Kitisak Sintiprungrat

Alterations in cellular proteome and secretome upon differentiation from monocyte to macrophage by treatment with phorbol myristate acetate: Insights into biological processes

Monocyte and macrophage are mainly involved in immune response and inflammatory processes. Monocytes circulate in the bloodstream and migrate to various tissues where they can differentiate to macrophages. However, the molecular basis of biological processes involved in this cellular differentiation remains ambiguous. This study was to investigate alterations in cellular and secreted proteins after this differentiation phase. Macrophage was differentiated from U937 human monocytic cell line by treatment with 100 ng/ml phorbol myristate acetate (PMA) for 48 h. Cellular and secreted proteins extracted from PMA-treated cells (macrophages) were compared with those of untreated cells (monocytes) using 2-DE (n=5 gels/condition; stained with Deep Purple fluorescence dye). Quantitative intensity analysis revealed 81 and 67 protein spots whose levels were significantly altered in cellular proteome and secretome. These proteins were subsequently identified by Q-TOF MS and/or MS/MS analyses. The altered levels of cellular elongation factor-2 (EF-2) and secreted alpha-tubulin were confirmed by Western blot analysis. Global protein network analysis demonstrated that these altered proteins were involved in cell death, lipid metabolism, cell morphology, cellular movement, and protein folding. Our data may provide some insights into molecular mechanisms of biological processes upon differentiation from monocytes to macrophages.

Secreted products of macrophages exposed to calcium oxalate crystals induce epithelial mesenchymal transition of renal tubular cells via RhoA-dependent TGF-β1 pathway.

Kidney stone disease is associated with renal fibrosis by the unclear mechanisms. We hypothesized that calcium oxalate (CaOx), a major crystalline component of kidney stones, could induce secretion of fibrotic factors from macrophages leading to “epithelial mesenchymal transition/transdifferentiation” (EMT) of renal tubular cells. Western blot analysis revealed an increased level of vimentin (mesenchymal marker) but decreased levels of E-cadherin and cytokeratin (epithelial markers) in MDCK cells treated with “secreted products from CaOx-exposed macrophages” (CaOx-M-Sup). Immunofluorescence study confirmed the increased level of vimentin and decreased level of cytokeratin, and also revealed the increased level of fibronectin (another mesenchymal marker). The data also showed decreased levels and disorganization of F-actin (cytoskeletal marker) and zonula occludens-1 (ZO-1) (tight junction marker) induced by CaOx-M-Sup. ELISA demonstrated the increased level of transforming growth factor-β1 (TGF-β1), the well-defined EMT inducer, in CaOx-M-Sup. Downstream signaling of TGF-β1 was involved as demonstrated by the decreased level of RhoA. Interestingly, pretreatment with a proteasome inhibitor (MG132) could restore RhoA to its basal level, most likely through ubiquitin-proteasome pathway (UPP). Moreover, MG132 successfully sustained cytoskeletal assembly and tight junction, and could prevent the cells from EMT. Altogether, these data demonstrate for the first time that CaOx-M-Sup could induce EMT in renal tubular cells by TGF-β1 signaling cascade via RhoA and UPP. This may be, at least in part, the underlying mechanism for renal fibrosis in kidney stone disease.

Macropinocytosis is the Major Mechanism for Endocytosis of Calcium Oxalate Crystals into Renal Tubular Cells

During an initial phase of kidney stone formation, the internalization of calcium oxalate (CaOx) crystals by renal tubular cells has been thought to occur via endocytosis. However, the precise mechanism of CaOx crystal endocytosis remained unclear. In the present study, MDCK renal tubular cells were pretreated with inhibitors specific to individual endocytic pathways, including nystatin (lipid raft/caveolae-mediated), cytochalasin D (actin-dependent or macropinocytosis), and chlorpromazine (CPZ; clathrin-mediated) before exposure to plain (non-labeled), or fluorescence-labeled CaOx monohydrate (COM) crystals. Quantitative analysis by flow cytometry revealed that pretreatment with nystatin and CPZ slightly decreased the crystal internalization, whereas the cytochalasin D pretreatment caused a marked decrease in crystal uptake. Immunofluorescence study and laser-scanning confocal microscopic examination confirmed that the cytochalasin D-pretreated cells had dramatic decrease of the internalized crystals, whereas the total number of crystals interacted with the cells was unchanged (crystals could adhere but were not internalized). These data have demonstrated for the first time that renal tubular cells endocytose COM crystals mainly via macropinocytosis. These novel findings will be useful for further tracking the endocytosed crystals inside the cells during the course of kidney stone formation.

Alterations in macrophage cellular proteome induced by calcium oxalate crystals: the association of HSP90 and F-actin is important for phagosome formation.

The presence of macrophages in renal interstitium is the key feature of progressive renal inflammation in kidney stone disease. However, response of macrophages to calcium oxalate monohydrate (COM) crystals, the major crystalline composition of kidney stone, remained unclear. This study aimed to investigate alterations in the cellular proteome of macrophages induced by COM crystals using a proteomics approach. U937-derived macrophages (by phorbol-12-myristate-13-acetate activation) were incubated without or with 100 μg/mL COM crystals for 24 h. Their cellular proteins were resolved by 2-DE (n = 10 gels; 5 were derived from 5 independent cultures in each group) and visualized with Deep Purple fluorescent dye. Spot matching, quantitative intensity analysis, and statistics revealed 18 differentially expressed protein spots, which were successfully identified by Q-TOF MS and MS/MS analyses. The altered levels of α-tubulin, β-actin and ezrin were validated by Western blot analysis. Protein interaction network analysis using STRING software showed that 90 kDa heat shock protein (HSP90) was associated with β-actin and α-tubulin (all these three proteins were increased in the COM-treated macrophages). Multiple immunofluorescence stainings confirmed the associations of HSP90 with filamentous form of actin (F-actin) and α-tubulin. However, only the association between HSP90 and F-actin was found on the phagosome membrane surrounding COM crystal, indicating that the association of HSP90 with F-actin, but not with α-tubulin, is important for phagosome formation. Silencing of HSP90 (siHSP90) reduced expression of cytoskeletal proteins and phagosome marker (Rab5) and successfully diminished COM crystal-induced phagocytosis and migration of macrophages. Our findings enlightened the significant role of these altered proteins, especially HSP90, in enhanced phagocytic activity of the COM-exposed macrophages.

Proteome changes in human monocytes upon interaction with calcium oxalate monohydrate crystals.

Monocytic infiltration in renal interstitium is commonly found surrounding the site of calcium oxalate (CaOx) crystal deposition in the kidney. Monocytes are supposed to eliminate the deposited crystals. However, effects of CaOx crystals on the infiltrating monocytes remain unknown. Therefore, this study investigated the altered cellular proteome of human monocytes in response to interaction with CaOx monohydrate (COM) crystals. After 24-h culture with or without 100 microg/mL COM crystals, U937 cells were harvested and subjected to 2-DE analysis with Deep Purple fluorescence staining (n = 5 gels/group; each was derived from independent culture). Spot matching, quantitative intensity analysis, and statistics revealed 22 differentially expressed proteins (9 up-regulated and 13 down-regulated proteins), which were successfully identified by Q-TOF MS and MS/MS analyses, including those involved in cell cycle, cellular structure, carbohydrate metabolism, lipid metabolism, mRNA processing, and protein synthesis, stabilization, and degradation. Randomly selected changes [up-regulated ALG-2 interacting protein 1 (Alix), elongation factor-2 (EF-2), and down-regulated beta-actin] were confirmed by Western blot analysis. Our data may help to understand how monocytes interact with COM crystals. These processes are proposed to cause subsequent inflammatory response in kidney stone disease through oxidative stress pathway(s).

Comparison of O-polysaccharide and hemolysin co-regulated protein as target antigens for serodiagnosis of melioidosis.

Background Melioidosis is a severe disease caused by Burkholderia pseudomallei. Clinical manifestations are diverse and acute infections require immediate treatment with effective antibiotics. While culture is the current diagnostic standard, it is time-consuming and has low sensitivity. In endemic areas, inaccessibility to biosafety level 3 facilities and a lack of good serodiagnostic tools can impede diagnosis and disease surveillance. Recent studies have suggested that O-polysaccharide (OPS) and hemolysin co-regulated protein 1 (Hcp1) are promising target antigens for serodiagnosis of melioidosis. Methodology/Principle findings We evaluated rapid ELISAs using crude antigens, purified OPS and Hcp1 to measure antibody levels in three sets of sera: (i) 419 serum samples from melioidosis patients, Thai and U.S. healthy donors, (ii) 120 serum samples from patients with other bacterial infections, and (iii) 423 serum samples from 200 melioidosis patients obtained upon admission and at 12 and 52 weeks post-recovery. We observed significantly higher antibody levels using the crude antigen prepared from wild type B. pseudomallei K96243 compared to that of an OPS-mutant. The areas under receiver operator characteristics (AUROCCs) for diagnosis were compared for individual Hcp1-ELISA or OPS-ELISA or combined Hcp1/OPS-ELISA. For Thai donors, AUROCCs were highest and comparable between the Hcp1-ELISA and the combined Hcp1/OPS-ELISA (0.95 versus 0.94). For U.S. donors, the AUROCC was highest for the combined Hcp1/OPS-ELISA (0.96). Significantly higher seropositivity was observed in diabetic patients compared to those without diabetes for both the Hcp1-ELISA (87.3% versus 69.7%) and OPS-ELISA (88.1% versus 60.6%). Although antibody levels for Hcp1 were highest upon admission, the titers declined by week 52 post-recovery. Conclusions/Significance Hcp1 and OPS are promising candidates for serodiagnosis of melioidosis in different groups of patients. The Hcp1-ELISA performed better than the OPS-ELISA in endemic areas, thus, Hcp1 represents a promising target antigen for the development of POC tests for acute melioidosis.

Deciphering minimal antigenic epitopes associated with Burkholderia pseudomallei and Burkholderia mallei lipopolysaccharide O-antigens

Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm), the etiologic agents of melioidosis and glanders, respectively, cause severe disease in both humans and animals. Studies have highlighted the importance of Bp and Bm lipopolysaccharides (LPS) as vaccine candidates. Here we describe the synthesis of seven oligosaccharides as the minimal structures featuring all of the reported acetylation/methylation patterns associated with Bp and Bm LPS O-antigens (OAgs). Our approach is based on the conversion of an l-rhamnose into a 6-deoxy-l-talose residue at a late stage of the synthetic sequence. Using biochemical and biophysical methods, we demonstrate the binding of several Bp and Bm LPS-specific monoclonal antibodies with terminal OAg residues. Mice immunized with terminal disaccharide–CRM197 constructs produced high-titer antibody responses that crossreacted with Bm-like OAgs. Collectively, these studies serve as foundation for the development of novel therapeutics, diagnostics, and vaccine candidates to combat diseases caused by Bp and Bm.Melioidosis and glanders are multifaceted infections caused by gram-negative bacteria. Here, the authors synthesize a series of oligosaccharides that mimic the lipopolysaccharides present on the pathogens’ surface and use them to develop novel glycoconjugates for vaccine development.

Effect of temperature on Burkholderia pseudomallei growth, proteomic changes, motility and resistance to stress environments

Burkholderia pseudomallei is a flagellated, gram-negative environmental bacterium that causes melioidosis, a severe infectious disease of humans and animals in tropical areas. We hypothesised that B. pseudomallei may undergo phenotypic adaptation in response to an increase in growth temperature. We analysed the growth curves of B. pseudomallei strain 153 cultured in Luria–Bertani broth at five different temperatures (25 °C–42 °C) and compared the proteomes of bacteria cultured at 37 °C and 42 °C. B. pseudomallei exhibited the highest growth rate at 37 °C with modest reductions at 30 °C, 40 °C and 42 °C but a more marked delay at 25 °C. Proteome analysis revealed 34 differentially expressed protein spots between bacterial cultures at 42 °C versus 37 °C. These were identified as chaperones (7 spots), metabolic enzymes (12 spots), antioxidants (10 spots), motility proteins (2 spots), structural proteins (2 spots) and hypothetical proteins (1 spot). Of the 22 down-regulated proteins at 42 °C, redundancy in motility and antioxidant proteins was observed. qRT-PCR confirmed decreased expression of fliC and katE. Experiments on three B. pseudomallei strains demonstrated that these had the highest motility, greatest resistance to H2O2 and greatest tolerance to salt stress at 37 °C. Our data suggest that temperature affects B. pseudomallei motility and resistance to stress.

Plasma proteome profiling of von Hippel-Lindau disease after total and subtotal nephrectomy: A preliminary study

OBJECTIVES Common treatment of renal cell carcinoma associated with von Hippel-Lindau (VHL) disease is total (bilateral) or subtotal nephrectomy. Whereas total nephrectomy is associated with absolutely no residual renal function, subtotal nephrectomy frequently leads to chronic kidney disease (CKD) with some residual renal functions. However, molecular mechanisms underlying CKD remain unclear and the diagnosis of CKD is frequently accomplished at its late stage. DESIGN AND METHODS We performed a plasma proteomics study to compare the plasma proteome profile of VHL patient who underwent total nephrectomy to the profiles of VHL patient with subtotal nephrectomy and healthy control. Totally 100 mug proteins from each sample was resolved by two-dimensional electrophoresis (2-DE) in triplicate and visualized with SYPRO Ruby fluorescence stain. RESULTS The normal plasma proteome profile markedly differed from the profiles of VHL patients. Comparative analysis between total versus subtotal nephrectomized patients revealed significant differences in levels of 20 plasma proteins. Pathway analysis revealed two important networks involving in lipid metabolism, molecular transport, carbohydrate metabolism, cellular growth and proliferation, and small molecule biochemistry, in which these identified and other proteins interplayed. CONCLUSIONS Our data identified potential biomarkers for CKD. Further characterization of these identified proteins might also lead to better understanding of molecular mechanisms underlying CKD.