data_sheet_1_Increased iNKT17 Cell Frequency in the Intestine of Non-Obese Diabetic Mice Correlates With High Bacterioidales and Low Clostridiales Abundance.PDF

<p>iNKT cells play different immune function depending on their cytokine-secretion phenotype. iNKT17 cells predominantly secrete IL-17 and have an effector and pathogenic role in the pathogenesis of autoimmune diseases such as type 1 diabetes (T1D). In line with this notion, non-obese diabetic (NOD) mice that spontaneously develop T1D have an increased percentage of iNKT17 cells compared to non-autoimmune strains of mice. The factors that regulate iNKT cell expansion and acquisition of a specific iNKT17 cell phenotype are unclear. Here, we demonstrate that the percentage of iNKT17 cells is increased in the gut more than peripheral lymphoid organs of NOD mice, thus suggesting that the intestinal environment promotes iNKT17 cell differentiation in these mice. Increased intestinal iNKT17 cell differentiation in NOD mice is associated with the presence of pro-inflammatory IL-6-secreting dendritic cells that could contribute to iNKT cell expansion and iNKT17 cell differentiation. In addition, we found that increased iNKT17 cell differentiation in the large intestine of NOD mice is associated with a specific gut microbiota profile. We demonstrated a positive correlation between percentage of intestinal iNKT17 cells and bacterial strain richness (α-diversity) and relative abundance of Bacterioidales strains. On the contrary, the relative abundance of the anti-inflammatory Clostridiales strains negatively correlates with the intestinal iNKT17 cell frequency. Considering that iNKT17 cells play a key pathogenic role in T1D, our data support the notion that modulation of iNKT17 cell differentiation through gut microbiota changes could have a beneficial effect in T1D.</p

Suppression of lamellocyte production by a mutant allele of <i>kay</i>, which encodes dFos.

<p><i>A</i>. Supernumerary lamellocytes produced in a <i>hop^Tum-l/+</i> larva. <i>B</i>. Substantial reduction in the lamellocyte population in a <i>hop^Tum-l/+</i>; <i>kay¹/+</i> larva. <i>C</i>. Quantification of the number of lamellocytes and non-lamellocytes produced in larva of <i>Canton S</i> control versus mutant larvae. Asterisk (*) denotes a significant difference in values from those observed in <i>hop^Tum-l</i> animals.</p

Fine mapping of the location of the <i>msn</i> lamellocyte enhancer and its essential DNA sequence elements.

<p><i>A</i>. Schematic of the various truncated and sequence mutated <i>msn</i> intron-3 DNAs tested for enhancer function in lamellocytes of <i>hop^Tum-l</i> larvae as compared to hemocytes of control larvae. <i>B</i>. Sequence of the 590-bp MSNF9e DNA with putative binding elements for the STAT, Srp, and AP-1 transcription factors highlighted.</p

<i>MSNF9-GFP</i> expression in larvae post <i>L. boulardi</i> infestation.

<p><i>A</i>. Lack of <i>MSNF9-GFP</i> activity in lymph glands dissected from a control, non-wasp infested larva. <i>B</i>. <i>MSNF9-GFP</i> expression in lymph glands dissected from a wasp infested larva. <i>C</i>. <i>MSNF9-GFP</i>-positive lamellocyte present in the hemolymph of a wasp infested larva. <i>D</i>. Detection of numerous <i>MSNF9-GFP</i>-positive lamellocytes encapsulating a wasp egg (asterisk).</p

<i>MSNF9-GFP</i> expression in circulating hemocytes obtained from third-instar larvae of different genetic backgrounds that induce lamellocyte production.

<p><i>A</i>. <i>w¹¹¹⁸; He-Gal4</i> control. <i>B</i>. <i>He-Gal4>UAS-dTCF^DN</i>. <i>C</i>. <i>He-Gal4>UAS-dRAC1</i>. <i>D</i>. <i>He-Gal4>UAS-hep^CA</i>. <i>E</i>. <i>Cg-Gal4>UAS-col</i>. <i>F</i>. <i>Cg-Gal4>Ush^DN</i>. Hemolymph samples were also stained for DNA (DAPI) and filamentous actin (phalloidin).</p

Temporal activation of the <i>msn</i> lamellocyte transcriptional enhancer.

<p><i>A</i>. Inactivity of the <i>MSNF9-GFP</i> transgene in lymph glands dissected from a <i>Basc/Y</i> control larva at 72 hr of development. <i>B</i>. Inactivity of the <i>MSNF9-GFP</i> transgene in circulating hemocytes present in hemolymph obtained from a <i>Basc/Y</i> control larva at 72 hr of development. <i>C</i>. <i>MSNF9-GFP</i>-positive hemocytes observed in lymph glands dissected from a <i>hop^Tum-l/Y</i> mutant larva at 72 hr of development. <i>D</i>. <i>MSNF9-GFP</i>-positive lamellocytes present in the hemolymph obtained from a <i>hop^Tum-l/Y</i> mutant larva at 72 hr of development. <i>E</i>. <i>MSNF9-GFP</i>-positive hemocytes observed in lymph glands dissected from a <i>hop^Tum-l/Y</i> mutant larva at 56 hr of development. <i>F</i>. <i>MSNF9-GFP</i>-positive lamellocytes present in the hemolymph obtained from a <i>hop^Tum-l/Y</i> mutant larva at 42 hr of development.</p

Model for the regulation of the <i>msn</i> gene during lamellocyte induction and differentiation.

<p>The MSNF9 lamellocyte-active enhancer is depicted as serving as a transcriptional target within a JNK pathway auto-regulatory circuit that promotes the production of lamellocytes in immune-challenged or genetically compromised animals. The schematic of the JNK pathway is adapted from Xia and Karin (19).</p

The AP-1 recognition sequence in the <i>msn</i> lamellocyte enhancer can compete for dFos-dJun DNA binding.

<p><i>A</i>. Sequence of the hMTII AP-1 DNA probe, MSNF9 AP-1 recognition site, and mutated MSNF9 AP-1 recognition site. <i>B</i>. Electromobility shift competition assay with hMTII wt AP-1, MSNF9 wt AP-1, and MSNF9 mut AP-1 double-stranded oligonucleotide DNAs.</p

Synthesis of aldehyde 6 and derivatives 9a-f.

<p>Synthesis scheme of aldehyde <b>6</b>: <i>i)</i> Pd(PPh₃)₂Cl₂, Na₂CO₃, DMF/EtOH, RT, 1h; ii) 1N NaOH (aq), MeOH/THF, reflux 2h. Synthesis scheme of derivatives <b>9a-f</b>: <i>iii</i>) DME, Et₃N, MW (300 W), 90°C, 10 min. <i>iv</i>) aldehyde 6, MW (300 W), 110°C, 5 min.</p

2D structures of HIV-1 inhibitors previously published.

<p>2D structures of HIV-1 inhibitors previously published.</p