Neutrophil-derived Activin-A moderates their pro-NETotic activity and attenuates collateral tissue damage caused by Influenza A virus infection.

Background: Pre-neutrophils, while developing in the bone marrow, transcribe the Inhba gene and synthesize Activin-A protein, which they store and release at the earliest stage of their activation in the periphery. However, the role of neutrophil-derived Activin-A is not completely understood. Methods: To address this issue, we developed a neutrophil-specific Activin-A-deficient animal model (S100a8-Cre/Inhba fl/fl mice) and analyzed the immune response to Influenza A virus (IAV) infection. More specifically, evaluation of body weight and lung mechanics, molecular and cellular analyses of bronchoalveolar lavage fluids, flow cytometry and cell sorting of lung cells, as well as histopathological analysis of lung tissues, were performed in PBS-treated and IAV-infected transgenic animals. Results: We found that neutrophil-specific Activin-A deficiency led to exacerbated pulmonary inflammation and widespread hemorrhagic histopathology in the lungs of IAV-infected animals that was associated with an exuberant production of neutrophil extracellular traps (NETs). Moreover, deletion of the Activin-A receptor ALK4/ACVR1B in neutrophils exacerbated IAV-induced pathology as well, suggesting that neutrophils themselves are potential targets of Activin-A-mediated signaling. The pro-NETotic tendency of Activin-A-deficient neutrophils was further verified in the context of thioglycollate-induced peritonitis, a model characterized by robust peritoneal neutrophilia. Of importance, transcriptome analysis of Activin-A-deficient neutrophils revealed alterations consistent with a predisposition for NET release. Conclusion: Collectively, our data demonstrate that Activin-A, secreted by neutrophils upon their activation in the periphery, acts as a feedback mechanism to moderate their pro-NETotic tendency and limit the collateral tissue damage caused by neutrophil excess activation during the inflammatory response.

Specific myeloid signatures in peripheral blood differentiate active and rare clinical phenotypes of multiple sclerosis.

Current understanding of Multiple Sclerosis (MS) pathophysiology implicates perturbations in adaptive cellular immune responses, predominantly T cells, in Relapsing-Remitting forms (RRMS). Nevertheless, from a clinical perspective MS is a heterogeneous disease reflecting the heterogeneity of involved biological systems. This complexity requires advanced analysis tools at the single-cell level to discover biomarkers for better patient-group stratification. We designed a novel 44-parameter mass cytometry panel to interrogate predominantly the role of effector and regulatory subpopulations of peripheral blood myeloid subsets along with B and T-cells (excluding granulocytes) in MS, assessing three different patient cohorts: RRMS, PPMS (Primary Progressive) and Tumefactive MS patients (TMS) (n=10, 8, 14 respectively). We further subgrouped our cohort into inactive or active disease stages to capture the early underlying events in disease pathophysiology. Peripheral blood analysis showed that TMS cases belonged to the spectrum of RRMS, whereas PPMS cases displayed different features. In particular, TMS patients during a relapse stage were characterized by a specific subset of CD11c+CD14+ CD33+, CD192+, CD172+-myeloid cells with an alternative phenotype of monocyte-derived macrophages (high arginase-1, CD38, HLA-DR-low and endogenous TNF-a production). Moreover, TMS patients in relapse displayed a selective CD4 T-cell lymphopenia of cells with a Th2-like polarised phenotype. PPMS patients did not display substantial differences from healthy controls, apart from a trend toward higher expansion of NK cell subsets. Importantly, we found that myeloid cell populations are reshaped under effective disease-modifying therapy predominantly with glatiramer acetate and to a lesser extent with anti-CD20, suggesting that the identified cell signature represents a specific therapeutic target in TMS. The expanded myeloid signature in TMS patients was also confirmed by flow cytometry. Serum neurofilament light-chain levels confirmed the correlation of this myeloid cell signature with indices of axonal injury. More in-depth analysis of myeloid subsets revealed an increase of a subset of highly cytolytic and terminally differentiated NK cells in PPMS patients with leptomeningeal enhancement (active-PPMS), compared to those without (inactive-PPMS). We have identified previously uncharacterized subsets of circulating myeloid cells and shown them to correlate with distinct disease forms of MS as well as with specific disease states (relapse/remission).

Rubicon promotes rather than restricts murine lupus and is not required for LC3-associated phagocytosis.

NADPH oxidase deficiency exacerbates lupus in murine models and patients, but the mechanisms remain unknown. It is hypothesized that NADPH oxidase suppresses autoimmunity by facilitating dead cell clearance via LC3-associated phagocytosis (LAP). The absence of LAP reportedly causes an autoinflammatory syndrome in aged, nonautoimmune mice. Prior work implicated cytochrome b-245, ß polypeptide (CYBB), a component of the NADPH oxidase complex, and the RUN and cysteine-rich domain-containing Beclin 1-interacting protein (RUBICON) as requisite for LAP. To test the hypothesis that NADPH oxidase deficiency exacerbates lupus via a defect in LAP, we deleted Rubicon in the B6.Sle1.Yaa and MRL.Faslpr lupus mouse models. Under this hypothesis, RUBICON deficiency should phenocopy NADPH oxidase deficiency, as both work in the same pathway. However, we observed the opposite - RUBICON deficiency resulted in reduced mortality, renal disease, and autoantibody titers to RNA-associated autoantigens. Given that our data contradict the published role for LAP in autoimmunity, we assessed whether CYBB and RUBICON are requisite for LAP. We found that LAP is not dependent on either of these 2 pathways. To our knowledge, our data reveal RUBICON as a novel regulator of SLE, possibly by a B cell-intrinsic mechanism, but do not support a role for LAP in lupus.

Imaging Approaches for the Study of Metabolism in Real Time Using Genetically Encoded Reporters.

Metabolism comprises of two axes in order to serve homeostasis: anabolism and catabolism. Both axes are interbranched with the so-called bioenergetics aspect of metabolism. There is a plethora of analytical biochemical methods to monitor metabolites and reactions in lysates, yet there is a rising need to monitor, quantify and elucidate in real time the spatiotemporal orchestration of complex biochemical reactions in living systems and furthermore to analyze the metabolic effect of chemical compounds that are destined for the clinic. The ongoing technological burst in the field of imaging creates opportunities to establish new tools that will allow investigators to monitor dynamics of biochemical reactions and kinetics of metabolites at a resolution that ranges from subcellular organelle to whole system for some key metabolites. This article provides a mini review of available toolkits to achieve this goal but also presents a perspective on the open space that can be exploited to develop novel methodologies that will merge classic biochemistry of metabolism with advanced imaging. In other words, a perspective of "watching metabolism in real time."

Visualizing S1P-directed cellular egress by intravital imaging.

Sphingosine-1-phosphate (S1P) is a bioactive lipid that provides cellular signals through plasma membrane G protein-coupled receptors. The S1P receptor signaling system has a fundamental and widespread function in licensing the exit and release of hematopoietically derived cells from various tissues into the circulation. Although the outlines of the mechanism have been established through genetic and pharmacologic perturbations, the temporal and spatial dynamics of the cellular events involved have been unclear. Recently, two-photon intravital imaging has been applied to living tissues to visualize the cellular movements and interactions that occur during egress processes. Here we discuss how some of these recent findings provide a clearer picture regarding S1P receptor signaling in modulating cell egress into the circulation. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.

Compromise in mRNA processing machinery in senescent human fibroblasts: implications for a novel potential role of Phospho-ATR (ser428).

Ataxia-Telangiectasia and Rad3 related kinase (ATR) is a major gatekeeper of genomic stability and has been the subject of exhaustive study in the context of cell cycle progression and senescence as a DNA damage-induced response. Conditional knockout of the kinase in adult mice results in accelerated aging phenomena, such as such hair graying, alopecia, kyphosis, osteoporosis, thymic involution, fibrosis, and other abnormalities. In addition to that, recent reports strongly implicate signaling mediated by this kinase in the regulation of alternative splicing of certain, mostly cancer-associated transcripts. Interest to the function of mRNA synthesis and processing is constantly increasing as severe degenerative diseases, such as cancer, cystic fibrosis and Hutchinson-Gilford progeria syndrome are at least partly attributed to these abnormalities. In light of the above, we investigate the RNA processing machinery in senescent fibroblasts as opposed to young, either exponentially proliferating or quiescent, further focusing on the distribution and localization of active, phosphorylated ATR at ser428. This study implicates the spatiotemporal presence of the phosphorylated kinase in the regulation of mRNA splicing and polyadenylation. This function appears perturbed in senescent cells, accompanied by a distinct pattern of phospho-ATR in the senescent nucleus.

TGF-beta regulates differentially the proliferation of fetal and adult human skin fibroblasts via the activation of PKA and the autocrine action of FGF-2.

Transforming growth factor-beta (TGF-beta) is a potent regulator of cell proliferation; interestingly its action is clearly cell type-dependent. In particular, it inhibits epithelial and endothelial cells' proliferation, while its action on many mesenchymal cells has been reported to be stimulatory. In this direction, we have recently shown that TGF-beta regulates the proliferation of normal human skin fibroblasts according to their developmental origin: i.e. it inhibits fetal fibroblasts, while it stimulates the proliferation of adult ones. Here, we present evidence on the mechanisms underlying this differential action. Concerning fetal fibroblasts, we have found that TGF-beta activates Protein Kinase A (PKA) and induces the expression of the cyclin-dependent kinase inhibitors (CKIs) p21(CIP1/WAF1) and p15(INK4B). Moreover, the specific PKA inhibitor H-89 blocks the induction of both CKIs and annuls the TGF-beta-mediated inhibitory effect, indicating the central role of PKA in this process. In contrast, in adult cells no PKA activation is observed. Moreover, TGF-beta stimulates cell proliferation by activating the MEK-ERK pathway, as the MEK inhibitor PD98059 blocks this effect. A specific neutralizing antibody against Fibroblast Growth Factor-2 (FGF-2) inhibits both ERK activation and the mitogenic activity of TGF-beta, indicating that the latter establishes an autocrine loop, via FGF-2, leading to cell proliferation. This loop requires FGF receptor-1 (FGFR-1), as its down-regulation by siRNA approach prevents TGF-beta from stimulating ERK-1/2 activation and DNA synthesis. In conclusion, the differential proliferative response of fetal and adult normal human skin fibroblasts to TGF-beta is regulated by distinct signaling pathways and furthermore it may provide information on the bimodal effect of this factor on cell proliferation, in general.

Differential proliferative response of fetal and adult human skin fibroblasts to transforming growth factor-beta.

Since pronounced differences exist between the fetal and adult repair processes, we studied the proliferative response of skin fibroblasts from these two stages to transforming growth factor-beta (TGF-beta), a cytokine with a broad range of activities in tissue repair. Here, we present evidence that TGF-beta inhibits fetal human skin fibroblasts, while it is stimulatory for adult ones. This proliferative effect of TGF-beta was found to be concentration- dependent, but isoform-independent. Furthermore, even a transient exposure of the cells to this growth factor was sufficient to exert its stimulatory or inhibitory action. Accordingly, we have studied the immediate responses provoked by TGF-beta in major signaling pathways, and we have found that it induces a rapid activation of the SMAD pathway, i.e., phosphorylation and nuclear translocation of SMAD2, followed by dephosphorylation, most probably due to degradation by the proteasome. However, similar intensity and kinetics of this activation have been observed in both fetal and adult fibroblasts. On the other hand, curcumin, a natural product with wound healing properties that inhibits several intracellular signaling pathways, was found to completely abrogate the inhibitory effect of TGF-beta1 on human fetal skin fibroblasts, without affecting the stimulatory action on fibroblasts from adult donors. In conclusion, there is a major radical in the proliferative response of fetal and adult human skin fibroblasts to TGF-beta, possibly reflecting the different repair strategies followed in these two stages of development.

Fusion of a tumour-associated antigen to HIV-1 Tat improves protein-based immunotherapy of cancer.

BACKGROUND: The ultimate success of cancer vaccination is primarily dependent upon the generation of tumour-specific CTLs. Protein-based vaccination, while safe, poorly elicits such CTL responses. As fusion of an antigen to the HIV-1 Tat transduction domain was reported to increase MHC class I presentation and CTL responses in vitro, we tested the potency of this approach to augment tumour-directed responses. MATERIALS AND METHODS: Human papillomavirus type 16 (HPV16) E7 proteins, fused (E7-Tat) or not (E7) to Tat carboxy-terminal region, were produced and studied in vitro and in vivo. RESULTS: E7-Tat, not E7, penetrated the cell membrane and was transcriptionally active. In vitro, E7-Tat induced higher IFN-gamma production from E7-specific T-cells than E7. In C57BL/6 mice, E7-Tat mixed with Quil A generated enhanced prophylactic and therapeutic suppression of HPV16-positive C3 tumour outgrowth. Similar, but greatly enhanced E7-specific effector and helper T-cell responses were elicited following E7-Tat/Quil A rather than E7/Quil A vaccination. CONCLUSION: This study offers a new strategy for improving subunit cancer vaccines.