Cell Membrane-associated Molecules Research Articles

Salmonella Subpopulations Identified from Human Specimens Express Heterogenous Phenotypes That Are Relevant to Clinical Diagnosis.

Clonal bacterial cells can give rise to functionally heterogeneous subpopulations. This diversification is considered an adaptation strategy that has been demonstrated for several bacterial species, including Salmonella enterica serovar Typhimurium. In previous studies on mouse models infected orally with pure Salmonella cultures, derived bacterial cells collected from animal tissues were found to express heterogenous phenotypes. Here, we show mixed Salmonella populations, apparently derived from the same progenitor, present in human specimens collected at a single disease time point, and in a long-term-infected patient, these Salmonella were no longer expressing surface-exposed antigen epitopes by isolates collected at earlier days of the disease. The subpopulations express different phenotypes related to cell surface antigen expression, motility, biofilm formation, biochemical metabolism, and antibiotic resistance, which can all contribute to pathogenicity. Some of the phenotypes correlate with single nucleotide polymorphisms or other sequence changes in bacterial genomes. These genetic variations can alter synthesis of cell membrane-associated molecules such as lipopolysaccharides and lipoproteins, leading to changes in bacterial surface structure and function. This study demonstrates the limitation of Salmonella diagnostic methods that are based on a single-cell population which may not represent the heterogenous bacterial community in infected humans. IMPORTANCE In animal model systems, heterogenous Salmonella phenotypes were found previously to regulate bacterial infections. We describe in this communication that different Salmonella phenotypes also exist in infected humans at a single disease time point and that their phenotypic and molecular traits are associated with different aspects of pathogenicity. Notably, variation in genes encoding antibiotic resistance and two-component systems were observed from the subpopulations of a patient suffering from persistent salmonellosis. Therefore, clinical and public health interventions of the disease that are based on diagnosis of a single-cell population may miss other subpopulations that can cause residual human infections.

Abstract 934: “GPCR-ome” modulation upon PD1 / PDL1 axis blockade

Abstract Background: Immunotherapy represents a novel weapon within the cancer armamentarium with unprecedented response observed in several indications including among others metastatic melanoma and Lung cancer. However, the limited rate of response accounting for app. 30% requires further optimization and thus calls the interest in elucidating mechanisms underlying sensitivity to immune checkpoint inhibitors. G Protein-Coupled Receptors (GPCRs) are the largest gene family of cell membrane-associated molecules mediating signal transmission, involved in several physiological functions including the shaping of the immune response but their role in tumor biology and particularly in immune evasion and resistance to immunotherapy remains to be elucidated Methods: Using a preclinical syngeneic mouse model of sarcoma based on the inoculation of murine sarcoma MCA205 cell line, we investigated - besides the evaluation of tumor growth and survival - a large set of GPCRs upon PDL1 or PD1 blockade. Gene expression analysis was performed on tumor samples by mean of RNA sequencing. Data were analyzed using computational methodology including CIBERSORT and GSEA algorithms to evaluate immune cell subset within the tumor and specific gene datasets enrichment respectively. A large set of GPCRs (1412 in total) was then investigated through conventional Differential Gene Expression (DGE) analysis. Results: PDL1/PD1 axis blockade, using specific antibodies, exhibited a strong anti-tumor effect. As highlighted by CIBERSORT analysis, anti-PD1/PDL1 antibodies triggered a CD8 and activated NK cells infiltration within the tumor. The recruitment of such effectory populations was associated with enrichment in genes from different hallmarks datasets including allograft rejection and interferon signaling thus confirming the induction of an efficient anti-tumor immune response. Concomitantly, this study revealed a significant up-regulation of several GPCRs already known to be involved in immune evasion - including Adenosine, Prostaglandins and chemokines receptors - but also highlighted the modulation of unexpected orphan or peptidic receptors. Altogether, this novel dataset confirms the induction of an effective anti-tumor immune response upon PD1 / PDL1 blockade and highlights the modulation of GPCRs that warrants further investigation to delineate their exact participation in sensitivity to immunotherapy. Citation Format: Imane Nafia, Xavier Leroy, Loic Cerf, Jean-Philippe Guegan, Anne-Laure Blayo, Antoine Italiano, Stephan Schann, Alban Bessede. “GPCR-ome” modulation upon PD1 / PDL1 axis blockade [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 934.

Reciprocal crosstalk between dendritic cells and natural killer cells under the effects of PGE2 in immunity and immunopathology

The reciprocal activating crosstalk between dendritic cells (DCs) and natural killer (NK) cells plays a pivotal role in regulating immune defense against viruses and tumors. The cytokine-producing capacity, Th-cell polarizing ability and chemokine expression, migration and stimulatory functions of DCs are regulated by activated NK cells. Conversely, the innate and effector functions of NK cells require close interactions with activated DCs. Cell membrane-associated molecules and soluble mediators, including cytokines and prostaglandins (PGs), contribute to the bidirectional crosstalk between DCs and NK cells. One of the most well-known and well-studied PGs is PGE2. Produced by many cell types, PGE2 has been shown to affect various aspects of the immune and inflammatory responses by acting on all components of the immune system. There is emerging evidence that PGE2 plays crucial roles in DC and NK cell biology. Several studies have shown that DCs are not only a source of PGE2, but also a target of its immunomodulatory action in normal immune response and during immune disorders. Although NK cells appear to be unable to produce PGE2, they are described as powerful PGE2-responding cells, as they express all PGE2 E-prostanoid (EP) receptors. Several NK cell functions (lysis, migration, proliferation, cytokine production) are influenced by PGE2. This review highlights the effects of PGE2 on DC-NK cell crosstalk and its subsequent impact on immune regulations in normal and immunopathological processes.

Ectodomain Shedding of Interleukin-2 Receptor β and Generation of an Intracellular Functional Fragment

Interleukin-2 (IL-2) regulates different functions of various lymphoid cell subsets. These are mediated by its binding to the IL-2 receptor (IL-2R) composed of three subunits (IL2-Ralpha, -beta, and -gamma(c)). IL-2Rbeta is responsible for the activation of several signaling pathways. Ectodomain shedding of membrane receptors is thought to be an important mechanism for down-regulation of cell surface receptor abundance but is also emerging as a mechanism that cell membrane-associated molecules require for proper action in vivo. Here, we demonstrate that IL-2Rbeta is cleaved in cell lines of different origin, including T cells, generating an intracellular 37-kDa fragment (37beta ic) that comprises the full intracellular C-terminal and transmembrane domains. Ectodomain shedding of IL-2Rbeta decreases in a mutant deleted of the juxtamembrane region, where cleavage is predicted to occur, and is inhibited by tissue inhibitor of metalloproteases-3. 37Beta ic is tyrosine-phosphorylated and associates with STAT-5, a canonic signal transducer of IL-2R. Finally, lymphoid cell transfection with a truncated form of IL-2Rbeta mimicking 37beta ic increases their proliferation. These data indicate that IL-2Rbeta is subject to ectodomain shedding generating an intracellular fragment biologically functional, because (i) it is phosphorylated, (ii) it associates with STAT5A, and (iii) it increases cell proliferation.

Endothelial Activation. Sliding Door to Atherosclerosis

The vascular endothelium has been long viewed as a simply physical separation between blood and tissue [1]. Over the last two decades a consistent bulk of data demonstrated that endothelium is a widely distributed organ, with a variable degree of heterogeneity among and within tissues [2, 3] and deeply involved in vascular physiology and pathophysiology, rather than a cellophane-like membrane that lines the circulatory system [1]. Being positioned at the interface of blood and tissue, the vascular endothelium is able to sense changes in hemodynamic forces and biochemical stimuli and promptly responds to local changes in biological needs by modulating vasomotion, hemostasis, angiogenesis, and vascular growth [1]. The vascular endothelium also modulates the trafficking of circulating blood cells by up-regulating adhesion molecules on cell surface [1, 4, 5]. These cell membrane-associated molecules are critic for leukocyte migration into specific organs under physiologic conditions and accelerate migration towards sites of inflammation [1, 4, 5]. Recently, the multi-step cascade of events that results in the local recruitment of leukocytes to sites of inflammatory challenge, also known as endothelial activation, has been considered as a crucial step in the initiation of atherosclerosis process, as well as in the development of advanced atherosclerosis [6].

Genomic and non-genomic effects of estrogens on endothelial cells

Estrogen receptors act via the regulation of transcriptional processes, involving nuclear translocation and binding on specific response elements, thus leading to regulation of target gene expression. However, novel non-transcriptional mechanisms of signal transduction through steroid hormone receptors have been identified. These so-called “non-genomic” effects are independent by gene transcription or protein synthesis and involve steroid-induced modulation of cytoplasmic or of cell membrane-bound regulatory proteins. Relevant biological actions of steroids have been associated with this signaling in different tissues. Ubiquitary regulatory cascades such as mitogen-activated protein kinases (MAPK), the phosphatidylinositol 3-OH kinase (PI3K) and tyrosine kinases are modulated through non-transcriptional mechanisms by steroid hormones. Furthermore, steroid hormone receptors modulation of cell membrane-associated molecules such as ion channels and G-protein-coupled receptors has been shown in diverse tissues. The vascular wall is a site where non-genomic steroid hormones actions are particularly prominent. For instance, estrogens and glucocorticoids trigger rapid vasodilatation due to rapid induction of nitric oxide synthesis in endothelial cells via the estrogen receptor-dependent activation of MAPK and PI3K, leading to relevant pathophysiological consequences, in vitro and in vivo. The growing amount of evidence collected in the last years claims that non-transcriptional signaling mechanisms play a primary role in the generation of the effects of steroids on endothelial cells, which may turn out to be of relevance for clinical purposes.

Ex vivo expansion of limbal epithelial stem cells: amniotic membrane serving as a stem cell niche

Identification, maintenance, and expansion of stem cells for subsequent transplantation has become a new strategy for treating many diseases in most medical subspecialties. The stem cells of the corneal epithelium are located in the limbal basal layer and are the ultimate source for constant corneal epithelial renewal. Like those in other tissues, limbal stem cells are supported by a unique stromal microenvironment called the stem cell niche, which consists of certain extracellular matrix components, cell membrane-associated molecules, and cytokine dialogues. Destructive loss of limbal stem cells or dysfunction of their stromal environment renders many corneas with a clinical entity called limbal stem cell deficiency, which is characterized by variable extents of conjunctival ingrowth depending on the severity of limbal damage. A new strategy of treating limbal stem cell deficiency is to transplant a bio-engineered graft by expanding limbal epithelial stem cells ex vivo on amniotic membrane. This review summarizes the published literature data collectively explaining how amniotic membrane is an ideal biological substrate that can help maintain and support the expansion of limbal epithelial stem cells.

Non-genomic actions of sex steroid hormones

Steroid hormone receptors have been traditionally considered to act via the regulation of transcriptional processes, involving nuclear translocation and binding to specific response elements, and ultimately leading to regulation of gene expression. However, novel non-transcriptional mechanisms of signal transduction through steroid hormone receptors have been identified. These so-called 'non-genomic' effects do not depend on gene transcription or protein synthesis and involve steroid-induced modulation of cytoplasmic or cell membrane-bound regulatory proteins. Several relevant biological actions of steroids have been associated with this kind of signaling. Ubiquitous regulatory cascades such as mitogen-activated protein kinases, the phosphatidylinositol 3-OH kinase and tyrosine kinases are modulated through non-transcriptional mechanisms by steroid hormones. Furthermore, steroid hormone receptor modulation of cell membrane-associated molecules such as ion channels and G-protein-coupled receptors has been shown. TIssues traditionally considered as 'non-targets' for classical steroid actions are instead found to be vividly regulated by non-genomic mechanisms. To this aim, the cardiovascular and the central nervous system provide excellent examples, where steroid hormones induce rapid vasodilatation and neuronal survival via non-genomic mechanisms, leading to relevant pathophysiological consequences. The evidence collected in the past Years indicates that target cells and organs are regulated by a complex interplay of genomic and non-genomic signaling mechanisms of steroid hormones, and the integrated action of these machineries has important functional roles in a variety of pathophysiological processes. The understanding of the molecular basis of the rapid effects of steroids is therefore important, and may in the future turn out to be of relevance for clinical purposes.

Assays for antibody-dependent cell-mediated cytotoxicity (ADCC) and reverse ADCC (redirected cytotoxicity) in human natural killer cells.

Crosslinking of molecules involved in the recognition of target cells elicits natural killer (NK) cell cytotoxicity, inducing either release of preformed cytotoxic mediators via granule exocytosis, or (enhanced) expression of ligands for target cell membrane-associated molecules that transduce apoptotic signals, e.g., Fas Ligand (FasL) or TRAIL (reviewed in refs. ,). Target cell recognition by NK cells occurs via either of two ways: (1) receptors capable of binding molecules on the target cells that include, but are not limited to, adhesion molecules and the identities of which are incompletely defined (reviewed in ref. ); and (2) receptors for the Fc fragment of IgG (FcγRIIIA, CD 16) (, reviewed in ref. ). The latter bind immunecomplexed IgG and therefore target cells that, not recognized spontaneously by NK cells, are sensitized with anti-target cell antibodies (Abs) of the IgG class. The two types of cytotoxicity elicited are referred to as “spontaneous” and “Ab-dependent’ cell-mediated cytotoxicity (ADCC).