Cd8+ T-Cell Responses in a Th2-Polarized Inflammatory Setting

A variety of external factors can affect the activation status, effector function, and memory differentiation of T cells. Such factors include resident microbiota and the cytokine milieu produced in a host. Loss of tolerance to commensal bacteria and excess inflammatory cytokines such as IL-4 have been implicated in the pathogenesis of many diseases, including inflammatory bowel disease (IBD) and allergy. Although these diseases are traditionally associated with CD4+ T cells, it is increasingly appreciated that CD8+ T cells in these settings may either contribute to or reduce pathology. This work explores the influence of various factors on CD8+ T-cell responses in an inflammatory setting, mice deficient in Nedd4-family interacting protein 1 (Ndfip1). Ndfip1 restricts IL-4 production in CD4+ T cells by facilitating degradation of the transcription factor JunB. Ndfip1-deficient CD4+ T cells have increased JunB levels and consequently overproduce IL-4. This excess IL-4 impairs Th17 and iTreg differentiation. Mice lacking Ndfip1 develop severe TH2-mediated inflammation at sites of environmental antigen exposure, including skin, GI tract, and lung, and develop IBD-like symptoms. We first tested the role of bacterial antigens in triggering this phenotype by treating mice lacking Ndfip1 in T cells with a cocktail of antibiotics to deplete intestinal bacteria. We then analyzed T cells for activation markers and examined tissues for signs of inflammation in the GI tract. Next, we analyzed whether exposure to the high levels of IL-4 in Ndfip1KO mice affects CD8+ T cell differentiation. We concluded that IL-4, but not intestinal bacteria, is required for the increase in activated/memory phenotype CD8+ T cells observed in Ndfip1KO mice. These findings add to the growing body of literature describing the importance of extrinsic cytokines to the development of memory-phenotype CD8+ T cells, and raise the possibility that such cells may be clinically relevant in diseases which are characterized by local increases in IL-4

Dynamic evolution of V1R putative pheromone receptors between Mus musculus and Mus spretus

<p>Abstract</p> <p>Background</p> <p>The mammalian vomeronasal organ (VNO) expresses two G-protein coupled receptor gene families that mediate pheromone responses, the V1R and V2R receptor genes. In rodents, there are ~150 V1R genes comprising 12 subfamilies organized in gene clusters at multiple chromosomal locations. Previously, we showed that several of these subfamilies had been extensively modulated by gene duplications, deletions, and gene conversions around the time of the evolutionary split of the mouse and rat lineages, consistent with the hypothesis that V1R repertoires might be involved in reinforcing speciation events. Here, we generated genome sequence for one large cluster containing two V1R subfamilies in Mus spretus, a closely related and sympatric species to Mus musculus, and investigated evolutionary change in these repertoires along the two mouse lineages.</p> <p>Results</p> <p>We describe a comparison of spretus and musculus with respect to genome organization and synteny, as well as V1R gene content and phylogeny, with reference to previous observations made between mouse and rat. Unlike the mouse-rat comparisons, synteny seems to be largely conserved between the two mouse species. Disruption of local synteny is generally associated with differences in repeat content, although these differences appear to arise more from deletion than new integrations. Even though unambiguous V1R orthology is evident, we observe dynamic modulation of the functional repertoires, with two of seven <it>V1Rb </it>and one of eleven <it>V1Ra </it>genes lost in spretus, two <it>V1Ra </it>genes becoming pseudogenes in musculus, two additional orthologous pairs apparently subject to strong adaptive selection, and another divergent orthologous pair that apparently was subjected to gene conversion.</p> <p>Conclusion</p> <p>Therefore, eight of the 18 (~44%) presumptive <it>V1Ra/V1Rb </it>genes in the musculus-spretus ancestor appear to have undergone functional modulation since these two species diverged. As compared to the rat-mouse split, where modulation is evident by independent expansions of these two V1R subfamilies, divergence between musculus and spretus has arisen more by mutations within coding sequences. These results support the hypothesis that adaptive changes in functional V1R repertoires contribute to the delineation of very closely related species.</p

Antibiotic treatment of wild type mice decreases bacterial load and does not produce overt immunological changes.

<p>(A) The average fold change in 16 s rDNA copies was quantified by qPCR from stool of wildtype mice treated with antibiotics and sucralose (n=5) or sucralose alone (n=3). Data shown is the fold change in 16S copies after 13 days of treatment. Error bars represent the SD. (B) The weight of mice treated with sucralose only (black line) or sucralose and antibiotics (dotted line) is shown over the course of treatment. Dot represents the mean of the population and error bars are the SD from the mean. (C) Percentages of activated splenic CD4+ T cells (defined as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034478#pone-0034478-g001" target="_blank">Figure 1D</a>) in mice treated with sucralose only or sucralose and antibiotics is shown. Each dot represents a single mouse. (D–E) Histological sections of the esophagus (D) and small bowel (E) are shown for mice treated with sucralose only (−antibiotics) or sucralose and antibiotics (+antibiotics). A 10× objective was used for esophagus and a 40× objective was used to analyze small bowel.</p

Antibiotic Treatment of Ndfip1 cKOs from Birth Does Not Reduce Inflammation.

<p>(A–D) Representative flow cytometry plots of cells isolated from the esophagus (A) and small bowel (C) of Ndfip1-cKO and control animals treated from birth to 5 weeks. Graphs of the percentages of eosinophils (Siglec F+) and CD4+ T cells in esophagus (B) or small bowel (D) from all mice in the experiment are shown. (E) Representative flow plots illustrating the percentage of activiated cells among splenic CD4+ gated T cells. (F) Percentages of activated T cells in the spleens of all mice treated with antibiotics from birth to 5 weeks are shown.</p

Ndfip1 CD4-cKO mice treated with antibiotics for 2 weeks do not show decreased eosinophilia or reduced inflammation in the esophagus.

<p>(A) The average 16s rDNA copies/nanogram of total stool DNA was quantified by qPCR at day 0 and day 13 of treatment with sucralose alone (n=5) or sucralose and antibiotics (n=4). Error bars illustrate SD. (B) Weights of mice treated with sucralose alone (−ABX=solid line) or antibiotics and sucralose (+ABX=dashed line) over the course of the 13 day treatment. (C) Representative H & E stained histological sections of the esophagus and small bowel of Ndfip1-cKO mice after 13 days of treatment with sucralose alone or antibiotics and sucralose are shown. (D) Percentages of activated CD4+ T cells in the spleens of mice treated with sucralose only (untreated) or with antibiotics and sucralose (treated) as determined by flow cytometric analysis. Each dot represents a single mouse.</p

Ndfip1 CD4-cKO mice treated with antibiotics from birth do not show changes in eosinophilia or inflammation in the esophagus, or splenic T cell activation.

<p>(A) Weights of control mice (n=4) (closed circles) or Ndfip1-cKO mice(n=4) (open circles) between weeks 3 to 5 of antibiotic treatment. (B) H & E stains of histological sections of esophagus taken from control and Ndfip1-cKO mice after antibiotic treatment from birth to 5 weeks. Images were taken using a 20× objective. Inset of panel outlined by the box is shown in the images on the right.</p