Infiltrating macrophages amplify doxorubicin-induced cardiac damage: role of catecholamines.

BACKGROUND: The functional contribution of non-myocyte cardiac cells, such as inflammatory cells, in the setup of heart failure in response to doxorubicin (Dox) is recently becoming of growing interest. OBJECTIVES: The study aims to evaluate the role of macrophages in cardiac damage elicited by Dox treatment. METHODS: C57BL/6 mice were treated with one intraperitoneal injection of Dox (20 mg/kg) and followed up for 5 days by cardiac ultrasounds (CUS), histological, and flow cytometry evaluations. We also tested the impact of Dox in macrophage-depleted mice. Rat cardiomyoblasts were directly treated with Dox (D-Dox) or with a conditioned medium from cultured murine macrophages treated with Dox (M-Dox). RESULTS: In response to Dox, macrophage infiltration preceded cardiac damage. Macrophage depletion prevents Dox-induced damage, suggesting a key role of these cells in promoting cardiotoxicity. To evaluate the crosstalk between macrophages and cardiac cells in response to DOX, we compared the effects of D-Dox and M-Dox in vitro. Cell vitality was lower in cardiomyoblasts and apoptosis was higher in response to M-Dox compared with D-Dox. These events were linked to p53-induced mitochondria morphology, function, and autophagy alterations. We identify a mechanistic role of catecholamines released by Dox-activated macrophages that lead to mitochondrial apoptosis of cardiac cells through ß-AR stimulation. CONCLUSIONS: Our data indicate that crosstalk between macrophages and cardiac cells participates in cardiac damage in response to Dox.

Unleashing the power of formyl peptide receptor 2 in cardiovascular disease.

N-formyl peptide receptors (FPRs) are seven-transmembrane, G protein-coupled receptors with a wide distribution in immune and non-immune cells, recognizing N-formyl peptides from bacterial and mitochondrial origin and several endogenous signals. Three FPRs have been identified in humans: FPR1, FPR2, and FPR3. Most FPR ligands can activate a pro-inflammatory response, while a limited group of FPR agonists can elicit anti-inflammatory and homeostatic responses. Annexin A1 (AnxA1), a glucocorticoid-induced protein, its N-terminal peptide Ac2-26, and lipoxin A4 (LXA4), a lipoxygenase-derived eicosanoid mediator, exert significant immunomodulatory effects by interacting with FPR2 and/or FPR1. The ability of FPRs to recognize both ligands with pro-inflammatory or inflammation-resolving properties places them in a crucial position in the balance between activation against harmful events and maintaince of tissue integrity. A new field of investigation focused on the role of FPRs in the setting of heart injury. FPRs are expressed on cardiac macrophages, which are the predominant immune cells in the myocardium and play a key role in heart diseases. Several endogenous (AnxA1, LXA4) and synthetic compounds (compound 43, BMS-986235) reduced infarct size and promoted the resolution of inflammation via the activation of FPR2 on cardiac macrophages. Further studies should evaluate FPR2 role in other cardiovascular disorders.

Immunoglobulins G modulate endothelial function and affect insulin sensitivity in humans.

BACKGROUND AND AIMS: Data from animals suggest that immunoglobulins G (IgG) play a mechanistic role in atherosclerosis and diabetes through endothelial dysfunction and insulin resistance. Patients with common variable immunodeficiency (CVID), who have low circulating levels of IgG and are treated with intravenous polyclonal IgG (IVIgG), may provide an ideal model to clarify whether circulating IgG modulate endothelial function and affect insulin sensitivity in humans. METHODS AND RESULTS: We studied 24 patients with CVID and 17 matched healthy controls (HC). Endothelial function was evaluated as flow mediated dilation (FMD) of the brachial artery at baseline and 1, 7, 14, and 21 days after IVIgG infusion in the CVID patients. We measured also plasma glucose, insulin, and calculated the HOMA-IR index. We also investigated the role of human IgG on the production of Nitric Oxide (NO) in vitro in Human Coronary Artery Endothelial Cells (HCAEC). Compared to HC, FMD of CVID patients was significantly impaired at baseline (9.4 ± 0.9 and 7.6 ± 0.6% respectively, p < 0.05) but rose above normal levels 1 and 7 days after IVIgG infusion to return at baseline at 14 and 21 days. Serum insulin concentration and HOMA-IR index dropped by 50% in CVID patients after IVIgG (p < 0.002 vs. baseline). In vitro IgG stimulated NO production in HCAEC. CONCLUSIONS: Reduced IgG levels are associated with endothelial dysfunction and IVIgG stimulates endothelial function directly while improving insulin sensitivity. The current findings may suggest an anti-atherogenic role of human IgG.

Role of the HIF-1α/Nur77 axis in the regulation of the tyrosine hydroxylase expression by insulin in PC12 cells.

Tyrosine hydroxylase (TH), catalyzing the conversion of tyrosine into l-DOPA, is the rate-limiting enzyme in dopamine synthesis. Defects in insulin action contribute to alterations of TH expression and/or activity in the brain and insulin increases TH levels in 1-methyl-4-phenylpyridinium (MPP+)-treated neuronal cells. However, the molecular mechanisms underlying the regulation of TH by insulin have not been elucidated yet. Using PC12 cells, we show for the first time that insulin increases TH expression in a biphasic manner, with a transient peak at 2 hr and a delayed response at 16 hr, which persists for up to 24 hr. The use of a dominant negative hypoxia-inducible factor 1-alpha (HIF-1α) and its pharmacological inhibitor chetomin, together with chromatin immunoprecipitation (ChIP) experiments for the specific binding to TH promoter, demonstrate the direct role of HIF-1α in the early phase. Moreover, ChIP experiments and transfection of a dominant negative of the nerve growth factor IB (Nur77) indicate the involvement of Nur77 in the late phase insulin response, which is mediated by HIF-1α. In conclusion, the present study shows that insulin regulates TH expression through HIF-1α and Nur77 in PC12 cells, supporting the critical role of insulin signaling in maintaining an appropriate dopaminergic tone by regulating TH expression in the central nervous system.

Interleukin-8, but not the Related Chemokine CXCL1, Sustains an Autocrine Circuit Necessary for the Properties and Functions of Thyroid Cancer Stem Cells.

Interleukin-8 (IL-8/CXCL8) mediates its biological effects through two receptors, CXCR1 and CXCR2. While CXCR1 recognizes IL-8 and granulocyte chemotactic protein-2, CXCR2 binds to multiple chemokines including IL-8, CXCL1, 2 and 3. Both IL-8 and CXCL1 have been implicated in the neoplastic features of thyroid cancer (TC). Here, we assessed the role of the autocrine circuits sustained by IL-8 and CXCL1 in determining TC stem cell (TC SC) features. Using immunohistochemistry, we found that thyroid epithelial cancerous, but not normal, cells stained positive for IL-8, whose levels correlated with lymph-nodal metastases. We assessed the expression of endogenous IL-8 and CXCL1, by ELISA assays, and of their receptors CXCR1 and CXCR2, by flow cytometry, in a panel of TC cell lines. These molecules were expressed in TC cell lines grown in adherence, and at higher levels also in thyrospheres enriched in stem-like cells. RNA interference demonstrated that IL-8/CXCR1, but not CXCL1/CXCR2, is crucial for the sphere-forming, self-renewal and tumor-initiating ability of TC cells. Accordingly, treatment of TC cells with IL-8, but not with CXCL1, potentiated cell stemness. We identified CD34 as an IL-8-induced gene and as a TC SC marker, since it was overexpressed in thyrospheres compared to adherent cells. Moreover, CD34 is required for the efficient sphere-forming ability and tumorigenicity of TC cells. Our data indicate that IL-8, but not the CXCL1 circuit, is critical for the regulation of TC SCs, and unveils novel potential targets for the therapy of as yet untreatable forms of TC. Stem Cells 2017;35:135-146.

New perspectives in cancer: Modulation of lipid metabolism and inflammation resolution.

Inflammation is considered an enabling feature of cancer. Besides the persistence of inflammatory stimuli, also defective mechanisms of resolution can lead to chronic inflammation. Inflammation resolution is an active process controlled by lipidic specialized pro-resolving mediators (SPMs), derived from ω-3 or ω-6 essential polyunsaturated fatty acids (PUFA) through the activity of lipoxygenases (ALOX5 and 15). Thus, a lack or defect in resolution mechanisms may affect cancer development and progression by prolonging inflammation. Components of pro-resolving pathways (PUFA, enzymes, or SPMs) have been reported to modulate various cancer features by affecting both cancer cells and cancer-associated stroma. Here, we will review the most important mechanisms by which SPMs, ω-3/6 PUFA, and ALOXs affect cancer biology, paying particular attention to their role in the inhibition of inflammation and angiogenesis, two of the most important hallmarks of cancer. The collection of these results may suggest novel perspectives in cancer management based on the modulation of lipid metabolism and the production of SPMs.

HIV-1 Nef promotes migration and chemokine synthesis of human basophils and mast cells through the interaction with CXCR4.

BACKGROUND: The Nef protein can be detected in plasma of HIV-1-infected patients and plays a role in the pathogenesis of HIV-1. Nef produced during the early stages of infection is fundamental in creating the ideal environment for viral replication, e.g. by reducing the ability of infected cells to induce an immune response. AIM: Based on previous experience showing that both Tat and gp41 of HIV-1 are potent chemotactic factors for basophils and mast cells, and gp120 is a powerful stimulus for the release of histamine and cytokines (IL-4 and IL-13) from basophils, in this study we aimed to verify if the HIV Nef protein can exert some effects on basophils and mast cells purified from healthy volunteers through the interaction with the CXCL12 receptor, CXCR4. METHODS: Basophils purified from peripheral blood cells of 30 healthy volunteers and mast cells obtained from lung tissue of ten healthy volunteers were tested by flow cytometric analysis, chemotaxis and chemokine production by ELISA assays. RESULTS: Nef is a potent chemoattractant for basophils and lung mast cells obtained from healthy, HIV-1 and HIV-2 seronegative individuals. Incubation of basophils and mast cells with Nef induces the release of chemokines (CXCL8/IL-8 and CCL3/MIP-1α). The chemotactic activity of Nef on basophils and mast cells is mediated by the interaction with CXCR4 receptors, being blocked by preincubation of FcεRI+ cells with an anti-CXCR4 Ab. Stimulation with Nef or CXCL12/SDF-1α, a CXCR4 ligand, desensitizes basophils to a subsequent challenge with an autologous or heterologous stimulus. CONCLUSIONS: These results indicate that Nef, a HIV-1-encoded α-chemokine homolog protein, plays a direct role in basophils and mast cell recruitment and activation at sites of HIV-1 replication, by promoting directional migration of human FcεRI+ cells and the release of chemokines from these cells. Together with our previous results, these data suggest that FcεRI+ cells contribute to the dysregulation of the immune system in HIV-1 infection.

ß2-Adrenergic receptor stimulation improves endothelial progenitor cell-mediated ischemic neoangiogenesis.

RATIONALE: Endothelial progenitor cells (EPCs) are present in the systemic circulation and home to sites of ischemic injury where they promote neoangiogenesis. ß2-Adrenergic receptor (ß2AR) plays a critical role in vascular tone regulation and neoangiogenesis. OBJECTIVE: We aimed to evaluate the role of ß2AR on EPCs' function. METHODS AND RESULTS: We firstly performed in vitro analysis showing the expression of ß2AR on EPCs. Stimulation of wild-type EPCs with ß-agonist isoproterenol induced a significant increase of Flk-1 expression on EPCs as assessed by fluorescence-activated cell sorter. Moreover, ß2AR stimulation induced a significant increase of cell proliferation, improved the EPCs migratory activity, and enhanced the EPCs' ability to promote endothelial cell network formation in vitro. Then, we performed in vivo studies in animals model of hindlimb ischemia. Consistent with our in vitro results, in vivo EPCs' treatment resulted in an improvement of impaired angiogenic phenotype in ß2AR KO mice after induction of ischemia, whereas no significant amelioration was observed when ß2AR knock out (KO) EPCs were injected. Indeed, wild-type-derived EPCs' injection resulted in a significantly higher blood flow restoration in ischemic hindlimb and higher capillaries density at histological analysis as compared with not treated or ß2AR KO EPC-treated mice. CONCLUSIONS: The present study provides the first evidence that EPCs express a functional ß2AR. Moreover, ß2AR stimulation results in EPCs proliferation, migration, and differentiation, enhancing their angiogenic ability, both in vitro and in vivo, leading to an improved response to ischemic injury in animal models of hindlimb ischemia.

Formyl peptide receptors at the interface of inflammation, angiogenesis and tumor growth.

N-formyl peptide receptors (FPRs) belong to the family of pattern recognition receptors (PRRs) that regulate innate immune responses. Three FPRs have been identified in humans: FPR1-FPR3. FPR expression was initially described in immune cells and subsequently in non-hematopoietic cells and certain tissues. Besides their involvement in inflammatory disorders, FPRs have been implicated in the regulation of tissue repair and angiogenesis. Angiogenesis is not only a key component of pathogen defence during acute infection and of chronic inflammatory disorders, but also plays a critical role in wound healing and tissue regeneration. Moreover, pathologic uncontrolled angiogenesis is central for tumour growth, progression, and the formation of metastases. In this review, we summarise the evidence for a central role of FPRs at the intersection between inflammation, physiologic angiogenesis and pathologic neovascularisation linked to cancer. These findings provide insights into the potential clinical relevance of new treatment regimens involving FPR modulation.

KLHL14 is a tumor suppressor downregulated in undifferentiated thyroid cancer.

KLHL14 is a substrate-binding subunit of Cullin-RING ligase 3 ubiquitin ligase complex, highly enriched in thyroid since early embryonic development, together with its antisense RNA KLHL14-AS. We have previously demonstrated that Klhl14-AS is a competing endogenous RNA regulating several differentiation and survival factors in thyroid cancer, acting as tumor suppressor. Recently, also KLHL14 has been shown to function as tumor suppressor in diffuse large B-cell lymphoma and in malignant mesothelioma. Here we show that KLHL14 expression is strongly reduced in anaplastic thyroid cancer, the less differentiated and most aggressive type of thyroid neoplasia. Such reduction is reproduced in different in vivo and in vitro models of thyroid cancer, being invariably associated with loss of differentiation. When Klhl14 expression is rescued in thyroid transformed cells, it reduces the cell proliferation rate and increase the number of apoptotic cells. On the other side, Klhl14 loss of function in normal thyroid cells affects the expression of several regulatory as well as functional thyroid markers. All these findings suggest that KLHL14 could be considered as a novel tumor suppressor in thyroid cancer, by also revealing its physiological role in the maintenance of a fully differentiated and functional thyroid phenotype.

Ketone Bodies Rescue Mitochondrial Dysfunction Via Epigenetic Remodeling.

Ischemic cardiac disease is a major cause of mortality worldwide. However, the exact molecular processes underlying this disorder are not fully known. This study includes a comprehensive and coordinated set of in vivo and in vitro experiments using human cardiac specimens from patients with postischemic heart failure (HF) and healthy control subjects, a murine model of HF, and cellular systems. These approaches identified for the first time a specific pattern of maladaptive chromatin remodeling, namely a double methylation of histone 3 at lysine 27 and a single methylation at lysine 36 (H3_K27me2K36me1) consistently induced by ischemic injury in all these settings: human HF; murine HF; and in vitro models. Mechanistically, this work demonstrates that this histone modification mediates the ischemia-induced transcriptional repression of PPARG coactivator 1α (PGC1α), master regulator of mitochondrial function and biogenesis. Intriguingly, both the augmented H3_K27me2K36me1 and the mitochondrial dysfunction ensued by PGC1α down-regulation were significantly attenuated by the treatment with ß-hydroxybutyrate, the most abundant ketone body in humans, revealing a novel pathway coupling metabolism to gene expression. Taken together, these findings establish maladaptive chromatin remodeling as a key mechanism in postischemic heart injury, functionally modulated by ketone bodies.

The lncRNA RMST is drastically downregulated in anaplastic thyroid carcinomas where exerts a tumor suppressor activity impairing epithelial-mesenchymal transition and stemness.

Thyroid cancer is the most prevalent endocrine malignancy and comprises a wide range of lesions subdivided into differentiated (DTC) and undifferentiated thyroid cancer (UTC), mainly represented by the anaplastic thyroid carcinoma (ATC). This is one of the most lethal malignancies in humankind leading invariably to patient death in few months. Then, a better comprehension of the mechanisms underlying the development of ATC is required to set up new therapeutic approaches. Long non-coding RNAs (lncRNAs) are transcripts over 200 nucleotides in length that do not code for proteins. They show a strong regulatory function at both transcriptional and post-transcriptional level and are emerging as key players in regulating developmental processes. Their aberrant expression has been linked to several biological processes, including cancer, making them potential diagnostic and prognostic markers. We have recently analyzed the lncRNA expression profile in ATC through a microarray technique and have identified rhabdomyosarcoma 2-associated transcript (RMST) as one of the most downregulated lncRNA in ATC. RMST has been reported to be deregulated in a series of human cancers, to play an anti-oncogenic role in triple-negative breast cancer, and to modulate neurogenesis by interacting with SOX2. Therefore, these findings prompted us to investigate the role of RMST in ATC development. In this study we show that RMST levels are strongly decreased in ATC, but only slightly in DTC, indicating that the loss of this lncRNA could be related to the loss of the differentiation and high aggressiveness. We also report a concomitant increase of SOX2 levels in the same subset of ATC, that inversely correlated with RMST levels, further supporting the RMST/SOX2 relationship. Finally, functional studies demonstrate that the restoration of RMST in ATC cells reduces cell growth, migration and the stemness properties of ATC stem cells. In conclusion, these findings support a critical role of RMST downregulation in ATC development.

The Impact of Resolution of Inflammation on Tumor Microenvironment: Exploring New Ways to Control Cancer Progression.

Non-resolving inflammation is an enabling feature of cancer. A novel super-family of lipid mediators termed Specialized Pro-resolving Mediators (SPMs) have a role as bioactive molecules mediating the resolution of inflammation in cancer biology. SPMs are derived from ω-3 and ω-6 polyunsaturated fatty acids through the activity of lipoxygenases. SPMs have been described to directly modulate cancer progression by interfering with the epithelial to mesenchymal transition and invasion of cancer cells. SPMs have also been demonstrated to act on several components of the tumor microenvironment (TME). Consistently with their natural immunomodulatory and anti-inflammatory properties, SPMs are able to reprogram macrophages to favor phagocytosis of cell debris, which are an important source of pro-inflammatory and pro-angiogenic signals; sustain a direct cytotoxic immune response against cancer cells; stimulate neutrophils anti-tumor activities; and inhibit the development of regulatory T and B cells, thus indirectly leading to enhanced anti-tumor immunity. Furthermore, the resolution pathways exert crucial anti-angiogenic functions in lung, liver, and gastrointestinal cancers, and inhibit cancer-associated fibroblast differentiation and functions in hepatocellular carcinoma and pancreatic cancer. The present review will be focused on the potential protective effects of resolution pathways against cancer, exerted by modulating different components of the TME.

Probiotic Lactobacillus rhamnosus GG (LGG) restrains the angiogenic potential of colorectal carcinoma cells by activating a proresolving program via formyl peptide receptor 1.

Formyl peptide receptors (FPR1, FPR2 and FPR3) are innate immune sensors of pathogen and commensal bacteria and have a role in colonic mucosa homeostasis. We identified FPR1 as a tumour suppressor in gastric cancer cells due to its ability to sustain an inflammation resolution response with antiangiogenic potential. Here, we investigate whether FPR1 exerts similar functions in colorectal carcinoma (CRC) cells. Since it has been shown that the commensal bacterium Lactobacillus rhamnosus GG (LGG) can promote intestinal epithelial homeostasis through FPR1, we explored the possibility that it could induce proresolving and antiangiogenic effects in CRC cells. We demonstrated that pharmacologic inhibition or genetic deletion of FPR1 in CRC cells caused a reduction of proresolving mediators and a consequent upregulation of angiogenic factors. The activation of FPR1 mediates opposite effects. Proresolving, antiangiogenic and homeostatic functions were also observed upon treatment of CRC cells with supernatant of LGG culture, but not of other lactic acid or nonprobiotic bacteria (i.e. Bifidobacterium bifidum or Escherichia coli). These activities of LGG are dependent on FPR1 expression and on the subsequent MAPK signalling activation. Thus, the innate immune receptor FPR1 could be a regulator of the balance between microbiota, inflammation and cancer in CRC models.

Glucose Enhances Pro-Tumorigenic Functions of Mammary Adipose-Derived Mesenchymal Stromal/Stem Cells on Breast Cancer Cell Lines.

Adiposity and diabetes affect breast cancer (BC) progression. We addressed whether glucose may affect the interaction between mammary adipose tissue-derived mesenchymal stromal/stem cells (MAT-MSCs) and BC cells. Two-dimensional co-cultures and spheroids were established in 25 mM or 5.5 mM glucose (High Glucose-HG or Low Glucose-LG) by using MAT-MSCs and MCF7 or MDA-MB231 BC cells. Gene expression was measured by qPCR, while protein levels were measured by cytofluorimetry and ELISA. CD44high/CD24low BC stem-like sub-population was quantified by cytofluorimetry. An in vivo zebrafish model was assessed by injecting spheroid-derived labeled cells. MAT-MSCs co-cultured with BC cells showed an inflammatory/senescent phenotype with increased abundance of IL-6, IL-8, VEGF and p16INK4a, accompanied by altered levels of CDKN2A and LMNB1. BC cells reduced multipotency and increased fibrotic features modulating OCT4, SOX2, NANOG, αSMA and FAP in MAT-MSCs. Of note, these co-culture-mediated changes in MAT-MSCs were partially reverted in LG. Only in HG, MAT-MSCs increased CD44high/CD24low MCF7 sub-population and promoted their ability to form mammospheres. Injection in zebrafish embryos of HG spheroid-derived MCF7 and MAT-MSCs was followed by a significant cellular migration and caudal dissemination. Thus, MAT-MSCs enhance the aggressiveness of BC cells in a HG environment.

In PD-1+ human colon cancer cells NIVOLUMAB promotes survival and could protect tumor cells from conventional therapies.

BACKGROUND: Colorectal cancer (CRC) is one of the most prevalent and deadly tumors worldwide. The majority of CRC is resistant to anti-programmed cell death-1 (PD-1)-based cancer immunotherapy, with approximately 15% with high-microsatellite instability, high tumor mutation burden, and intratumoral lymphocytic infiltration. Programmed death-ligand 1 (PD-L1)/PD-1 signaling was described in solid tumor cells. In melanoma, liver, and thyroid cancer cells, intrinsic PD-1 signaling activates oncogenic functions, while in lung cancer cells, it has a tumor suppressor effect. Our work aimed to evaluate the effects of the anti-PD-1 nivolumab (NIVO) on CRC cells. METHODS: In vitro NIVO-treated human colon cancer cells (HT29, HCT116, and LoVo) were evaluated for cell growth, chemo/radiotherapeutic sensitivity, apoptosis, and spheroid growth. Total RNA-seq was assessed in 6-24 hours NIVO-treated human colon cancer cells HT29 and HCT116 as compared with NIVO-treated PES43 human melanoma cells. In vivo mice carrying HT29 xenograft were intraperitoneally treated with NIVO, OXA (oxaliplatin), and NIVO+OXA, and the tumors were characterized for growth, apoptosis, and pERK1/2/pP38. Forty-eight human primary colon cancers were evaluated for PD-1 expression through immunohistochemistry. RESULTS: In PD-1+ human colon cancer cells, intrinsic PD-1 signaling significantly decreased proliferation and promoted apoptosis. On the contrary, NIVO promoted proliferation, reduced apoptosis, and protected PD-1+ cells from chemo/radiotherapy. Transcriptional profile of NIVO-treated HT29 and HCT116 human colon cancer cells revealed downregulation of BATF2, DRAM1, FXYD3, IFIT3, MT-TN, and TNFRSF11A, and upregulation of CLK1, DCAF13, DNAJC2, MTHFD1L, PRPF3, PSMD7, and SCFD1; the opposite regulation was described in NIVO-treated human melanoma PES43 cells. Differentially expressed genes (DEGs) were significantly enriched for interferon pathway, innate immune, cytokine-mediated signaling pathways. In vivo, NIVO promoted HT29 tumor growth, thus reducing OXA efficacy as revealed through significant Ki-67 increase, pERK1/2 and pP38 increase, and apoptotic cell reduction. Eleven out of 48 primary human colon cancer biopsies expressed PD-1 (22.9%). PD-1 expression is significantly associated with lower pT stage. CONCLUSIONS: In PD-1+ human colon cancer cells, NIVO activates tumor survival pathways and could protect tumor cells from conventional therapies.

Helicobacter pylori Hp(2-20) promotes migration and proliferation of gastric epithelial cells by interacting with formyl peptide receptors in vitro and accelerates gastric mucosal healing in vivo.

Helicobacter pylori-derived peptide RpL1 aa 2-20 (Hp(2-20)) in addition to its antimicrobial action exerts several immunomodulatory effects in eukaryotic cells by interacting with formyl peptide receptors (FPRs). It has recently been shown that activation of FPRs facilitates intestinal epithelial cell restitution. We investigated whether Hp(2-20) induces healing of injured gastric mucosa and assessed the mechanisms underlying any such effect. We investigated the expression of FPRs in two gastric epithelial cell lines (MKN-28 and AGS) at mRNA and protein level. To determine whether FPRs were functional we performed chemotaxis experiments and proliferation assays and studied the Hp(2-20)-activated downstream signaling pathway. The effect of Hp(2-20) on mucosal healing was evaluated in rats after indomethacin-induced injury. Here we show that: (1) FPRs were expressed in both cell lines; (2) Hp(2-20) stimulated migration and proliferation of gastric epithelial cells; (3) this effect was specifically mediated by formyl peptide receptor-like 1 (FPRL1) and FPRL2 and was associated with activation of FPR-related downstream signaling pathways; (4) Hp(2-20) up-regulated the expression and secretion of vascular endothelial growth factor; and (5) Hp(2-20) accelerated healing of rat gastric mucosa after injury brought about by indomethacin at both the macroscopic and microscopic levels. In conclusion, by interacting with FRPL1 and FPRL2, H. pylori-derived Hp(2-20) induces cell migration and proliferation, as well as the expression of vascular endothelial growth factor, thereby promoting gastric mucosal healing. This study provides further evidence of the complexity of the relationship between H. pylori and human gastric mucosa, and it suggests that a bacterial product may be used to heal gastric mucosal injury.

The N-Formyl Peptide Receptors and Rheumatoid Arthritis: A Dangerous Liaison or Confusing Relationship?

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by a progressive symmetric inflammation of the joints resulting in bone erosion and cartilage destruction with a progressive loss of function and joint deformity. An increased number of findings support the role of innate immunity in RA: many innate immune mechanisms are responsible for producing several cytokines and chemokines involved in RA pathogenesis, such as Tumor Necrosis Factor (TNF)-α, interleukin (IL)-6, and IL-1. Pattern recognition receptors (PRRs) play a crucial role in modulating the activity of the innate arm of the immune response. We focused our attention over the years on the expression and functions of a specific class of PRR, namely formyl peptide receptors (FPRs), which exert a key function in both sustaining and resolving the inflammatory response, depending on the context and/or the agonist. We performed a broad review of the data available in the literature on the role of FPRs and their ligands in RA. Furthermore, we queried a publicly available database collecting data from 90 RA patients with different clinic features to evaluate the possible association between FPRs and clinic-pathologic parameters of RA patients.

Toll-Like Receptor 7 Mediates Inflammation Resolution and Inhibition of Angiogenesis in Non-Small Cell Lung Cancer.

Pattern recognition receptors (PRR) promote inflammation but also its resolution. We demonstrated that a specific PRR-formyl peptide receptor 1 (FPR1)-sustains an inflammation resolution response with anti-angiogenic and antitumor potential in gastric cancer. Since toll-like receptor 7 (TLR7) is crucial in the physiologic resolution of airway inflammation, we asked whether it could be responsible for pro-resolving and anti-angiogenic responses in non-small cell lung cancer (NSCLC). TLR7 correlated directly with pro-resolving and inversely with angiogenic mediators in NSCLC patients, as revealed by a publicly available RNAseq analysis. In NSCLC cells, depletion of TLR7 caused an upregulation of angiogenic mediators and a stronger vasculogenic response of endothelial cells compared to controls, assessed by qPCR, ELISA, protein array, and endothelial cell responses. TLR7 activation induced the opposite effects. TLR7 silencing reduced, while its activation increased, the pro-resolving potential of NSCLC cells, evaluated by qPCR, flow cytometry, and EIA. The increased angiogenic potential of TLR7-silenced NSCLC cells is due to the lack of pro-resolving mediators. MAPK and STAT3 signaling are responsible for these activities, as demonstrated through Western blotting and inhibitors. Our data indicate that TLR7 sustains a pro-resolving signaling in lung cancer that inhibits angiogenesis. This opens new possibilities to be exploited for cancer treatment.

PD-1 blockade delays tumor growth by inhibiting an intrinsic SHP2/Ras/MAPK signalling in thyroid cancer cells.

BACKGROUND: The programmed cell death-1 (PD-1) receptor and its ligands PD-L1 and PD-L2 are immune checkpoints that suppress anti-cancer immunity. Typically, cancer cells express the PD-Ls that bind PD-1 on immune cells, inhibiting their activity. Recently, PD-1 expression has also been found in cancer cells. Here, we analysed expression and functions of PD-1 in thyroid cancer (TC). METHODS: PD-1 expression was evaluated by immunohistochemistry on human TC samples and by RT-PCR, western blot and FACS on TC cell lines. Proliferation and migration of TC cells in culture were assessed by BrdU incorporation and Boyden chamber assays. Biochemical studies were performed by western blot, immunoprecipitation, pull-down and phosphatase assays. TC cell tumorigenicity was assessed by xenotransplants in nude mice. RESULTS: Human TC specimens (47%), but not normal thyroids, displayed PD-1 expression in epithelial cells, which significantly correlated with tumour stage and lymph-node metastasis. PD-1 was also constitutively expressed on TC cell lines. PD-1 overexpression/stimulation promoted TC cell proliferation and migration. Accordingly, PD-1 genetic/pharmacologic inhibition caused the opposite effects. Mechanistically, PD-1 recruited the SHP2 phosphatase to the plasma membrane and potentiated its phosphatase activity. SHP2 enhanced Ras activation by dephosphorylating its inhibitory tyrosine 32, thus triggering the MAPK cascade. SHP2, BRAF and MEK were necessary for PD-1-mediated biologic functions. PD-1 inhibition decreased, while PD-1 enforced expression facilitated, TC cell xenograft growth in mice by affecting tumour cell proliferation. CONCLUSIONS: PD-1 circuit blockade in TC, besides restoring anti-cancer immunity, could also directly impair TC cell growth by inhibiting the SHP2/Ras/MAPK signalling pathway.