Diana Bareyan

TLR-Induced Local Metabolism of Vitamin D3 Plays an Important Role in the Diversification of Adaptive Immune Responses

The addition of monophosphoryl lipid A, a minimally toxic derivative of LPS, to nonmucosally administered vaccines induced both systemic and mucosal immune responses to coadministered Ags. This was dependent on an up-regulated expression of 1α-hydroxylase (CYP27B1, 1αOHase), the enzyme that converts 25-hydroxycholecalciferol, a circulating inactive metabolite of vitamin D3, into 1,25(OH)2D3 (calcitriol). In response to locally produced calcitriol, myeloid dendritic cells (DCs) migrated from cutaneous vaccination sites into multiple secondary lymphoid organs, including classical inductive sites of mucosal immunity, where they effectively stimulated B and T cell immune responses. The endogenous production of calcitriol by monophosphoryl lipid A-stimulated DCs appeared to be Toll-IL-1R domain-containing adapter-inducing IFN-β-dependent, mediated through a type 1 IFN-induced expression of 1αOHase. Responsiveness to calcitriol was essential to promote the trafficking of mobilized DCs to nondraining lymphoid organs. Collectively, these studies help to expand our understanding of the physiologically important roles played by locally metabolized vitamin D3 in the initiation and diversification of adaptive immune responses. The influences of locally produced calcitriol on the migration of activated DCs from sites of vaccination/infection into both draining and nondraining lymphoid organs create a condition whereby Ag-responsive B and T cells residing in multiple lymphoid organs are able to simultaneously engage in the induction of adaptive immune responses to peripherally administered Ags as if they were responding to an infection of peripheral or mucosal tissues they were designed to protect.

A Role for Immature Myeloid Cells in Immune Senescence

The reduced efficiency of the mammalian immune system with aging increases host susceptibility to infectious and autoimmune diseases. However, the mechanisms responsible for these pathologic changes are not well understood. In this study, we demonstrate that the bone marrow, blood, and secondary lymphoid organs of healthy aged mice possess increased numbers of immature myeloid cells that are phenotypically similar to myeloid-derived suppressor cells found in lymphoid organs of mice with progressive tumors and other pathologic conditions associated with chronic inflammation. These cells are characterized by the presence of Gr1 and CD11b markers on their surfaces. Gr1+CD11b+ cells isolated from aged mice possess an ability to suppress T cell proliferation/activation and produce heightened levels of proinflammatory cytokines, both constitutively and upon activation, including IL-12, which promotes an excessive production of IFN-γ. IFN-γ priming is essential for excessive proinflammatory cytokine production and the suppressive activities by Gr1+CD11b+ cells from aged mice. These cells suppress T cell proliferation through an NO-dependent mechanism, as depletion of splenic Gr1+ cells reduces NO levels and restores T cell proliferation. Insights into mechanisms responsible for the proinflammatory and immune suppressive activities of Gr1+CD11b+ cells from aged mice have uncovered a defective PI3K–Akt signaling pathway, leading to a reduced Akt-dependent inactivation of GSK3β. Our data demonstrate that abnormal activities of the Gr1+CD11b+ myeloid cell population from aged mice could play a significant role in the mechanisms responsible for immune senescence.

TLR ligands that stimulate the metabolism of vitamin D3 in activated murine dendritic cells can function as effective mucosal adjuvants to subcutaneously administered vaccines

Cathelicidin production by human myeloid cells stimulated through toll-like receptor (TLR) 2/1, the migration of human CD8+ T cells to inflamed skin sites, and the ability of murine dendritic cells (DCs) to migrate from skin sites of vaccination to mucosal lymphoid organs all occur via calcitriol-dependent mechanisms. Herein, we report that murine DCs exposed to TLR3/TLR4 ligands upregulate their expression of 1 alpha-hydroxylase, the enzyme that converts circulating 25(OH)D3 to calcitriol, the active form of vitamin D3. TLR3/TLR4 ligands injected subcutaneously affect DC migration in vivo, allowing their trafficking to both draining and non-draining systemic and mucosal lymphoid organs. Subcutaneously delivered vaccines containing TLR3/TLR4 ligands and antigen stimulate the induction of both systemic and mucosal immune responses. Vaccines containing TLR9 ligands fail to stimulate 1 alpha-hydroxylase protein expression, are incapable of redirecting DC migration into Peyers patches and do not induce mucosal immune responses. These findings support a hypothesis that active metabolites of vitamin D3 produced locally are able to affect various aspects of innate and acquired immune responses.

Conditions That Diminish Myeloid-Derived Suppressor Cell Activities Stimulate Cross-Protective Immunity

ABSTRACT Immunity conferred by conventional vaccines is restricted to a narrow range of closely related strains, highlighting the unmet medical need for the development of vaccines that elicit protection against multiple pathogenic serotypes. Here we show that a Salmonella bivalent vaccine comprised of strains that lack and overproduce DNA adenine methylase (Dam) conferred cross-protective immunity to salmonella clinical isolates of human and animal origin. Protective immunity directly correlated with increased levels of cross-reactive opsonizing antibodies and memory T cells and a diminished expansion of myeloid-derived suppressor cells (MDSCs) that are responsible for the immune suppression linked to several conditions of host stress, including chronic microbial infections, traumatic insults, and many forms of cancer. Further, aged mice contained increased numbers of MDSCs and were more susceptible to Salmonella infection than young mice, suggesting a role for these cells in the immune declines associated with the natural aging process. These data suggest that interventions capable of reducing MDSC presence and activities may allow corresponding increases in B- and T-cell stimulation and benefit the ability of immunologically diverse populations to be effectively vaccinated as well as reducing the risk of susceptible individuals to infectious disease.

Intrauterine Growth Restriction Alters T-Lymphocyte Cell Number and Dual Specificity Phosphatase 1 Levels in the Thymus of Newborn and Juvenile Rats

Intrauterine growth restricted (IUGR) infants have increased susceptibility to infection associated with higher risk of illness and death. Dual specificity phosphatase 1 (DUSP1), which is transcribed in the thymus, increases in quantity as T cells mature and differentiate into CD4+ cells. Little is known about how IUGR affects DUSP1 levels and T-cell subpopulations over time. We hypothesized that IUGR would decrease cell count, CD4+ and CD8+ subpopulations of T lymphocytes, and DUSP1 levels in IUGR rat thymus and spleen. Bilateral uterine artery ligation produced IUGR rats. Thymus and spleen were harvested at P0 and P21. Flow cytometry was used to compare CD4+ and CD8+ lymphocyte populations. Real-time RT-PCR and Western blotting were used to determine DUSP1 quantity. IUGR significantly decreased total cell count in P0 and P21 IUGR male and female thymus. IUGR significantly increased CD4+ cells in IUGR P0 males and females, significantly decreased CD4+ cells in P21 female thymus, and significantly altered DUSP1 levels in the IUGR female thymus at P0 and P21, although it is not yet known whether the change in DUSP1 levels is due to a change in the level per cell or to a change in cellular composition of the thymus.

SUMOylation Regulates Growth Factor Independence 1 in Transcriptional Control and Hematopoiesis

ABSTRACT Cell fate specification requires precise coordination of transcription factors and their regulators to achieve fidelity and flexibility in lineage allocation. The transcriptional repressor growth factor independence 1 (GFI1) is comprised of conserved Snail/Slug/Gfi1 (SNAG) and zinc finger motifs separated by a linker region poorly conserved with GFI1B, its closest homolog. Moreover, GFI1 and GFI1B coordinate distinct developmental fates in hematopoiesis, suggesting that their functional differences may derive from structures within their linkers. We show a binding interface between the GFI1 linker and the SP-RING domain of PIAS3, an E3-SUMO (small ubiquitin-related modifier) ligase. The PIAS3 binding region in GFI1 contains a conserved type I SUMOylation consensus element, centered on lysine-239 (K239). In silico prediction algorithms identify K239 as the only high-probability site for SUMO modification. We show that GFI1 is modified by SUMO at K239. SUMOylation-resistant derivatives of GFI1 fail to complement Gfi1 depletion phenotypes in zebrafish primitive erythropoiesis and granulocytic differentiation in cultured human cells. LSD1/CoREST recruitment and MYC repression by GFI1 are profoundly impaired for SUMOylation-resistant GFI1 derivatives, while enforced expression of MYC blocks granulocytic differentiation. These findings suggest that SUMOylation within the GFI1 linker favors LSD1/CoREST recruitment and MYC repression to govern hematopoietic differentiation.

Anti-MTG16 antibodies reveal MTG16 subcellular distribution and nucleocytoplasmic transport in erythroleukemia cells

License. The full terms of the License are available at http://creativecommons.org/licenses/by-nc/3.0/. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. Permissions beyond the scope of the License are administered by Dove Medical Press Limited. Information on how to request permission may be found at: http://www.dovepress.com/permissions.php Antibody Technology Journal 2015:5 27–41 Antibody Technology Journal Dovepress

Abstract 3089: Regulation of GFI proteins by Notch intracellular domains

Growth factor independence (GFI)-1 and its homolog, GFI1B, are transcriptional regulators that play critical roles during hematopoiesis. GFI family proteins are characterized by near-invariant SNAG and zinc finger domains that direct transcriptional repression and DNA-binding, respectively. A linker region with limited homology and unknown function separates the conserved SNAG and zinc-finger regions. GFI proteins display distinct expression patterns within the myelo-erythroid compartment and make similarly distinct contributions to cell fate specification. GFI1 expression is necessary for hematopoietic stem cell maintenance and is required for granulocytic differentiation beyond the promyelocyte stage. Enforced expression of GFI1 promotes IL-2-independent growth of hematopoietic progenitors and inhibits apoptosis. Mutations in GFI1 lead to a severe congenital neutropenia syndrome that carries an increased risk of myeloid leukemia development. GFI1B expression predominates in erythroid and megakaryocytic precursors and is required for terminal differentiation along both lineages. Moreover, elevated GFI1B expression occurs preferentially in erythroid and megakaryoblastic leukemias, and contributes to the malignant phenotype by supporting growth and preventing apoptosis. Given their complementary roles as growth and survival factors, yet distinct contributions to lineage allocation in hematopoiesis, it is essential to define how GFI family proteins are integrated into the signaling machinery governing cell fate specification. We show that both GFI1 and GFI1B interact with intracellular domains of Notch receptors. Enforced expression of the Notch1 intracellular domain (N1-ICD) impairs GFI-mediated transcriptional repression. Using deletion mutagenesis, we have mapped the regions required for the N1-ICD–GFI1 interaction. Both the ankyrin repeat and transactivation domains of N1-ICD contribute binding surfaces for GFI1. In reciprocal mapping experiments, we show that N1-ICD binding to GFI1 requires a 128-amino acid motif in the divergent linker region near the zinc finger repeat elements. This region contains a high probability consensus sumoylation element that is absolutely conserved in mammalian species but not present in GFI1B. These data suggest that Notch activation may directly impact transcriptional targets regulated by GFI family proteins and expand the potential sphere of influence for Notch in hematopoietic development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3089. doi:1538-7445.AM2012-3089