Phenotypic Plasticity in Morphological Traits of Abelmoschus esculentus L. Induced by Histone Deacetylase Inhibitor, Trichostatin A

Epigenetic changes such as DNA methylation and histone modifications, when meiotically inherited across generations, can act as a stable evolutionary force that is independent of any accompanying DNA mutations. Certain histone deacetylase (HDAC)-inhibiting chemicals such as Trichostatin A (TSA) and sodium butyrate are known to regulate the total acetylated histones in the genome, which is important for regulating the expression of various traits in all organisms. This study investigated all morphological variations in traits of Abelmoschus esculentus L. (okra) induced by different doses of Trichostatin A in a popular okra variety, Arka Abhay. Two sets of seeds were treated with two doses (0.4 µM and 1.2 µM) of TSA and were incubated in the chemical for three and five days, respectively, to record the effects of dose and incubation periods on various agronomic characters of okra. The treatment of TSA had a negative impact on the majority of the characters under evaluation. Total seedlings emerged, and mean shoot and root length were retarded following the TSA treatment. Extremely dwarfed plants with malformed leaves and flowers were a common observation. Pollen sterility combined with distortion of the reproductive whorls of the flowers were particularly pronounced at high doses with a prolonged incubation period. Treated plants had a significantly delayed first flowering and produced short fruits with altered morphology. Variations in seeds with respect to the number, colour and size were also recorded. Total reduction with respect to seedling parameters, total pollen production, the number of fertile pollens, plant height and other damaging effects on leaves, flowers, fruits and seeds increased as the dose and incubation period increased. Statistical analysis revealed the significant negative effect of TSA treatments on plant height, the number of ridges and locules per fruit, number of seeds per fruit and test weight. The treatment, 1.2 µM Trichostatin A incubated for three days, showed a remarkable difference as traits such as total field emergence, seedling parameters and days to first flowering plant height, number of seeds per fruit and test weight deviated from the expected trend of decreasing growth parameters as the dose and incubation period increased. The study further revealed that the treatment (1.2 µM TSA incubated for three days) can be suggested for use in okra to induce epigenetic variations without significantly compromising the growth and vigour parameters of okra

Structural basis of Blastomyces Endoglucanase-2 adjuvancy in anti-fungal and -viral immunity.

The development of safe subunit vaccines requires adjuvants that augment immunogenicity of non-replicating protein-based antigens. Current vaccines against infectious diseases preferentially induce protective antibodies driven by adjuvants such as alum. However, the contribution of antibody to host defense is limited for certain classes of infectious diseases such as fungi, whereas animal studies and clinical observations implicate cellular immunity as an essential component of the resolution of fungal pathogens. Here, we decipher the structural bases of a newly identified glycoprotein ligand of Dectin-2 with potent adjuvancy, Blastomyces endoglucanase-2 (Bl-Eng2). We also pinpoint the developmental steps of antigen-specific CD4+ and CD8+ T responses augmented by Bl-Eng2 including expansion, differentiation and tissue residency. Dectin-2 ligation led to successful systemic and mucosal vaccination against invasive fungal infection and Influenza A infection, respectively. O-linked glycans on Bl-Eng2 applied at the skin and respiratory mucosa greatly augment vaccine subunit- induced protective immunity against lethal influenza and fungal pulmonary challenge

MyD88 Shapes Vaccine Immunity by Extrinsically Regulating Survival of CD4⁺ T Cells during the Contraction Phase

<div><p>Soaring rates of systemic fungal infections worldwide underscore the need for vaccine prevention. An understanding of the elements that promote vaccine immunity is essential. We previously reported that Th17 cells are required for vaccine immunity to the systemic dimorphic fungi of North America, and that Card9 and MyD88 signaling are required for the development of protective Th17 cells. Herein, we investigated where, when and how MyD88 regulates T cell development. We uncovered a novel mechanism in which MyD88 extrinsically regulates the survival of activated T cells during the contraction phase and in the absence of inflammation, but is dispensable for the expansion and differentiation of the cells. The poor survival of activated T cells in <i>Myd88</i>^-/- mice is linked to increased caspase3-mediated apoptosis, but not to Fas- or Bim-dependent apoptotic pathways, nor to reduced expression of the anti-apoptotic molecules Bcl-2 or Bcl-xL. Moreover, TLR3, 7, and/or 9, but not TLR2 or 4, also were required extrinsically for MyD88-dependent Th17 cell responses and vaccine immunity. Similar MyD88 requirements governed the survival of virus primed T cells. Our data identify unappreciated new requirements for eliciting adaptive immunity and have implications for designing vaccines.</p></div

<i>Myd88</i>^-/- mice fail to mount Th17 and Th1 cells and resistance after vaccination.

<p>Wild type C57BL6 and <i>Myd88</i>^-/- mice were s.c. vaccinated with 10⁵ or 10⁶ live <i>B</i>.<i>dermatitidis</i> yeast and followed for survival <b>(A)</b>. To circumvent dissemination, mice were vaccinated s.c. with 10⁶ heat-killed yeast and challenged i.t. with 2 x 10³ 26199 yeast. Two weeks post-infection, lung CFU were enumerated <b>(B).</b> Data are the mean ± SEM (n = 8–10 mice/group). Data are representative of three independent experiments. The number of Th17 and Th1 cells recalled to the lung were assessed at day 4 post-infection. Data are the mean ± SEM (n = 4–6 mice/group) <b>(C).</b> The numbers indicate the mean ± SEM of cytokine producing CD4⁺ T cells per mouse. * <i>P</i> <0.05 vs. vaccinated wild-type controls. Dot plots show concatenated samples of 4–6 mice/ group. Data are representative of three independent experiments. <b>(D)</b> Wild-type and <i>Myd88</i>^-/- mice were vaccinated s.c. with 10⁷ live <i>H</i>. <i>capsulatum</i> yeast and challenged with 5 x 10⁵ Hc G217B yeast. Two weeks post-infection, lung CFU were enumerated. * <i>P</i> <0.05 vs. vaccinated wild-type controls. <b>(E)</b> The frequencies of Th17 and Th1 cells were enumerated at day 4 post-infection. Data are the mean ± SEM (n = 4–6 mice/group).</p

Extrinsic MyD88 regulates T cells during the contraction phase, but not the programming phase.

<p><b>(A)</b>. Schematic: One million purified CD4⁺ T cells from naïve 1807 mice were transferred into <i>Myd88</i>^-/- and wild type mice prior to vaccination. Seven days post-vaccination, CD4⁺ T cells were magnetic bead-purified from the sdLN and spleen of the vaccinated mice and adoptively transferred into new sets of naïve <i>Myd88</i>^-/- and wild type mice as shown in the schematic and activated effector T cells were rested for 4wks. <b>(B)</b> Recipient mice were challenged and 1807 T cells enumerated in the lung at day 4 post-infection. Data are representative of two independent experiments. * <i>P</i> < 0.05 vs. groups with no asterisk. <b>(C)</b> Congenic Thy1.1⁺ wild type mice were vaccinated and at day 7 post-vaccination the CD4⁺ T were sorted for CD44⁺ T cells, transferred into naïve <i>Myd88</i>^-/- and wild type mice, and the mice rested for 4 wks as above. At day 4 post-challenge, the numbers of congenic Thy 1.1⁺ T cells were determined by FACS. Data are the mean ± SEM (n = 4–6 mice/group). Data are representative of two independent experiments. * <i>P</i> < 0.05 vs. corresponding wild type controls.</p

The role of extrinsic MyD88 for effector CD4⁺ T cell survival.

<p><b>(A-C)</b> Mice received 10⁶ purified CD4⁺ T cells from naïve 1807 mice and were vaccinated with heat-killed yeast. <b>(A)</b> 1807 T cell contraction. At serial time intervals post-vaccination, the numbers of activated 1807 T cells were enumerated from the sdLN and spleen by FACS. * <i>P</i> < 0.05 vs. wild type control recipient mice. <b>(B)</b> Caspase 3 expression. Numbers in the dot plot indicate the numbers (top line) and frequencies (2^nd line from top) of intracellular caspase 3 positive 1807 T cells within the CD4⁺ T cell gate at day 12 post-vaccination. * <i>P</i> < 0.05 vs. wild type control recipient mice. <b>(C)</b> The mean fluorescence intensity of Bcl2 and Bcl-xL expression in 1807 T cells transferred into wild type and <i>Myd88</i>^-/- recipients. Isotype controls are shown on the left of the graphs (unlabeled). <b>(D)</b> Adoptive transfer of naïve Bim^-/- 1807 T cells and Fas^-/- 1807 T cells into <i>Myd88</i>^-/- and wild type controls prior to vaccination. At days 7 and 35 post-vaccination, the numbers of adoptively transferred 1807 T cells were enumerated in the sdLN and spleen. Data are the mean ± SEM (n = 4–6 mice/group). Data are representative of two independent experiments. * <i>P</i> < 0.05 vs. corresponding wild type recipient mice. ** <i>P</i> < 0.05 vs. corresponding wild type 1807 cells.</p

Intrinsic MyD88 is dispensable for the development of Th17 and Th1 cells.

<p>Naïve <i>Myd88</i>^-/- and <i>Myd88</i>^+/+ 1807 T cells (indicated by -/- and +/+) were adoptively transferred into wild type C57BL6 recipient mice prior to s.c. vaccination (Panels A-C). Expansion <b>(A)</b>, contraction <b>(B)</b> and migration <b>(C)</b> of transferred 1807 cells and endogenous CD4⁺ T cells was determined by FACS in the skin draining lymph nodes (sdLN) on days 7 and 35 post-vaccination and in the lung at day 4 post-infection. Data are representative of five independent experiments. * <i>P</i> < 0.05 vs. wild type 1807 T cells. <b>(D)</b> <i>Myd88</i>∆T mice that are CD4-Cre⁺ <i>Myd88</i>fl/fl lack MyD88 in αβT cells and CD4-Cre^- <i>Myd88</i>fl/fl controls were vaccinated with 10⁶ heat-killed vaccine yeast and challenged with wild-type yeast. The number of activated (CD44⁺) and cytokine producing CD4⁺ T cells were enumerated at day 4 post-infection. Lung CFU were determined at day 4 and two weeks post-infection. Data from day 4 post-infection are an average of three independent experiments and day 14 post-infection data are representative of two independent experiments. The numbers in the graph indicate the n-fold change in lung CFU vs. unvaccinated control mice.</p

TLR3, 7 and 9 upstream of MyD88 extrinsically regulate T cell contraction.

<p>One million CD4⁺ T cells from naïve 1807 mice were transferred into wild type mice prior to vaccination. Seven days post-vaccination, effector CD4⁺ T cells were magnetic bead-purified from the sdLN and spleen of the vaccinated mice and adoptively transferred into naïve <i>Myd88</i>^-/-, IL-1R^-/-, TLR23479^-/-, TLR379^-/- and TLR24^-/- and wild type mice. After 4 weeks of rest, primed 1807 T cells harvested from the lymph nodes and spleen were enumerated by FACS. <b>(A)</b> Data are expressed as the mean ± SD of 8–12 mice/group. Data represent the average from two independent experiments. * <i>P</i> < 0.05 vs. wild type control mice. <b>(B)</b> shows the dot plots of concatenated events from 8–12 mice/group from panel A. * <i>P</i> < 0.05 vs. wild type control mice. <b>(C)</b> <i>Myd88</i>^-/-, IL-1R^-/-, TLR23479^-/-, TLR379^-/- and wild type mice were vaccinated with 10⁶ heat-killed vaccine yeast. At days 7 and 49 post-vaccination, the number of Cnx-specific CD4⁺ T cells in the sdLN and spleen was enumerated using tetramer enrichment and FACS detection. Tetramer positive cells are shown within the gate in each dot plot. The numbers represent the geometric mean ± SEM of tetramer-positive cells of 5 mice. Data are expressed from a single experiment representative of three independent experiments. * <i>P</i> < 0.05 vs. wild type control mice. <b>(D)</b> <i>Myd88</i>^-/-, IL-1R^-/-, TLR23479^-/-, TLR379^-/-, TLR24^-/- and wild type mice were vaccinated as above, challenged and lung CFU enumerated at day 14 post-infection when unvaccinated mice were moribund. The numbers indicate the n-fold change vs. unvaccinated mice. Data are the mean ± SD of 10–20 mice/group from a single experiment representative of three independent experiments. * <i>P</i> < 0.05 vs. vaccinated wild type control mice.</p