studies have investigated the effects of cellular cross-talk within distinct tumor microenvironments, these interactions have yet to be fully examined in bone. It is well-established that many common cancers metastasize to bone, resulting in the development of tumor-induced bone disease (TIBD), a multi-facetted illness that is driven by complex cell interactions within the bone marrow. Our group has previously published that myeloid progenitor cells expand in the presence of tumors in bone, aligning with the notion that myeloid cells can act as tumor promotors. Several groups, including ours, have established that transforming growth factor β (TGF-β), an abundant growth factor in bone, can regulate both TIBD and myeloid expansion. TGF-β inhibitors have been shown to increase bone volume, decrease bone destruction, and reduce but not eliminate tumor. Therefore, we hypothesize that inhibiting TGF-β will reduce myeloid expansion leading to a reduction of tumor burden in bone and osteoclast-mediated bone loss, causing to an overall reduction in TIBD. To address this hypothesis, two different mouse models of breast cancer bone colonization were pre-treated with the TGF-β neutralizing antibody, 1D11, prior to tumor inoculation (athymic: MDA-MB-231, BALB/c: 4T1) and continuously treated until sacrifice. Additionally, a genetically modified mouse model with a myeloid specific deletion of transforming growth factor beta receptor II (TGF-βRII) (TGF-βRII) was utilized in our studies. Systemic inhibition of TGF-β lead to fewer osteolytic lesions, and reduced tumor burden in bone as expected from previous studies. Additionally, early TGF-β inhibition affected expansion of distinct myeloid populations and shifted the cytokine profile of pro-tumorigenic factors in bone, 4T1 tumor cells, and bone-marrow derived macrophages. Similar observations were seen in tumor-bearing TGF-βRII mice, where these mice contained fewer bone lesions and significantly less tumor burden in bone, suggesting that TGF-β inhibition regulates myeloid expansion leading to a significant reduction in TIBD.
Published by Elsevier Inc.
Proliferative vitreoretinopathy (PVR) is a common complication of open globe injury and the most common cause of failed retinal detachment surgery. The response by retinal pigment epithelial (RPE) cells liberated into the vitreous includes proliferation and migration; most importantly, epithelial to mesenchymal transition (EMT) of RPE plays a central role in the development and progress of PVR. For the first time, we show that knockdown of BIRC5, a member of the inhibitor of apoptosis protein family, using either lentiviral vector based CRISPR/Cas9 nickase gene editing or inhibition of survivin using the small-molecule inhibitor YM155, results in the suppression of EMT in RPE cells. Knockdown of survivin or inhibition of survivin significantly reduced TGFβ-induced cell proliferation and migration. We further demonstrated that knockdown or inhibition of survivin attenuated the TGFβ signaling by showing reduced phospho-SMAD2 in BIRC5 knockdown or YM155-treated cells compared to controls. Inhibition of the TGFβ pathway using TGFβ receptor inhibitor also suppressed survivin expression in RPE cells. Our studies demonstrate that survivin contributes to EMT by cross-talking with the TGFβ pathway in RPE cells. Targeting survivin using small-molecule inhibitors may provide a novel approach to treat PVR disease.
Copyright © 2018 Elsevier Inc. All rights reserved.
Endothelial-to-mesenchymal transition (EndoMT) is a cellular process often initiated by the transforming growth factor β (TGF-β) family of ligands. Although required for normal heart valve development, deregulated EndoMT is linked to a wide range of pathological conditions. Here, we demonstrate that endothelial fatty acid oxidation (FAO) is a critical in vitro and in vivo regulator of EndoMT. We further show that this FAO-dependent metabolic regulation of EndoMT occurs through alterations in intracellular acetyl-CoA levels. Disruption of FAO via conditional deletion of endothelial carnitine palmitoyltransferase II (Cpt2) augments the magnitude of embryonic EndoMT, resulting in thickening of cardiac valves. Consistent with the known pathological effects of EndoMT, adult Cpt2 mice demonstrate increased permeability in multiple vascular beds. Taken together, these results demonstrate that endothelial FAO is required to maintain endothelial cell fate and that therapeutic manipulation of endothelial metabolism could provide the basis for treating a growing number of EndoMT-linked pathological conditions.
Copyright © 2018 Elsevier Inc. All rights reserved.
Tissues stiffen during aging and during the pathological progression of cancer, fibrosis, and cardiovascular disease. Extracellular matrix stiffness is emerging as a prominent mechanical cue that precedes disease and drives its progression by altering cellular behaviors. Targeting extracellular matrix mechanics, by preventing or reversing tissue stiffening or interrupting the cellular response, is a therapeutic approach with clinical potential. Major drivers of changes to the mechanical properties of the extracellular matrix include phenotypically converted myofibroblasts, transforming growth factor β (TGFβ), and matrix cross-linking. Potential pharmacological interventions to overcome extracellular matrix stiffening are emerging clinically. Aside from targeting stiffening directly, alternative approaches to mitigate the effects of increased matrix stiffness aim to identify and inhibit the downstream cellular response to matrix stiffness. Therapeutic interventions that target tissue stiffening are discussed in the context of their limitations, preclinical drug development efforts, and clinical trials.
Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
The survival rate for pancreatic ductal adenocarcinoma (PDAC) remains low. More therapeutic options to treat this disease are needed, for the current standard of care is ineffective. Using an animal model of aggressive PDAC (Kras/p48), we discovered an effect of TGFβ signaling in regulation of G-CSF secretion in pancreatic epithelium. Elevated concentrations of G-CSF in PDAC promoted differentiation of Ly6G cells from progenitors, stimulated IL10 secretion from myeloid cells, and decreased T-cell proliferation via upregulation of Arg, iNOS, VEGF, IL6, and IL1b from CD11b cells. Deletion of in PDAC cells or use of a G-CSF-blocking antibody decreased tumor growth. Anti-G-CSF treatment in combination with the DNA synthesis inhibitor gemcitabine reduced tumor size, increased the number of infiltrating T cells, and decreased the number of Ly6G cells more effectively than gemcitabine alone. Human analysis of human datasets from The Cancer Genome Atlas and tissue microarrays correlated with observations from our mouse model experiments, especially in patients with grade 1, stage II disease. We propose that in aggressive PDAC, elevated G-CSF contributes to tumor progression through promoting increases in infiltration of neutrophil-like cells with high immunosuppressive activity. Such a mechanism provides an avenue for a neoadjuvant therapeutic approach for this devastating disease. .
©2017 American Association for Cancer Research.
The TGF- and Wnt/-catenin pathways have important roles in modulating CKD, but how these growth factors affect the epithelial response to CKD is not well studied. TGF- has strong profibrotic effects, but this pleiotropic factor has many different cellular effects depending on the target cell type. To investigate how TGF- signaling in the proximal tubule, a key target and mediator of CKD, alters the response to CKD, we injured mice lacking the TGF- type 2 receptor specifically in this epithelial segment. Compared with littermate controls, mice lacking the proximal tubular TGF- receptor had significantly increased tubular injury and tubulointerstitial fibrosis in two different models of CKD. RNA sequencing indicated that deleting the TGF- receptor in proximal tubule cells modulated many growth factor pathways, but Wnt/-catenin signaling was the pathway most affected. We validated that deleting the proximal tubular TGF- receptor impaired -catenin activity and Genetically restoring -catenin activity in proximal tubules lacking the TGF- receptor dramatically improved the tubular response to CKD in mice. Deleting the TGF- receptor alters many growth factors, and therefore, this ameliorated response may be a direct effect of -catenin activity or an indirect effect of -catenin interacting with other growth factors. In conclusion, blocking TGF- and -catenin crosstalk in proximal tubules exacerbates tubular injury in two models of CKD.
Copyright © 2017 by the American Society of Nephrology.
BACKGROUND - The challenging biological and mechanical environment of posterolateral fusion (PLF) requires a carrier that spans the transverse processes and resists the compressive forces of the posterior musculature. The less traumatic posterolateral approach enabled by minimally invasive surgical techniques has prompted investigations into alternative rhBMP-2 carriers that are injectable, settable, and compression-resistant. In this pilot study, we investigated injectable low-viscosity (LV) polymer/composite bone grafts as compression-resistant carriers for rhBMP-2 in a single-level rabbit PLF model.
METHODS - LV grafts were augmented with ceramic microparticles: (1) hydrolytically degradable bioactive glass (BG), or (2) cell-degradable 85% β-tricalcium phosphate/15% hydroxyapatite (CM). Material properties, such as pore size, viscosity, working time, and bulk modulus upon curing, were measured for each LV polymer/ceramic material. An in vivo model of posterolateral fusion in a rabbit was used to assess the grafts' capability to encourage spinal fusion.
RESULTS - These materials maintained a working time between 9.6 and 10.3 min, with a final bulk modulus between 1.2 and 3.1 MPa. The LV polymer/composite bone grafts released 55% of their rhBMP-2 over a 14-day period. As assessed by manual palpation in vivo, fusion was achieved in all (n = 3) animals treated with LV/BG or LV/CM carriers incorporating 430 μg rhBMP-2/ml. Images of μCT and histological sections revealed evidence of bone fusion near the transverse processes.
CONCLUSION - This study highlights the potential of LV grafts as injectable and compression-resistant rhBMP-2 carriers for posterolateral spinal fusion.
We previously reported that single cells from a human colorectal cancer (CRC) cell line (HCA-7) formed either hollow single-layered polarized cysts or solid spiky masses when plated in 3D in type-I collagen. To begin in-depth analyses into whether clonal cysts and spiky masses possessed divergent properties, individual colonies of each morphology were isolated and expanded. The lines thus derived faithfully retained their parental cystic and spiky morphologies and were termed CC (cystic) and SC (spiky), respectively. Although both CC and SC expressed EGF receptor (EGFR), the EGFR-neutralizing monoclonal antibody, cetuximab, strongly inhibited growth of CC, whereas SC was resistant to growth inhibition, and this was coupled to increased tyrosine phosphorylation of MET and RON. Addition of the dual MET/RON tyrosine kinase inhibitor, crizotinib, restored cetuximab sensitivity in SC. To further characterize these two lines, we performed comprehensive genomic and transcriptomic analysis of CC and SC in 3D. One of the most up-regulated genes in CC was the tumor suppressor , and the most up-regulated gene in SC was () in 3D and xenografts. Analysis of a CRC tissue microarray showed that epithelial, but not stromal, VCAN staining strongly correlated with reduced survival, and combined epithelial VCAN and absent HPGD staining portended a poorer prognosis. Thus, with this 3D system, we have identified a mode of cetuximab resistance and a potential prognostic marker in CRC. As such, this represents a potentially powerful system to identify additional therapeutic strategies and disease-relevant genes in CRC and possibly other solid tumors.