Adenosine A(2A) receptor (A(2A)R)-dependent signaling in macrophages plays a key role in the regulation of inflammation. However, the processes regulating A(2A)R targeting to the cell surface and degradation in macrophages are incompletely understood. For example, the C-terminal domain of the A(2A)R and proteins interacting with it are known to regulate receptor recycling, although it is unclear what role potential A(2A)R-interacting partners have in macrophages. Here, we aimed to identify A(2A)R-interacting partners in macrophages that may effect receptor trafficking and activity. To this end, we performed a yeast two-hybrid screen using the C-terminal tail of A(2A)R as the "bait" and a macrophage expression library as the "prey." We found that the lysosomal protease cathepsin D (CtsD) was a robust hit. The A(2A)R-CtsD interaction was validated in vitro and in cellular models, including RAW 264.7 and mouse peritoneal macrophage (IPM) cells. We also demonstrated that the A(2A)R is a substrate of CtsD and that the blockade of CtsD activity increases the density and cell surface targeting of A(2A)R in macrophages. Conversely, we demonstrate that A(2A)R activation prompts the maturation and enzymatic activity of CtsD in macrophages. In summary, we conclude that CtsD is a novel A(2A)R-interacting partner and thus describe molecular and functional interplay that may be crucial for adenosine-mediated macrophage regulation in inflammatory processes.

Many autoimmune and infectious diseases are characterized by the formation of granulomas which are inflammatory lesions that consist of spatially organized immune cells. These sites protect the host and control pathogens like Mycobacterium tuberculosis (Mtb), but are highly inflammatory and cause pathology. Using bacille Calmette-Guerin (BCG) and Mtb infection in mice that induce sarcoid or caseating granulomas, we show that a subpopulation of granuloma macrophages produces vascular endothelial growth factor (VEGF-A), which recruits immune cells to the granuloma by a non-angiogenic pathway. Selective blockade of VEGF-A in myeloid cells, combined with granuloma transplantation, shows that granuloma VEGF-A regulates granulomatous inflammation. The severity of granuloma-related inflammation can be ameliorated by pharmaceutical or genetic inhibition of VEGF-A, which improves survival of mice infected with virulent Mtb without altering host protection. These data show that VEGF-A inhibitors could be used as a host-directed therapy against granulomatous diseases like tuberculosis and sarcoidosis, thereby expanding the value of already existing and approved anti-VEGF-A drugs.

In “The role of TAMIS (transanal minimally invasive surgery) in the management of advanced rectal cancer—one shared story of three exceptional cases,” the authors aim to raise awareness for the use of TAMIS in patients with advanced rectal cancer, when the gold standard of treatment (total mesorectal excision, TME) is either not feasible or refused by the patient. The results of the above mentioned paper encourage the consideration of TAMIS in certain clinical scenarios.1 In the United States, colorectal cancer is estimated to have the third highest mortality rate among all types of cancers for women, and the second highest mortality rate among all types of cancers for men in 2017. Furthermore, colorectal cancer is one of the more common forms of cancer in the United States.2 Approximately 135,430 new cases of colorectal cancer are estimated for 2017.2 The incidence of rectal cancer in men and women under the age of 50 has been increasing at an alarming rate of 2.1% per year from 1992 through 2012.3 It is especially important to offer treatments with positive clinical outcomes that also consider quality of life in younger patients. Multiple studies have shown that this minimally invasive approach can preserve both fecal continence and allow for a higher quality of life.4,5 Research on minimally invasive procedures for rectal cancer is desirable since studies analyzing large sample populations with significant conclusions are lacking. TME is known to have various complications such as wound infections, fistulas, leaks, bleeding, and extensive postoperative morbidity and mortality, so an

Nemeth, Zoltan H. MD, PhD; Hasko, Gyorgy MD, PhD; DiFazio, Louis T. MD, FACS; Kong, Karen; Bilaniuk, Jaroslaw W. MD, FACS; Csoka, Balazs PhD Author Information

The macrophage is a major phagocytic cell type, and its impaired function is a primary cause of immune paralysis, organ injury, and death in sepsis. An incomplete understanding of the endogenous molecules that regulate macrophage bactericidal activity is a major barrier for developing effective therapies for sepsis. Using an in vitro killing assay, we report here that the endogenous purine ATP augments the killing of sepsis-causing bacteria by macrophages through P2X4 receptors (P2X4Rs). Using newly developed transgenic mice expressing a bioluminescent ATP probe on the cell surface, we found that extracellular ATP levels increase during sepsis, indicating that ATP may contribute to bacterial killing in vivo. Studies with P2X4R-deficient mice subjected to sepsis confirm the role of extracellular ATP acting on P2X4Rs in killing bacteria and protecting against organ injury and death. Results with adoptive transfer of macrophages, myeloid-specific P2X4R-deficient mice, and P2rx4 tdTomato reporter mice indicate that macrophages are essential for the antibacterial, antiinflammatory, and organ protective effects of P2X4Rs in sepsis. Pharmacological targeting of P2X4Rs with the allosteric activator ivermectin protects against bacterial dissemination and mortality in sepsis. We propose that P2X4Rs represent a promising target for drug development to control bacterial growth in sepsis and other infections.

Nemeth, Zoltan H. MD, PhD; Csoka, Balazs PhD; Hakakian, Daniel; Hasko, Gyorgy MD, PhD Author Information

BACKGROUND:The murine model of high fat diet (HFD)-induced obesity is characterized by an increment of intestinal permeability, secondary to an impairment of mucosal epithelial barrier and enteric inflammation, followed by morphofunctional rearrangement of the enteric nervous system. The present study investigated the involvement of abdominal macrophages in the mechanisms underlying the development of enteric dysmotility associated with obesity.METHODS:Wild type C57BL/6J mice were fed with HFD (60% kcal from fat) or normocaloric diet (NCD, 18% kcal from fat) for 8 weeks. Groups of mice fed with NCD or HFD were treated with clodronate encapsulated into liposomes to deplete abdominal macrophages. Tachykininergic contractions, elicited by electrical stimulation or exogenous substance P (SP), were recorded in vitro from longitudinal muscle colonic preparations. Substance P distribution was examined by confocal immunohistochemistry. The density of macrophages in the colonic wall was examined by immunohistochemical analysis. Malondialdehyde (MDA, colorimetric assay) and IL-1β (ELISA assay) levels were also evaluated.RESULTS:MDA and IL-1β levels were increased in colonic tissues from HFD-treated animals. In colonic preparations, electrically evoked tachykininergic contractions were enhanced in HFD mice. Immunohistochemistry displayed an increase in substance P immunoreactivity in myenteric ganglia, as well as in the muscular layers of colonic cryosections from obese mice. Macrophage depletion in HFD mice was associated with a significant reduction of colonic inflammation. In addition, the decrease in macrophage density attenuated the morphofunctional alterations of tachykininergic pathways observed in obese mice.CONCLUSION:Obesity elicited by HFD determines a condition of colonic inflammation, followed by a marked rearrangement of motor excitatory tachykininergic enteric nerves. Macrophage depletion counteracted the morphofunctional changes of colonic neuromuscular compartment, suggesting a critical role for these immune cells in the onset of enteric dysmotility associated with obesity.

Group 2 innate lymphoid cells (ILC2s) represent a rapid source of type 2 cytokines, such as IL-5 and IL-13, and play an important role in orchestrating type 2 immune response. Adenosine is an endogenous purine nucleoside, a catabolite of ATP that binds and activates >= 1 of 4 transmembrane G protein-coupled cell-surface adenosine receptors (ARs)-A(1), A(2A), A(2B), and A(3). Here, we studied the role of ARs in the regulation of cytokine production by ILC2s. We found that A(2B)ARs suppress the production of both IL-5 and IL-13 by ILC2s, whereas A(2A)ARs augment IL-5 production and fail to affect IL-13 release. Combined stimulation of all ARs led to the suppression of both IL-5 and IL-13 production, which indicated that A(2B)ARs dominate A(2A)ARs. Both pre-and post-transcriptional processes may be involved in the AR modulation of ILC2 IL-5 and IL-13 production. Thus, we identify adenosine as a novel negative regulator of ILC2 activation.

Intestinal helminth infections trigger potent type 2 immune responses, although the mechanisms that initiate these host protective responses against these multicellular parasites remain uncertain. We have previously reported that inoculation with the murine intestinal nematode parasite Heligmosomoides polygyrus (Hp) results in rapid increases in intestinal epithelial surface expression of the ectonucleotidases, CD39 and CD73, which in turn degrade extracellular ATP into adenosine. Adenosine binds to adenosine G-protein-coupled cell surface receptors (A1, A2A, A2B and A3 –AR). Mice deficient in A2BAR have an impaired type 2 immune response and delayed worm expulsion during helminth infection. We now report that mice lacking A2BAR specifically on intestinal epithelial cells (Villin-Cre-A2BARfl/fl), but not on myeloid cells (LysMCre-A2BARfl/fl), have an impaired type 2 memory immune response with delayed worm expulsion and decreased egg production after Hp secondary inoculation. pSTAT6 levels in CD4+ T cells and B cell MHC-II surface expression (an indicator of IL-4 activity) from mesenteric lymph nodes of Villin-Cre-A2BARfl/fl mice were significantly decreased after primary Hp inoculation. Taken together, our data demonstrate a key role for epithelial cell specific A2BAR signaling in the initiation of the protective type 2 immune response to helminth infection. These studies indicate that extracellular adenosine binding to epithelial cell A2BAR acts as an endogenous danger signal that is released following helminth infection, which triggers host protective type 2 immune responses.

Background and PurposeGlycogen phosphorylase (GP) is the key enzyme for glycogen degradation. GP inhibitors (GPi‐s) are glucose lowering agents that cause the accumulation of glucose in the liver as glycogen. Glycogen metabolism has implications in beta cell function. Glycogen degradation can maintain cellular glucose levels, which feeds into catabolism to maintain insulin secretion, and elevated glycogen degradation levels contribute to glucotoxicity. The purpose of this study was to assess whether influencing glycogen metabolism in beta cells by GPi‐s affects the function of these cells.Experimental ApproachThe effects of structurally different GPi‐s were investigated on MIN6 insulinoma cells and in a mouse model of diabetes.Key ResultsGPi treatment increased glycogen content and, consequently, the surface area of glycogen in MIN6 cells. Furthermore, GPi treatment induced insulin receptor β (InsRβ), Akt and p70S6K phosphorylation, as well as pancreatic and duodenal homeobox 1(PDX1) and insulin expression. In line with these findings, GPi‐s enhanced non‐stimulated and glucose‐stimulated insulin secretion in MIN6 cells. The InsRβ was shown to co‐localize with glycogen particles as confirmed by in silico screening, where components of InsR signalling were identified as glycogen‐bound proteins. GPi‐s also activated the pathway of insulin secretion, indicated by enhanced glycolysis, mitochondrial oxidation and calcium signalling. Finally, GPi‐s increased the size of islets of Langerhans and improved glucose‐induced insulin release in mice.Conclusion and ImplicationsThese data suggest that GPi‐s also target beta cells and can be repurposed as agents to preserve beta cell function or even ameliorate beta cell dysfunction in different forms of diabetes.Linked ArticlesThis article is part of a themed section on Inventing New Therapies Without Reinventing the Wheel: The Power of Drug Repurposing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.2/issuetoc

Nemeth, Zoltan H. MD, PhD; Csoka, Balazs PhD; Hakakian, Daniel BS; DiFazio, Louis T. MD; Hasko, Gyorgy MD, PhD Author Information

Our study aimed at finding a mechanistic relationship between the gut microbiome and breast cancer. Breast cancer cells are not in direct contact with these microbes, but disease could be influenced by bacterial metabolites including secondary bile acids that are exclusively synthesized by the microbiome and known to enter the human circulation. In murine and bench experiments, a secondary bile acid, lithocholic acid (LCA) in concentrations corresponding to its tissue reference concentrations (< 1 μM), reduced cancer cell proliferation (by 10–20%) and VEGF production (by 37%), aggressiveness and metastatic potential of primary tumors through inducing mesenchymal-to-epithelial transition, increased antitumor immune response, OXPHOS and the TCA cycle. Part of these effects was due to activation of TGR5 by LCA. Early stage breast cancer patients, versus control women, had reduced serum LCA levels, reduced chenodeoxycholic acid to LCA ratio, and reduced abundance of the baiH (7α/β-hydroxysteroid dehydroxylase, the key enzyme in LCA generation) gene in fecal DNA, all suggesting reduced microbial generation of LCA in early breast cancer.

Adenosine, a key extracellular signaling mediator, regulates several aspects of metabolism by activating 4 G-protein-coupled receptors, the A1, A2A, A2B, and A3 adenosine receptors (ARs). The role of A2AARs in regulating high-fat-diet (HFD)-induced metabolic derangements is unknown. To evaluate the role of A2AARs in regulating glucose and insulin homeostasis in obesity, we fed A2AAR-knockout (KO) and control mice an HFD for 16 wk to initiate HFD-induced metabolic disorder. We found that genetic deletion of A2AARs caused impaired glucose tolerance in mice fed an HFD. This impaired glucose tolerance was caused by a decrease in insulin secretion but not in insulin sensitivity. Islet size and insulin content in pancreata of A2AAR-deficient mice were decreased compared with control mice after consuming an HFD. A2AAR-KO mice had decreased expression of the β-cell-specific markers pdx1, glut2, mafA, and nkx6.1 and increased expression of the dedifferentiation markers sox2 and hes1. Ex vivo islet experiments confirmed the role of A2AARs in protecting against decreased insulin content and release caused by HFD. Other experiments with bone marrow chimeras revealed that inflammation was not the primary cause of decreased insulin secretion in A2AAR-KO mice. Altogether, our data showed that A2AARs control pancreatic dysfunction in HFD-induced obesity.-Csóka, B., Törő, G., Vindeirinho, J., Varga, Z. V., Koscsó, B., Németh, Z. H., Kókai, E., Antonioli, L., Suleiman, M., Marchetti, P., Cseri, K., Deák, Á., Virág, L., Pacher, P., Bai, P., Haskó, G. A2A adenosine receptors control pancreatic dysfunction in high-fat-diet-induced obesity.

HIF-1 is a ubiquitous signaling molecule constantly expressed by the body, but is degraded during normoxic conditions. In hypoxic conditions, it persists and is active. Hypoxia is often associated with trauma due to interrupted blood flow, inflammation or other reasons, causing HIF-1 to be active in signaling and recovery. In this review, the function of HIF-1 is examined, as well as its clinical significance with regard to trauma and critical care. Using this information, we then identify potential points of treatment and intervention.

Adenosine A(2B) receptors (A(2B)R) regulate several enteric functions. However, their implication in the pathophysiology of intestinal dysmotility associated with high-fat diet (HFD)-induced obesity has not been elucidated. We investigated the expression of A(2B)R in mouse colon and their role in the mechanisms underlying the development of enteric dysmotility associated with obesity. Wild-type C57BL/6J mice were fed with HFD (60% kcal from fat) or normocaloric diet (NCD; 18% kcal from fat) for 8 weeks. Colonic A(2B)R localization was examined by immunofluorescence. The role of A(2B)R in the control of colonic motility was examined in functional experiments on longitudinal muscle preparations (LMPs). In NCD mice, A(2B)R were predominantly located in myenteric neurons; in HFD animals, their expression increased throughout the neuromuscular layer. Functionally, the A(2B)R antagonist MRS1754 enhanced electrically induced NK1-mediated tachykininergic contractions in LMPs from HFD mice, while it was less effective in tissues from NCD mice. The A(2B) receptor agonist BAY 60-6583 decreased colonic tachykininergic contractions in LMPs, with higher efficacy in preparations from obese mice. Both A(2B)R ligands did not affect contractions elicited by exogenous substance P. Obesity is related with a condition of colonic inflammation, leading to an increase of A(2B)R expression. A(2B)R, modulating the activity of excitatory tachykininergic nerves, participate to the enteric dysmotility associated with obesity.

Nemeth, Zoltan H. MD, PhD; Csoka, Balazs PhD; Spolarics, Zoltan MD, PhD; DiFazio, Louis T. MD; Rolandelli, Rolando H. MD, FACS; Hasko, Gyorgy MD, PhD Author Information

The global epidemic of obesity is constantly growing and represents an enormous challenge for health care systems worldwide. Obesity fuels the development of metabolic syndrome that includes components such as elevated glucose levels, insulin resistance, elevated blood pressure, and increased levels of triglycerides (1). Obesity and metabolic syndrome increase the risk of metabolic diseases, such as type 2 diabetes (T2D), cardiovascular disease, and atherosclerosis, and contribute to a reduction in life expectancy (2). In the U.S., the obesity rate in adults has reached 36% and obesity affects more than 1 billion people worldwide (3). Currently, 9.3% of the U.S. population has diabetes. In adults, T2D accounts for 90–95% of all diagnosed cases of diabetes, and the estimated total economic burden of diabetes in the U.S. is $245 billion (4). Thus, it is imperative that we increase our understanding of the mechanisms that lead to the development of obesity-induced insulin resistance and T2D, which will help identify therapeutic targets to reduce the impact of these syndromes on morbidity and mortality. The first piece of evidence that obesity, insulin resistance, and inflammation are interconnected was provided more than a century ago when Dr. R.T. Williamson (5) observed that the anti-inflammatory drug sodium salicylate improved glucose control in patients with diabetes. Almost 90 years later, Hotamisligil et al. (6) revisited this observation as they found that the neutralization of tumor necrosis factor (TNF)-α improved insulin resistance, and thus a link between inflammation and diet-induced insulin resistance was established. Subsequent studies elucidated that a complex immune cellular network regulates inflammation and insulin responsiveness in metabolic tissues. Recently, interleukin (IL)-1β antibodies in monotherapy or in …

Abstract Extracellular ATP binds to and signals through P2X7 receptors (P2X7R)s to modulate immune function in both inflammasome-dependent and independent manners. We show here using P2X7-/- mice as well as pharmacological receptor and channel ligands that ATP release through connexin/pannexin channels and subsequent P2X7R activation are crucial for the control of mortality, bacterial dissemination and inflammation following sepsis induced by cecal ligation and puncture. Our results with P2X7-/- bone-marrow chimeras, adoptive transfer of macrophages, and myeloid-specific P2X7-/- mice indicate that P2X7R signaling on macrophages is required for the protective effect of P2X7Rs. P2X7R signaling protects through enhancing bacterial killing by macrophages, but independently of the inflammasome. In summary, targeting P2X7Rs provides a new opportunity for harnessing an endogenous protective immune mechanism in the therapy of sepsis.

In this issue of Biochemical Journal, Chen and colleagues characterize an interaction between ACBD3 (acyl-CoA-binding domain-containing 3) protein and PARP [poly(ADP-ribose) polymerase]-1 through the activation of ERKs (extracellular-signal-regulated kinases). This study envisages a pathway through which ABCD3 translates enhanced fatty acid levels to ERK and consequently PARP-1 activation. The consequences of PARP-1 activation lead to cellular and tissue damage, implying that the ACBD3/PARP-1 pathway is an important pathway in lipotoxicity events.