Involvement of CCL2 in Salivary Gland Response to Hyperosmolar Stress Related to Sjögren's Syndrome.

In primary Sjögren's syndrome (pSS) patients, salivary gland (SG) epithelial cells (SGECs) could be exposed to chronic hyperosmotic stress (HOS), consecutive to their destruction and deregulation, that exacerbates an inflammatory response. The aims of this study were to assess the mechanism accounting for C-C motif chemokine ligand 2 (CCL2) expression in an immortalized human salivary gland epithelial acinar cell line (NS-SV-AC) subjected to HOS, as well as the involvement of CCL2 in pSS. CCL2 mRNA and protein levels were determined via RT-qPCR and ELISA. Reporter plasmids and a promoter pull-down assay were used to identify transcription factors associated with CCL2 mRNA increase. Our data showed that HOS-induced CCL2 mRNA increase was independent of the nuclear factor of activated T-cells 5 (NFAT5) and nuclear factor-kappa B (NFkB) but involved Kruppel-like factor 5 (KLF5). CCL2 protein levels, quantified by enzyme-linked immunosorbent assay (ELISA) in sera samples from pSS patients, correlated with the European Alliance of Associations for Rheumatology's Sjogren's syndrome disease activity index (ESSDAI) score for systemic activity. In addition, CCL2 protein levels were higher in patients with biological activity, cutaneous manifestations, and ESSDAI score superior or equal to five. Our data suggest that chronic HOS could exacerbate pSS disease by contributing to the inflammatory process induced by the expression and secretion of CCL2.

Lipopolysaccharide Modifies Glycerol Permeability and Metabolism in 3T3-L1 Adipocytes.

Aquaglyceroporins-aquaporin membrane channels (AQP) that conduct glycerol and other small neutral solutes in addition to water-play major roles in obesity. In adipocytes, aquaglyceroporins mediate glycerol uptake and release across the plasma membrane, which are two key steps for triacylglycerols (TAGs) synthesis (lipogenesis) and hydrolysis (lipolysis). The aim of this study was to assess both glycerol permeability and metabolism in undifferentiated 3T3-L1 cells (UDCs) as well as in untreated (CTL-DCs) versus lipopolysaccharide (LPS-DCs)-treated differentiated 3T3-L1 adipocytes. Glycerol release, TAGs content and whole membrane glycerol permeability were significantly increased in DCs as compared to UDCs. Moreover, in DCs, LPS treatment significantly increased TAGs content and decreased glycerol permeability. In addition, a significant reduction in whole membrane glycerol permeability was observed in LPS-DCs as compared to CTL-DCs. The relative contributions of AQP3, AQP7 and AQP9 (facilitated diffusion), as well as that of the phospholipid bilayer (simple diffusion), to the whole membrane glycerol permeability, were estimated biophysically in UDCs, CTL-DCs and LPS-DCs, using selective AQP inhibitors. Further studies will be required to determine if modifications in either subcellular localization and/or activity of aquaglyceroporins could account for the data herein. Nevertheless, our findings provide novel insights in understanding the LPS-induced adipocyte hypertrophy that accompanies obesity.

Forskolin Inhibits Lipopolysaccharide-Induced Modulation of MCP-1 and GPR120 in 3T3-L1 Adipocytes through an Inhibition of NFκB.

In an obese state, Toll-like receptor-4 (TLR-4) upregulates proinflammatory adipokines secretion including monocyte chemotactic protein-1 (MCP-1) in adipose tissue. In contrast, G-protein coupled receptor 120 (GPR120) mediates antiobesity effects. The aim of this study was to determine the signaling pathway by which Forskolin (FK), a cyclic adenosine monophosphate- (cAMP-) promoting agent causing positive changes in body composition in overweight and obese adult men, affects MCP-1 and GPR120 expression during an inflammatory response induced by lipopolysaccharide (LPS) in adipocytes, such as in an obese state. 3T3-L1 cells differentiated into adipocytes (DC) were stimulated with LPS in the absence or presence of FK and inhibitors of TLR-4 and inhibitor of kappa B (IκBα). In DC, LPS increased MCP-1, TLR-4, and nuclear factor-κB1 (NFκB1) mRNA levels, whereas it decreased GPR120 mRNA levels. In DC, FK inhibited the LPS-induced increase in MCP-1, TLR-4, and NFκB1 mRNA levels and the LPS-induced decrease in GPR120 mRNA. BAY11-7082 and CLI-095 abolished these LPS-induced effects. In conclusion, FK inhibits LPS-induced increase in MCP-1 mRNA levels and decrease in GPR120 mRNA levels in adipocytes and may be a potential treatment for inflammation in obesity. Furthermore, TLR-4-induced activation of NFκB may be involved in the LPS-induced regulation of these genes.

Involvement of JNK/NFκB Signaling Pathways in the Lipopolysaccharide-Induced Modulation of Aquaglyceroporin Expression in 3T3-L1 Cells Differentiated into Adipocytes.

Aquaglyceroporins, belonging to the family of aquaporins (AQPs), are integral plasma membrane proteins permeable to water and glycerol that have emerged as key players in obesity. The aim of this study was to investigate the expression profile of AQPs in undifferentiated and differentiated 3T3-L1 cells and to investigate the changes in expression of aquaglyceroporins in 3T3-L1 cells differentiated into adipocytes and subjected to lipopolysaccharide (LPS) mimicking inflammation occurring during obesity. Furthermore, the study aimed at identifying the signaling cascade involved in the regulation of aquaglyceroporins expression upon LPS stimulation. 3T3-L1 cells were grown as undifferentiated cells (UDC; preadipocytes) or cells differentiated into adipocytes (DC, adipocytes). DC were incubated in the presence or absence of LPS with or without inhibitors of various protein kinases. AQPs mRNA expression levels were measured by real-time quantitative polymerase chain reaction (RT-qPCR). AQP1, AQP2, AQP3, AQP9 and AQP11 mRNA were expressed in both UDC and DC, whereas AQP4, AQP7 and AQP8 mRNA were expressed only in DC. In DC, LPS up-regulated AQP3 mRNA levels (p < 0.05) compared to control; these effects were inhibited by CLI095, SP600125 and BAY11-7082 (p < 0.05). LPS decreased both AQP7 and AQP11 mRNA levels (p < 0.01) in DC as compared to control; this decrease was inhibited by CLI095 and BAY11-7082 (p < 0.05) and additionally by SP00125 for AQP7 (p < 0.05). SB203580 had no effect on LPS-induced AQP3, AQP7 and AQP11 mRNA levels modulations. In conclusion, our results clearly show that many AQPs are expressed in murine 3T3-L1 adipocytes. Moreover, in DCs, LPS led to decreased AQP7 and AQP11 mRNA levels but to increased AQP3 mRNA levels, resulting from the Toll-like receptor 4 (TLR4)-induced activation of JNK and/or NFκB pathway.

Analysis of aquaporin expression in liver with a focus on hepatocytes.

A deeper understanding of aquaporins (AQPs) expression and transcriptional regulation will provide useful information for liver pathophysiology. We established a complete AQPs mRNA expression profile in human and mouse liver, as well as protein localization of expressed AQPs. Additionally, the modulation of AQPs mRNA levels in response to various agents was determined in human HuH7 cells and in primary culture of mouse hepatocytes. AQP1, AQP3, AQP7, AQP8, and AQP9 mRNA and protein expressions were detected in human liver, while only AQP6 and AQP11 mRNAs were detected. We reported for the first time the localization of AQP3 in Kupffer cells, AQP7 in hepatocytes and endothelial cells, and AQP9 in cholangiocytes. In addition, we confirmed the localization of AQP1 in endothelial cells, and of AQP8 and AQP9 in hepatocytes. On HuH7 cells, we reported the presence of AQP4 mRNA, confirmed the presence of AQP3, AQP7, and AQP11 mRNAs, but not of AQP8 mRNA. On primary culture of murine hepatocytes, AQP1 and AQP7 mRNAs were identified, while the presence of AQP3, AQP8, AQP9, and AQP11 mRNAs was confirmed. At the protein level, murine endothelial liver cells expressed AQP1 and AQP9, while hepatocytes expressed AQP3, AQP7, AQP8, and AQP9, and macrophages expressed AQP3. Dexamethasone, forskolin, AICAR, rosiglitazone, octanoylated, and non-octanoylated ghrelin regulated some AQP expression in primary culture of murine hepatocytes and human HuH7 cells. Additional studies will be required to further assess the role of AQPs expression in human and murine liver and understand the transcriptional regulation of AQPs in hepatocytes under pathophysiological conditions.

Molecular Epidemiology of Group A Streptococcus Infections in The Gambia.

Molecular epidemiological data on Group A Streptococcus (GAS) infection in Africa is scarce. We characterized the emm-types and emm-clusters of 433 stored clinical GAS isolates from The Gambia collected between 2004 and 2018. To reduce the potential for strain mistyping, we used a newly published primer for emm-typing. There was considerable strain diversity, highlighting the need for vaccine development offering broad strain protection.

Unraveling Human AQP5-PIP Molecular Interaction and Effect on AQP5 Salivary Glands Localization in SS Patients.

Saliva secretion requires effective translocation of aquaporin 5 (AQP5) water channel to the salivary glands (SGs) acinar apical membrane. Patients with Sjögren's syndrome (SS) display abnormal AQP5 localization within acinar cells from SGs that correlate with sicca manifestation and glands hypofunction. Several proteins such as Prolactin-inducible protein (PIP) may regulate AQP5 trafficking as observed in lacrimal glands from mice. However, the role of the AQP5-PIP complex remains poorly understood. In the present study, we show that PIP interacts with AQP5 in vitro and in mice as well as in human SGs and that PIP misexpression correlates with an altered AQP5 distribution at the acinar apical membrane in PIP knockout mice and SS hMSG. Furthermore, our data show that the protein-protein interaction involves the AQP5 C-terminus and the N-terminal of PIP (one molecule of PIP per AQP5 tetramer). In conclusion, our findings highlight for the first time the role of PIP as a protein controlling AQP5 localization in human salivary glands but extend beyond due to the PIP-AQP5 interaction described in lung and breast cancers.

Analysis of Global Collection of Group A Streptococcus Genomes Reveals that the Majority Encode a Trio of M and M-Like Proteins.

The core Mga (multiple gene activator) regulon of group A Streptococcus (GAS) contains genes encoding proteins involved in adhesion and immune evasion. While all GAS genomes contain genes for Mga and C5a peptidase, the intervening genes encoding M and M-like proteins vary between strains. The genetic make-up of the Mga regulon of GAS was characterized by utilizing a collection of 1,688 GAS genomes that are representative of the global GAS population. Sequence variations were examined with multiple alignments, and the expression of all core Mga regulon genes was examined by quantitative reverse transcription-PCR in a representative strain collection. In 85.2% of the sampled genomes, the Mga locus contained genes encoding Mga, Mrp, M, Enn, and C5a peptidase proteins. These isolates account for 53% of global infections. Only 9.1% of genomes did not contain either an mrp or an enn gene. The pairwise identity within Enn (68.6%) and Mrp (83.2%) protein sequences was higher than within M proteins (44.7%). Gene expression varied between strains tested, but high expression was recorded for all genes in at least one strain. Previous nomenclature issues were clarified with molecular gene definitions. Our findings support a shift in focus in the GAS research field to further consider the role of Mrp and Enn in virulence and vaccine development.IMPORTANCE While the GAS M protein has been the leading vaccine target for decades, the bacteria encode many other virulence factors of interest for vaccine development. In this work, we show that emm-like genes are encoded in a remarkable majority of GAS genomes and expressed at a level similar to that for the emm gene. In collaboration with the U.S. Centers for Disease Control, we developed molecular definitions of the different emm and emm-like gene families. This clarification should abrogate mistyping of strains, especially in the area of whole-genome typing. We have also updated the emm-typing collection by removing emm-like gene sequences and provided in-depth analysis of Mrp and Enn protein sequence structure and diversity.

Multiple proteins arising from a single gene: The role of the Spa33 variants in Shigella T3SS regulation.

Shigella invasion and dissemination in intestinal epithelial cells relies on a type 3 secretion system (T3SS), which mediates translocation of virulence proteins into host cells. T3SSs are composed of three major parts: an extracellular needle, a basal body, and a cytoplasmic complex. Three categories of proteins are hierarchically secreted: (a) the needle components, (b) the translocator proteins which form a pore (translocon) inside the host cell membrane and (c) the effectors interfering with the host cell signaling pathways. In the absence of host cell contact, the T3SS is maintained in an "off" state by the presence of a tip complex. Secretion is activated by host cell contact which allows the release of a gatekeeper protein called MxiC. In this work, we have investigated the role of Spa33, a component of the cytoplasmic complex, in the regulation of secretion. The spa33 gene encodes a 33-kDa protein and a smaller fragment of 12 kDa (Spa33C ) which are both essential components of the cytoplasmic complex. We have shown that the spa33 gene gives rise to 5 fragments of various sizes. Among them, three are necessary for T3SS. Interestingly, we have shown that Spa33 is implicated in the regulation of secretion. Indeed, the mutation of a single residue in Spa33 induces an effector mutant phenotype, in which MxiC is sequestered. Moreover, we have shown a direct interaction between Spa33 and MxiC.

Streptococcus pyogenes infections with limited emm-type diversity in the homeless population of Brussels, 2016-2018.

OBJECTIVES: The aim was to characterize the clinical features, outcomes, and strain diversity of laboratory-confirmed Streptococcus pyogenes (group A Streptococcus, GAS) infections among inpatients hospitalized at a tertiary level hospital in Brussels, Belgium, according to the patients' housing status (homeless vs. not homeless). METHODS: Between August 2016 and January 2018, all patients hospitalized with a laboratory-confirmed GAS infection were prospectively enrolled and risk factors were recorded. GAS strains were characterized using emm-typing and emm-clustering in both inpatients and outpatients. Analyses were performed according to homelessness status. RESULTS: During the study period, 48% (28/58) of adults hospitalized with a GAS infection at the tertiary hospital were homeless. The estimated incidence rate was 100 times higher for homeless persons. Skin abscesses were more frequent in the homeless group (21.4% vs. 3.3%) and mortality was high (10.7%). Limited emm-type diversity was found in this group, with four emm-types (64, 77, 83, and 101) accounting for 76.1% of the infections, and the majority of these emm-types belonged to the D4 emm-cluster. Pooled analyses of inpatient and outpatient strains indicated lower diversity in the homeless group. CONCLUSIONS: The homeless are disproportionately affected by GAS and have a higher rate of abscesses and high mortality. The lower emm-type diversity and preferential infection with four emm-types likely reflects endemic circulation of GAS in this population. Preventive strategies are warranted in this fragile population.

Modification of aquaporin expression in response to fenretinide-induced transdifferentiation of ARPE-19 cells into neuronal-like cells.

PURPOSE: The goal of this study was to investigate the modifications of aquaporin (AQP) expression in ARPE-19 cells in response to fenretinide-induced transdifferentiation into neuronal-like cells METHODS: ARPE-19 cells were treated daily for 7 days with 3 µm fenretinide or dimethyl sulphoxide as control. mRNA and protein expression were evaluated by real-time quantitative PCR, Western blot analysis and immunofluorescence. RESULTS: Control ARPE-19 cells expressed AQP1, AQP4, AQP6 and AQP11 at the mRNA level, but only AQP4, AQP6 and AQP11 at the protein level. Fenretinide induced the transdifferentiation of ARPE-19 cells into neuronal-like cells. Indeed, fenretinide induced morphological changes similar to neurons characterized by elongated cell body and the formation of neurite branching. Moreover, ARPE-19 cells transdifferentiated to neuron-like cells were characterized by significant decrease in retinal pigmented epithelium markers, for example cytokeratin 8 and cellular retinaldehyde-binding protein, as well as an increase in neuronal markers such as synaptophysin and calretinin. AQP4 expression, at both mRNA and protein levels, and AQP6 expression, only at protein level, were significantly decreased in ARPE-19 cells transdifferentiated into neuronal-like cells. CONCLUSIONS: The expression of AQP4 and AQP6 is downregulated during fenretinide-induced transdifferentiation.

Murine 3T3-L1 adipocyte cell differentiation model: validated reference genes for qPCR gene expression analysis.

BACKGROUND: Analysis of gene expression at the mRNA level, using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), mandatorily requires reference genes (RGs) as internal controls. However, increasing evidences have shown that RG expression may vary considerably under experimental conditions. We sought for an appropriate panel of RGs to be used in the 3T3-L1 cell line model during their terminal differentiation into adipocytes. To this end, the expression levels of a panel of seven widely used RG mRNAs were measured by qRT-PCR. The 7 RGs evaluated were ß-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyl-transferase I (HPRT), ATP synthase H+ transporting mitochondrial F1 complex beta subunit (ATP-5b), tyrosine 3-monooxygenase/tryptophan 5- monooxygenase activation protein, zeta polypeptide (Ywhaz), Non-POU-domain containing octamer binding protein (NoNo), and large ribosomal protein L13a (RPL). METHODOLOGY/PRINCIPAL FINDINGS: Using three Excel applications, GeNorm, NormFinder and BestKeeper, we observed that the number and the stability of potential RGs vary significantly during differentiation of 3T3-L1 cells into adipocytes. mRNA expression analyses using qRT-PCR revealed that during the entire differentiation program, only NoNo expression is relatively stable. Moreover, the RG sets that were acceptably stable were different depending on the phase of the overall differentiation process (i.e. mitotic clonal expansion versus the terminal differentiation phase). RPL, ACTB, and Ywhaz, are suitable for terminal differentiation, whereas ATP-5b and HPRT, are suitable during mitotic clonal expansion. CONCLUSION: Our results demonstrate that special attention must be given to the choice of suitable RGs during the various well defined phases of adipogenesis to ensure accurate data analysis and that the use of several RGs is absolutely required. Consequently, our data show for the first time, that during mitotic clonal expansion, the most suitable RGs are ATP-5b, NoNo and HPRT, while during terminal differentiation the most suitable RGs are, NoNo, RPL, ACTB and Ywhaz.

Perturbation of glycerol metabolism in hepatocytes from n3-PUFA-depleted rats.

Second generation n3-PUFA-depleted rats represent a good animal model of metabolic syndrome as they display several features of the disease such as liver steatosis, visceral obesity and insulin resistance. The goal of our study was to investigate the influence of n3-PUFA deficiency on hepatic glycerol metabolism. Aquaglyceroporin 9 (AQP9) allows hepatic glycerol transport and consequently contributes to neoglucogenesis. AQP9 knockout mice display hypertriacyl-glycerolemia, one of the hallmarks of the metabolic syndrome. Our data show reduced AQP9 expression at the protein level in n3-PUFA-depleted rats, without any changes at the mRNA levels. [U-¹4C]glycerol uptake was increased in hepatocytes from n3-PUFA-depleted animal cells. The apparent discrepancy between decreased AQP9 protein expression, and increased [U-¹4C]glycerol uptake could be explained by an observed increase in glycerol kinase activity.