Intergenerational transfer of antibiotic-perturbed microbiota enhances colitis in susceptible mice

Antibiotic exposure in children has been associated with the risk of Inflammatory Bowel Disease (IBD). Since antibiotic use in children or in their pregnant mother can affect how the intestinal microbiome develops, we asked whether the transfer of an antibiotic-perturbed microbiota from mothers to their children could affect their risk of developing IBD. Here we demonstrate that germ-free adult pregnant mice inoculated with a gut microbial community shaped by antibiotic exposure transmitted their perturbed microbiota to their offspring with high fidelity. Without any direct or continued exposure to antibiotics, this dysbiotic microbiota in the offspring remained distinct from controls for at least 21 weeks. By using both IL-10-deficient and wild type mothers, we showed that both inoculum and genotype shape the microbiota populations in the offspring. Since IL10−/− mice are genetically susceptible to colitis, we could assess the risk due to maternal transmission of an antibiotic-perturbed microbiota. We found that the IL10−/− offspring that had received the perturbed gut microbiota developed markedly increased colitis. Taken together, our findings indicate that antibiotic exposure shaping the maternal gut microbiota has effects that extend to their offspring with both ecological and long-term disease consequences

Human Th1 Cells That Express CD300a Are Polyfunctional and After Stimulation Up-Regulate the T-Box Transcription Factor Eomesodermin

Human naïve CD4 T cells express low levels of the immunomodulatory receptor CD300a, whereas effector/memory CD4 cells can be either CD300a+ or CD300a−. This suggested that CD300a expression could define a specific subset within the effector/memory CD4 T cell subpopulations. In fact, ex vivo analysis of the IFN-γ producing CD4 T cells showed that they are enriched in the CD300a+ subset. Moreover, stimulated CD4 T cells producing TNF-α and IL-2 besides IFN-γ (polyfunctional) are predominantly CD300a+. In addition to producing markedly higher levels of Th1-associated cytokines, the stimulated CD300a+ CD4 T cells are distinguished by a striking up-regulation of the T-box transcription factor eomesodermin (Eomes), whereas T-bet is up-regulated in both CD300a+ and CD300a− activated CD4 T cells to similar levels. The pleiotropic cytokine TGF-β1 has a determinant role in dictating the development of this Th1 subset, as its presence inhibits the expression of CD300a and down-regulates the expression of Eomes and IFN-γ. We conclude that CD300a+ human Th1 cells tend to be polyfunctional and after stimulation up-regulate Eomes

HIV Envelope gp120 Activates LFA-1 on CD4 T-Lymphocytes and Increases Cell Susceptibility to LFA-1-Targeting Leukotoxin (LtxA)

The cellular adhesion molecule LFA-1 and its ICAM-1 ligand play an important role in promoting HIV-1 infectivity and transmission. These molecules are present on the envelope of HIV-1 virions and are integral components of the HIV virological synapse. However, cellular activation is required to convert LFA-1 to the active conformation that has high affinity binding for ICAM-1. This study evaluates whether such activation can be induced by HIV itself. The data show that HIV-1 gp120 was sufficient to trigger LFA-1 activation in fully quiescent naïve CD4 T cells in a CD4-dependent manner, and these CD4 T cells became more susceptible to killing by LtxA, a bacterial leukotoxin that preferentially targets leukocytes expressing high levels of the active LFA-1. Moreover, virus p24-expressing CD4 T cells in the peripheral blood of HIV-infected subjects were found to have higher levels of surface LFA-1, and LtxA treatment led to significant reduction of the viral DNA burden. These results demonstrate for the first time the ability of HIV to directly induce LFA-1 activation on CD4 T cells. Although LFA-1 activation may enhance HIV infectivity and transmission, it also renders the cells more susceptible to an LFA-1-targeting bacterial toxin, which may be harnessed as a novel therapeutic strategy to deplete virus reservoir in HIV-infected individuals

CD300a⁺ CD4 T cells are more polyfunctional than CD300a⁻ CD4 T cells.

<p>(A) Purified CD4 T cells were stimulated with PMA and ionomycin for 4–5 h. Then, cells were stained for cell surface expression of CD300a and production of IFN-γ and TNF-α and/or IL-2. The percentage of double and triple producers in the CD300a⁺ and CD300a⁻ cells is shown. Each symbol represents a different donor. (B) Graphic representation of the average ± SEM of the MFI of IFN-γ, IL-2 or TNF-α for the triple producers shown in panel A. (C) Purified CD4 T cells were stimulated with PMA and ionomycin for 4–5 h. Then, cells were stained for cell surface expression of CD300a and the production of the indicated cytokines. The percentage of triple cytokine producers in the CD300a⁺ and CD300a⁻ subsets is shown. Each symbol is a different donor.</p

CD300a⁺ cells up-regulate Eomes after stimulation.

<p>(A) mRNAs for the indicated transcription factors, and IL-12Rβ2 and Jak3 were quantified by real-time PCR. Graph represents the average ± SEM. AU: arbitrary units. Data are from 5–6 donors. (B) Purified CD4 T cells were stimulated with plate bound anti-CD3 plus anti-CD28 mAb for 24 h. The lymphocyte gate was determined according to the forward and side scatter parameters. Then cells were stained for cell surface expression of CD300a and for the intracellular expression of IFN-γ. The expression of Eomes, T-bet and CD300a was determined in the IFN-γ⁺ cells. Results are representative of three donors.</p

Flow cytometric analysis of human peripheral blood CD4 T cells.

<p>In the upper panel, freshly isolated CD4 T cells were labeled with anti-CD4, -CD45RO, and -CD300a mAb. The lymphocyte gate was determined according to the forward and side scatter parameters. Then CD45RO expression was determined for the CD4⁺ cells. The expression of CD300a was assessed in naïve cells (CD45RO⁻, dashed line) and memory cells (CD45RO⁺, continuous line). In the lower panel, freshly isolated CD4 T cells were labeled with anti-CD4, -CD25, -CD127 and -CD300a mAb. After electronically gating the CD4⁺ cells, the expression of CD300a was assessed for the Treg cells (CD25⁺CD127^low, continuous line) and non Treg cells (dashed line). Results are representative of 12 healthy donors.</p

Human circulating IFN-γ producing CD4 T cells are enriched in the CD300a⁺ subset.

<p>(A) Flow cytometric analyses for the production of IFN-γ, IL-4 and IL-17 in combination with cell surface expression of CD300a by purified CD4 T cells stimulated with PMA and ionomycin for 4–5 h. The lymphocyte gate was determined according to the forward and side scatter parameters. The CD300a expression was determined for the cytokine producing cells. A representative healthy donor is shown. The bar graph represents the average ± SEM of the percentage of CD300a⁺ cells within each cytokine producing subset. Results are from 15–19 donors. (B) MFI of IFN-γ expression in the cytokine producing memory CD300a⁺ and CD300a⁻ cells. (C) Expression of CXCR3 and CD300a by circulating memory CD4 T cells. Freshly isolated CD4 T cells were labeled with anti-CD4,-CD45RO, -CD300a and -CXCR3. Dot plots are from CD4⁺CD45RO⁺ gated cells. Results from a representative donor are shown from four donors that were analyzed. (D) PBMCs from healthy donors were stimulated overnight with CMV pp65 or TT in the presence of brefeldin A and then analyzed for the production of IFN-γ and IL-2 by memory CD4 T cells (CD4⁺CD45RO⁺). For the cytokine positive cells, the cell surface expression of CD300a was measured. The response of two of the four donors analyzed is shown.</p

Proliferation and phenotype of CD45RO⁺CD300a⁺ and CD45RO⁺CD300a⁻ CD4 T cells.

<p>Proliferation, as measured by CFSE dilution, of sorted CD45RO⁺CD300a⁺ and CD45RO⁺CD300a⁻ cells in response to plate bound anti-CD3 plus anti-CD28 mAb. After three days of stimulation, cells were rested four additional days. After the resting period, the cell surface expression of CXCR3, CD25 and CD300a was measured. IFN-γ production was measured by intracellular staining after a brief stimulation with PMA and ionomycin for 4 h. The bar graph represents the percentage of IFN-γ⁺ cells for each cell generation in the CD300a⁺ and CD300a⁻ subsets. Results shown are representative of four different donors.</p