PDMS Micropatterns Coated with PDA and RGD Induce a Regulatory Macrophage-like Phenotype.

Regulatory macrophages (Mreg) are a special cell type that present a potential therapeutic strategy for various inflammatory diseases. In vitro, Mreg generation mainly takes 7-10 days of treatment with chemicals, including cytokines. In the present study, we established a new approach for Mreg generation using a three-dimensional (3D) micropatterned polydimethylsiloxane (PDMS) surface coated with a natural biopolymer adhesive polydopamine (PDA) and the common cell adhesion peptide motif arginylglycylaspartic acid (RGD). The 3D PDMS surfaces were fabricated by photolithography and soft lithography techniques and were subsequently coated with an RGD+PDA mixture to form a surface that facilitates cell adhesion. Human monocytes (THP-1 cells) were cultured on different types of 2D or 3D micropatterns for four days, and the cell morphology, elongation, and Mreg marker expression were assessed using microscopic and flow cytometric analyses. The cells grown on the PDA+RGD-coated 3D micropatterns (20-µm width/20-µm space) exhibited the most elongated morphology and strongest expression levels of Mreg markers, such as CD163, CD206, CD209, CD274, MER-TK, TREM2, and DHRS9. The present study demonstrated that PDA+RGD-coated 3D PDMS micropatterns successfully induced Mreg-like cells from THP-1 cells within four days without the use of cytokines, suggesting a time- and cost-effective method to generate Mreg-like cells in vitro.

Detection of Salmonella genes in stool samples of children aged 5 years and younger in urban and rural areas of Bangladesh.

INTRODUCTION: Typhoid incidence in children is higher in urban areas than in rural areas of Bangladesh. This study examined whether healthy urban children harboured higher levels of Salmonella genes than healthy rural children. METHODOLOGY: Stool samples from 140 children were studied: 70 from rural areas and 70 from urban metropolitan areas. RESULTS: The stool samples of urban children contained more Salmonella genes (median 4, IQR 3-4) than those of rural children (median 3, IQR 3-4). This suggests that urban Bangladeshi children have more Salmonella genes in their guts than rural children. Especially, in those under 12 months of age, the Salmonella gene prevalence in urban children was unique. They had more Salmonella genes (median 4, IQR 4-5) than rural children in the same age group (median 3, IQR 2.5-4). We also found more Salmonella genes in urban children who drank tap water (median 4, IQR 3-5) than in rural children whose water source was tube well water (median 3, IQR 2-4) and boiled pond water (median 3, IQR 3-3.5). However, there was no significant difference of Salmonella genes between urban children who drank tap-water and children whose water source was a tube well (median 4, IQR 3-4). CONCLUSIONS: These data suggest that the urban environment, including the drinking water supply system, increases the likelihood of healthy children in urban areas harbouring more potentially pathogenic Salmonella organisms in their gut than found in rural healthy children.

Differential interleukin-1ß induction by uropathogenic Escherichia coli correlates with its phylotype and serum C-reactive protein levels in Korean infants.

Urinary tract infection (UTI) is one of the most common bacterial infections in infants less than age 1 year. UTIs frequently recur and result in long-term effects include sepsis and renal scarring. Uropathogenic Escherichia coli (UPEC), the most prevalent organism found in UTIs, can cause host inflammation via various virulence factors including hemolysin and cytotoxic necrotizing factors by inducing inflammatory cytokines such as interleukin (IL)-1ß. However, the ability of each UPEC organism to induce IL-1ß production may differ by strain. Furthermore, the correlation between differential IL-1ß induction and its relevance in pathology has not been well studied. In this study, we isolated UPEC from children under age 24 months and infected bone-marrow derived macrophages with the isolates to investigate secretion of IL-1ß. We found that children with higher concentrations of C-reactive protein (CRP) were more likely to harbor phylotype B2 UPEC strains that induced more IL-1ß production than phylotype D. We also observed a significant correlation between serum CRP level and in vitro IL-1ß induction by phylotype B2 UPEC bacteria. Our results highlight the diversity of UPEC in terms of IL-1ß induction capacity in macrophages and suggest a potential pathogenic role in UTIs by inducing inflammation in infants.

All-Trans Retinoic Acid Enhances Bacterial Flagellin-Stimulated Proinflammatory Responses in Human Monocyte THP-1 Cells by Upregulating CD14.

All-trans retinoic acid (ATRA), an active form of vitamin A, exerts immunomodulatory functions. In this study, we examined the immune potentiating effect of ATRA on bacterial flagellin-induced NF-κB activation and proinflammatory cytokine production in human monocytic cell line THP-1. ATRA treatment significantly enhanced the flagellin-induced NF-κB/AP-1 activity in THP-1 via the RAR/RXR pathway. Similarly, ATRA enhanced the expression and production of TNF-α and IL-1ß in THP-1 cells upon flagellin challenge. The cell surface expression of toll-like receptor 5 (TLR5), which is the receptor for bacterial flagellin, was significantly reduced by ATRA in a concentration- and time-dependent manner. To determine the mechanisms underlying the ATRA-enhanced immune response against bacterial flagellin despite the reduced cell surface expression of TLR5 in ATRA-treated THP-1, we examined the cell surface expression of CD14, which has been proposed to be a TLR co-receptor that enhances the response to microbial components. The cell surface expression of CD14 was significantly enhanced by ATRA treatment, especially in the presence of flagellin. Anti-CD14 antibody treatment prior to ATRA and flagellin treatments completely abolished ATRA-enhanced TNF-α and IL-1ß production. Our results suggest that ATRA enhances flagellin-stimulated proinflammatory responses in human monocyte THP-1 cells by upregulating CD14 in a RAR/RXR-dependent manner.

1,25-Dihydroxyvitamin D3 up-regulates TLR10 while down-regulating TLR2, 4, and 5 in human monocyte THP-1.

In humans, there are ten Toll-like receptors (TLRs), among which TLR10 is the only orphan receptor whose function is unknown. In this study, we examined the effects of IFN-γ, LPS and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on TLR10 expression of human monocyte THP-1 and compared them with those of other surface TLRs such as TLR2, 4 and 5 to differentiate TLR10 from other TLRs. Surface TLR10 expression on THP-1 was significantly enhanced by the addition of IFN-γ or LPS in a fashion similar to that of other TLRs. However, TLR10 expression was differentially regulated by 1,25(OH)2D3. Surface TLR10 expression on THP-1 was significantly enhanced at 24h, reaching approximately two times the control level at 48h after treatment with 100nM 1,25(OH)2D3, while that of TLR2, 4 and 5 decreased gradually in response to treatment over time. 1,25(OH)2D3 at concentrations above 1nM markedly enhanced surface TLR10 expression, but concentrations below 1nM did not. TLR10 mRNA expression was also increased by 1,25(OH)2D3. We next screened for putative binding sites of nuclear vitamin D receptor (VDR) and its counterpart RXR-α within promoter of TLR genes using a transcription factor binding site-prediction program. The results revealed that TLR10 is the only receptor among the tested TLRs that has both a VDR and RXR-α binding site within its proximal promoter. To identify possible involvement of VDR/RXR in the 1,25(OH)2D3-induced TLR10 up-regulation, we engaged the VDR synthesis inhibitor, dexamethasone, and the RXR antagonist, 1,8-dihydroxyanthraquinone. We found that TLR10 up-regulation was significantly blocked with pre-treatment of these inhibitors. These findings indicate that surface TLR10 expression is differentially regulated by 1,25(OH)2D3 and mainly regulated at the transcriptional level via VDR/RXR-α. Overall, results presented herein suggest that TLR10 functions differently from other known surface TLRs under certain circumstances. Further study using primary cells is necessary to confirm the results of the present study.