Depletion of intestinal LTi-like cells increases susceptibility to <i>S.</i> Typhimurium infection.

<p>(A) Scatter plot demonstrating depletion of colonic IL-1R1⁺ CD4⁺ Lin⁻ LTi-like cells by injection of anti-CD4 antibodies. Number of CD4⁺ cells (<i>top</i>) and LTi-like cells (<i>bottom</i>) isolated from cLP of Rag1^−/− C57BL/6J mice is indicated. Data shown are representative of three experiments. (B-D) Weights (normalized to starting weight) (B), survival (C), and fecal burden of <i>S.</i> Typhimurium (D) over time following <i>S.</i> Typhimurium infection in isotype control (<i>square</i>) and anti-CD4 (αCD4; <i>triangle</i>) antibody-treated mice. (E) CFUs of <i>S.</i> Typhimurium in the liver or spleen at time of death in isotype control- and αCD4-treated mice. (F) Histological scores for the cecum and proximal colon in isotype control- and αCD4-treated mice. *, <i>p</i><0.05; **, <i>p</i><0.01; NS, not significant.</p

Number of colonic IL-1R1⁺ CD4⁺ LTi-like cells depends on the gut flora.

<p>(A) Number of IL-1R1⁺ CD4⁺ LTi-like cells in the cLP of Swiss-Webster mice that were either SPF, GF, monocolonized with <i>B. fragilis</i> (BF), or monocolonized with segmented filamentous bacteria (SFB). <i>n</i> = 8−10. (B) Number of IL-1R1⁺ CD4⁺ LTi-like cells in the cLP of SPF Swiss-Webster mice treated for five weeks with either vancomycin (vanco), neomycin (neo), or metronidazole (metro). N = 8−10. (C) Number of IL-1R1⁺ CD4⁺ LTi-like cells in the cLP of Swiss-Webster mice that were either SPF or born GF and co-housed at weaning age with SPF mice (GF→SPF). <i>n</i> = 4−5. *, <i>p</i><0.05; ***, <i>p</i><0.001; NS, not significant. For box and whisker plots, line represents median, box represents 25^th to 75^th percentile range, and whiskers represent range.</p

IL-1R1 is required for IL-23-stimulated IL-17 and IL-22 production by LTi-like cells <b><i>in vitro</i></b><b>.</b>

<p>(A and B) Box and whiskers plot depicting percent of WT (W) or IL-1R1^−/− (I) colonic CD4⁺ LTi-like cells that produce IL-22 (A) or IL-17 (B). (C) Box and whiskers plot depicting percent of colonic LTi-like cells isolated from Rag1^−/− (R) C57BL/6J mice that produce IL-22. (D) Box and whiskers plot depicting percent of WT (W) or IL-1R1^−/− (I) colonic CD4⁺ LTi-like cells that produce IFN-γ. Except in (C), cells were isolated from WT (<i>top panels</i>) or IL-1R1^−/− C57BL/6J mice (<i>bottom panels</i>). Cells were stimulated by rIL-23 (23; <i>right panels</i>) or medium (M; <i>left panels</i>). Box and whisker plots representative of at least three independent experiments. *, <i>p</i><0.05; NS, not significant. For box and whisker plots, line represents median, box represents 25^th to 75^th percentile range, and whiskers represent range.</p

Gut-associated IL-1R1⁺ CD4⁺ Lin⁻ LTi-like cells are located predominantly in the intestinal lamina propria.

<p>(A) Representative FACS scatter plots depicting IL-1R1⁺ cells as a percentage of total CD4⁺ or CD8⁺ cells in spleen and gut-associated compartments of WT C57BL/6J mice. Numbers represent percentage of CD4⁺ or CD8⁺ cells expressing IL-1R1. (B and C) Bar graph depicting IL-1R1⁺ cells as a percentage of total CD4⁺ (B) or CD8⁺ (C) cells in spleen and gut-associated compartments of WT C57BL/6J mice. Bar represents median; error bar represents standard error. Filled bars represent anti-IL-1R1 flurophore-conjugated antibodies; empty bars represent isotype control antibodies. Spl, spleen; MLN, mesenteric lymph nodes; SI, small intestine; IEL, intraepithelial lymphocytes; LPL, lamina propria lymphocytes. Data collected from three independent experiments. (D) Gating scheme for IL-1R1⁺ CD4⁺ cells. SSC, side scatter. FSC, forward scatter. Number is the percent of CD4⁺ CD3⁻ lymphocytes that are IL-1R1⁺ in WT C57BL/6J mice. (E) Scatter plot depicting IL-1R1⁺ CD4⁺ cells in Rag1^−/− C57BL/6J mice. Number is the percent of CD4⁺ lymphocytes that are IL-1R1⁺ in Rag1^−/− mice. Gated on CD4⁺ lymphocytes as in (D). (F) FACS histograms demonstrating expression of c-kit, CD127, RORγt, and Lin in WT C57BL/6J mice. Plots are gated on IL-1R1⁺ CD4⁺ cells. Empty, isotype control; filled, antibody. *, p<0.05; **, p<0.01; NS, not significant.</p

Colonic LTi-like cells are significant innate producers of IL-22.

<p>(A) Representative FACS histogram depicting percentage of CD4⁺ LTi-like cells that produce IL-22 under control conditions (<i>left panel</i>) and in DSS colitis (<i>right panel</i>) in Rag1^−/− C57BL/6J mice. (B) Graph summarizing percentage of CD4⁺ LTi-like cells producing IL-22. (C) Representative scatter plot depicting the phenotype of IL-22-producing lymphocytes under control conditions (<i>left panel</i>) and in DSS colitis (<i>right panel</i>) in Rag1^−/− C57BL/6J mice. Gated on IL-22⁺ lymphocytes. (D) Graph depicting percentage of IL-22-producing lymphocytes that are CD4⁺ LTi-like cells. All graphs represent three independent experiments. *, <i>p</i><0.05; NS, not significant. For box and whisker plots, line represents median, box represents 25^th to 75^th percentile range, and whiskers represent range.</p

Photoinduced C–S Bond Cleavage of Thioglycosides and Glycosylation

A glycosyl coupling reaction via photoinduced direct activation of thioglycosides and subsequent <i>O</i>-glycosylation in the absence of photosensitizer was developed for the first time. This reaction underwent a selectively homolytic cleavage of a C–S bond to generate a glycosyl radical, which was oxidized to an oxacarbenium ion by Cu­(OTf)₂, and a sequential <i>O</i>-glycosylation. A wide range of glycosides were synthesized in moderate to excellent yield using sugars, amino acids, or cholesterol as the acceptors

Inducement of Cytokine Release by GFPBW2, a Novel Polysaccharide from Fruit Bodies of Grifola frondosa, through Dectin‑1 in Macrophages

Polysaccharides, especially β-glucans isolated from various species of mushrooms, are considered as biological response modifiers (BRMs) to be widely used in the treatment of cancer, especially due to their immunostimulatory activity. We herein characterized the structure of a novel water-soluble homogeneous polysaccharide (GFPBW2) from the fruit bodies of mushroom Grifola frondosa and investigated its immunomodulatory activity in vitro. GFPBW2 was purified from the alkali-extracted fractions by stepwise elution with a molecular weight of 26.2 kDa. On the basis of infrared and NMR spectroscopy, methylation and monosaccharide composition analysis, partial acid hydrolysis, and Smith degradation, its structure was elucidated to possess a backbone consisting of β-d-1,3- and β-d-1,4-linked glucopyranosyl residues, with branches attached to <i>O</i>-6 of β-d-1,3-linked glucopyranosyl residues. Functionally, it is an effective inducer of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) secretion in murine resident peritoneal macrophages. Using quartz crystal microbalance (QCM) analysis, we found that GFPBW2 could bind dendritic cell-associated C-type lectin-1 (Dectin-1) with an affinity constant (<i>K</i>_d) value of 1.08 × 10 ^–7 M, while it could activate Syk and enhance TNF-α production in RAW264.7 cells overexpressing wild type but not mutant Dectin-1. Furthermore, Syk, NF-κB signaling, and cytokine release in resident peritoneal macrophages induced by GFPBW2 could be significantly inhibited by a specific Dectin-1 blocking reagent, Laminarin. These data suggested that GFPBW2 might be a potential ligand of Dectin-1, and the potential of GFPBW2 to activate macrophage through triggering cytokine secretion might be attributed, at least in part, to the involvement of Dectin-1

Homogalacturonans from Preinfused Green Tea: Structural Characterization and Anticomplementary Activity of Their Sulfated Derivatives

Two homogeneous water-soluble polysaccharides (TPSR4-2B and TPSR4-2C) were obtained from preinfused green tea. Their average molecular weights were estimated to be 41 kDa and 28 kDa, respectively. A combination of composition, methylation, and configuration analysis, as well as NMR spectroscopy, indicated that both TPSR4-2B and TPSR4-2C were poly-(1–4)-α-d-galactopyranosyluronic acid in which 30.5 ± 0.3% and 28.3 ± 0.5%, respectively, of uronic acid existed as methyl ester. Two sulfated derivatives (Sul-R4-2B and Sul-R4-2C) from TPSR4-2B and TPSR4-2C were prepared after sulfation with a 2:1 chlorosulfonic acid–pyridine ratio. The anticomplementary assay showed that Sul-R4-2B and Sul-R4-2C demonstrated a stronger inhibitory effect on the complement activation through the classic pathway, compared to that of heparin. Preliminary mechanism studies by using complement component depleted-sera indicated that both Sul-R4-2B and Sul-R4-2C selectively interact with C1q, C1r, C1s, C2, C5, and C9 but not with C3 and C4. The relationship between DS and the anticomplementary activity of sulfated derivatives of homogalacturonans showed that low sulfated derivatives of homogalacturonans also exhibited potent anticomplementary effect, which might greatly reduce the side effects related to heparin and oversulfated chondroitin sulfate, such as anticoagulant activity and allergic-type reaction. These results suggested that sulfated derivatives of homogalacturonans might be promising drug candidates for therapeutic complement inhibition