Age-associated temporal decline in butyrate-producing bacteria plays a key pathogenic role in the onset and progression of neuropathology and memory deficits in 3×Tg-AD mice.

Alterations in the gut-microbiome-brain axis are increasingly being recognized to be involved in Alzheimer's disease (AD) pathogenesis. However, the functional consequences of enteric dysbiosis linking gut microbiota and brain pathology in AD progression remain largely undetermined. The present work investigated the causal role of age-associated temporal decline in butyrate-producing bacteria and butyrate in the etiopathogenesis of AD. Longitudinal metagenomics, neuropathological, and memory analyses were performed in the 3×Tg-AD mouse model. Metataxonomic analyses showed a significant temporal decline in the alpha diversity marked by a decrease in butyrate-producing bacterial communities and a concurrent reduction in cecal butyrate production. Inferred metagenomics analysis identified the bacterial acetyl-CoA pathway as the main butyrate synthesis pathway impacted. Concomitantly, there was an age-associated decline in the transcriptionally permissive acetylation of histone 3 at lysines 9 and 14 (H3K9/K14-Ac) in hippocampal neurons. Importantly, these microbiome-gut-brain changes preceded AD-related neuropathology, including oxidative stress, tau hyperphosphorylation, memory deficits, and neuromuscular dysfunction, which manifest by 17-18 months. Initiation of oral administration of tributyrin, a butyrate prodrug, at 6 months of age mitigated the age-related decline in butyrate-producing bacteria, protected the H3K9/K14-Ac status, and attenuated the development of neuropathological and cognitive changes associated with AD pathogenesis. These data causally implicate age-associated decline in butyrate-producing bacteria as a key pathogenic feature of the microbiome-gut-brain axis affecting the onset and progression of AD. Importantly, the regulation of butyrate-producing bacteria and consequent butyrate synthesis could be a significant therapeutic strategy in the prevention and treatment of AD.

Absence of CCR2 reduces spontaneous intestinal tumorigenesis in the ApcMin /+ mouse model.

The biological activities of chemokine (C-C motif) ligand 2 (CCL2) are mediated via C-C chemokine receptor-2 (CCR2). Increased CCL2 level is associated with metastasis of many cancers. In our study, we investigated the role of the CCL2/CCR2 axis in the development of spontaneous intestinal tumorigenesis using the ApcMin/+ mouse model. Ablation of CCR2 in ApcMin/+ mice significantly increased the overall survival and reduced intestinal tumor burden. Immune cell analysis showed that CCR2-/- ApcMin/+ mice exhibited significant reduction in the myeloid cell population and increased interferon γ (IFN-γ) producing T cells both in spleen and mesenteric lymph nodes compared to ApcMin/+ mice. The CCR2-/- ApcMin/+ tumors showed significantly reduced levels of interleukin (IL)-17 and IL-23 and increased IFN-γ and Granzyme B compared to ApcMin/+ tumors. Transfer of CCR2+/+ ApcMin/+ CD4+ T cells into Rag2-/- mice led to development of colitis phenotype with increased CD4+ T cells hyper proliferation and IL-17 production. In contrast, adoptive transfer of CCR2-/- ApcMin/+ CD4+ T cells into Rag2-/- mice failed to enhance colonic inflammation or IL-17 production. These results a suggest novel additional role for CCR2, where it regulates migration of IL-17 producing cells mediating tumor-promoting inflammation in addition to its role in migration of tumor associated macrophages.

Epigenetic Mechanisms Underlying HIV-Infection Induced Susceptibility of CD4+ T Cells to Enhanced Activation-Induced FasL Expression and Cell Death.

BACKGROUND: Chronic immune activation and CD4 T cell depletion are significant pathogenic features of HIV infection. Expression of Fas ligand (FasL), a key mediator of activation-induced cell death in T cells, is elevated in people living with HIV-1 infection (PLWH). However, the epigenetic mechanisms underlying the enhanced induction of FasL expression in CD4 T lymphocytes in PLWH are not completely elucidated. Hence, the current work examined the effect of HIV infection on FasL promoter-associated histone modifications and transcriptional regulation in CD4 T lymphocytes in PLWH. METHOD: Flow cytometric analysis was performed to examine the Fas-FasL expression on total CD4 T cells and naïve/memory CD4 T cell subsets. Epigenetic FasL promoter histone modifications were investigated by chromatin immunoprecipitation-quantitative real-time polymerase chain reaction analysis using freshly isolated total CD4 T lymphocytes from HIV-1 infected and noninfected individuals. RESULTS: All naïve/memory CD4 T cell subsets from PLWH showed markedly greater frequency of FasL expression. Notably, examination of functional outcome of FasL/Fas co-expression demonstrated the preferential susceptibility of Tcm and Tem subsets to activation-induced apoptosis. Importantly, these CD4 T cells collectively demonstrated a distinct FasL promoter histone profile involving a coordinated cross-talk between histone H3 modifications leading to enhanced FasL gene expression. Specifically, levels of transcriptionally permissive histone H3K4-trimethylation (H3K4Me3) and histone H3K9-acetylation (H3K9Ac) were increased, with a concomitant decrease in the repressive H3K9-trimethylation (H3K9Me3). CONCLUSION: The present work demonstrates that epigenetic mechanisms involving promoter-histone modifications regulate transcriptional competence and FasL expression in CD4 T cells from PLWH and render them susceptible to activation-induced cell death.

Adipose-resident myeloid-derived suppressor cells modulate immune cell homeostasis in healthy mice.

The metabolically dynamic nature of healthy adipose places this tissue under regular inflammatory stress. A network of adipose-resident anti-inflammatory immune cells modulates and resolves this endogenous inflammation. Previous work in our laboratory identified a CD11b+ Gr1+ subset of these immunosuppressive adipose stromal cells in healthy mice. Myeloid-derived suppressor cells (MDSCs), typically associated with cancer and chronic inflammation, have a similar surface marker phenotype and accumulate in adipose of high-fat diet-fed mice. Given the routine inflammatory stresses on healthy adipose and the suppressive nature of the tissue-resident immune cells, we hypothesized that these CD11b+ Gr1+ cells were a genuine population of MDSCs involved in regulating tissue homeostasis. Flow cytometric analysis of these cells found that they were CD11b+ CD301- Ly6C+ Ly6G+/- and did not express traditional macrophage markers. Moreover, in vitro functional assays demonstrated that these cells suppressed αCD3/αCD28-induced T-cell proliferation, solidifying their identity as bona fide adipose-resident MDSCs. Systemic MDSC depletion altered adipose immune cell dynamics in otherwise healthy mice, increasing the number of CD4+ effector memory T cells and modifying the surface markers expressed by adipose-resident macrophages. In addition, transcription of various immunomodulatory cytokines was clearly dysregulated in the adipose of MDSC-depleted animals compared with controls. Altogether, our findings indicate that there is a population of bona fide MDSCs in the adipose of otherwise healthy mice that actively contribute to the health and immune homeostasis of this tissue.

Infrared imaging of lymphatic function in the upper extremity of normal controls and hand transplant recipients via subcutaneous indocyanine green injection.

OBJECTIVES: The goal of this study was to define the parameters of movement of indocyanine green in the upper extremity of normal control and hand transplant recipients. The purpose was to establish a non-invasive method of determining the level of lymphatic function in hand transplant recipients. In hand transplantation (and replantation), the deep lymphatic vessels are rarely repaired, resulting in altered lymphatic connections. In most cases, the relatively rapid inosculation of superficial lymphatic networks and drainage via the venous systems results in sufficient interstitial fluid and lymph drainage of the graft to prevent edema. However, our group and others have determined that some transplant recipients demonstrate chronic edema which is associated with lymphatic stasis. In one case, a patient with chronic edema has developed chronic rejection characterized by thinning of the skin, loss of adnexal structures, and fibrosis and contracture of the hand. METHODS: Lymphatic function was evaluated by intradermal administration of near-infrared fluorescent dye, indocyanine green, and dynamic imaging with an infrared camera system (LUNA). To date, the assessment of lymphatic drainage in the upper extremity by clearance of indocyanine green dye has been studied primarily in oncology patients with abnormal lymphatic function, making assessment of normal drainage problematic. To establish normal parameters, indocyanine green lymphatic clearance functional tests were performed in a series of normal controls, and subsequently compared with indocyanine green clearance in hand transplant recipients. RESULTS: The results demonstrate varied patterns of lymphatic drainage in the hand transplant patients that partially mimic normal hand lymphatic drainage, but also share characteristics of lymphedema patients defined in other studies. The study revealed significant deceleration of the dye drainage in the allograft of a patient with suspected chronic rejection and edema of the graft. Analysis of other hand transplant recipients revealed differing levels of dye deceleration, often localized at the level of surgical anastomosis. CONCLUSION: These studies suggest intradermal injection of indocyanine green and near-infrared imaging may be a useful clinical tool to assess adequacy of lymphatic function in hand transplant recipients.

Mast Cell-Dependent CD8+ T-cell Recruitment Mediates Immune Surveillance of Intestinal Tumors in ApcMin/+ Mice.

The presence of mast cells in some human colorectal cancers is a positive prognostic factor, but the basis for this association is incompletely understood. Here, we found that mice with a heterozygous mutation in the adenomatous polyposis coli gene (ApcMin/+) displayed reduced intestinal tumor burdens and increased survival in a chemokine decoy receptor, ACKR2-null background, which led to discovery of a critical role for mast cells in tumor defense. ACKR2-/-ApcMin/+ tumors showed increased infiltration of mast cells, their survival advantage was lost in mast cell-deficient ACKR2-/-SA-/-ApcMin/+ mice as the tumors grew rapidly, and adoptive transfer of mast cells restored control of tumor growth. Mast cells from ACKR2-/- mice showed elevated CCR2 and CCR5 expression and were also efficient in antigen presentation and activation of CD8+ T cells. Mast cell-derived leukotriene B4 (LTB4) was found to be required for CD8+ T lymphocyte recruitment, as mice lacking the LTB4 receptor (ACKR2-/-BLT1-/-ApcMin/+) were highly susceptible to intestinal tumor-induced mortality. Taken together, these data demonstrate that chemokine-mediated recruitment of mast cells is essential for initiating LTB4/BLT1-regulated CD8+ T-cell homing and generation of effective antitumor immunity against intestinal tumors. We speculate that the pathway reported here underlies the positive prognostic significance of mast cells in selected human tumors. Cancer Immunol Res; 6(3); 332-47. ©2018 AACR.

Adipose-derived cellular therapies in solid organ and vascularized-composite allotransplantation.

PURPOSE OF REVIEW: Controlling acute allograft rejection following vascularized composite allotransplantation requires strict adherence to courses of systemic immunosuppression. Discovering new methods to modulate the alloreactive immune response is essential for widespread application of vascularized composite allotransplantation. Here, we discuss how adipose-derived cellular therapies represent novel treatment options for immune modulation and tolerance induction in vascularized composite allotransplantation. RECENT FINDINGS: Adipose-derived mesenchymal stromal cells are cultured from autologous or allogeneic adipose tissue and possess immunomodulatory qualities capable of prolonging allograft survival in animal models of vascularized composite allotransplantation. Similar immunosuppressive and immunomodulatory effects have been observed with noncultured adipose stromal-vascular-fraction-derived therapies, albeit publication of in-vivo stromal vascular fraction cell modulation in transplantation models is lacking. However, both stromal vascular fraction and adipose derived mesenchymal stem cell therapies have the potential to effectively modulate acute allograft rejection via recruitment and induction of regulatory immune cells. SUMMARY: To date, most reports focus on adipose derived mesenchymal stem cells for immune modulation in transplantation despite their phenotypic plasticity and reliance upon culture expansion. Along with the capacity for immune modulation, the supplemental wound healing and vasculogenic properties of stromal vascular fraction, which are not shared by adipose derived mesenchymal stem cells, hint at the profound therapeutic impact stromal vascular fraction-derived treatments could have on controlling acute allograft rejection and tolerance induction in vascularized composite allotransplantation. Ongoing projects in the next few years will help design the best applications of these well tolerated and effective treatments that should reduce the risk/benefit ratio and allow more patients access to vascularized composite allotransplantation therapy.

T cell-mediated antitumor immune response eliminates skin tumors induced by mouse papillomavirus, MmuPV1.

Previous studies of naturally occurring mouse papillomavirus (PV) MmuPV1-induced tumors in B6.Cg-Foxn1nu/nu mice suggest that T cell deficiency is necessary and sufficient for the development of such tumors. To confirm this, MmuPV1-induced tumors were transplanted from T cell-deficient mice into immunocompetent congenic mice. Consequently, the tumors regressed and eventually disappeared. The elimination of MmuPV1-infected skin/tumors in immunocompetent mice was consistent with the induction of antitumor T cell immunity. This was confirmed by adoptive cell experiments using hyperimmune splenocytes collected from graft-recipient mice. In the present study, such splenocytes were injected into T cell-deficient mice infected with MmuPV1, and they eliminated both early-stage and fully formed tumors. We clearly show that anti-tumor T cell immunity activated during tumor regression in immunocompetent mice effectively eliminates tumors developing in T cell-deficient congenic mice. The results corroborate the notion that PV-induced tumors are strongly linked to the immune status of the host, and that PV antigens are major anti-tumor antigens. Successful anti-PV T cell responses should, therefore, lead to effective anti-tumor immune therapy in human PV-infected patients.

Discovery of a Broad-Spectrum Antiviral Compound That Inhibits Pyrimidine Biosynthesis and Establishes a Type 1 Interferon-Independent Antiviral State.

Viral emergence and reemergence underscore the importance of developing efficacious, broad-spectrum antivirals. Here, we report the discovery of tetrahydrobenzothiazole-based compound 1, a novel, broad-spectrum antiviral lead that was optimized from a hit compound derived from a cytopathic effect (CPE)-based antiviral screen using Venezuelan equine encephalitis virus. Compound 1 showed antiviral activity against a broad range of RNA viruses, including alphaviruses, flaviviruses, influenza virus, and ebolavirus. Mechanism-of-action studies with metabolomics and molecular approaches revealed that the compound inhibits host pyrimidine synthesis and establishes an antiviral state by inducing a variety of interferon-stimulated genes (ISGs). Notably, the induction of the ISGs by compound 1 was independent of the production of type 1 interferons. The antiviral activity of compound 1 was cell type dependent with a robust effect observed in human cell lines and no observed antiviral effect in mouse cell lines. Herein, we disclose tetrahydrobenzothiazole compound 1 as a novel lead for the development of a broad-spectrum, antiviral therapeutic and as a molecular probe to study the mechanism of the induction of ISGs that are independent of type 1 interferons.

Immunobiology in VCA.

Transplantation of vascularized composite tissue is a relatively new field that is an amalgamation of experience in solid organ transplantation and reconstructive plastic and orthopedic surgery. What is novel about the immunobiology of VCA is the addition of tissues with unique immunologic characteristics such as skin and vascularized bone, and the nature of VCA grafts, with direct exposure to the environment, and external forces of trauma. VCAs are distinguished from solid organ transplants by the requirement of rigorous physical therapy for optimal outcomes and the fact that these procedures are not lifesaving in most cases. In this review, we will discuss the immunobiology of these systems and how the interplay can result in pathology unique to VCA as well as provide potential targets for therapy.

MmuPV1 infection and tumor development of T cell-deficient mice is prevented by passively transferred hyperimmune sera from normal congenic mice immunized with MmuPV1 virus-like particles (VLPs).

Infection by mouse papillomavirus (PV), MmuPV1, of T cell-deficient, B6.Cg-Foxn1(nu)/J nude mice revealed that four, distinct squamous papilloma phenotypes developed simultaneously after infection of experimental mice. Papillomas appeared on the muzzle, vagina, and tail at or about day 42days post-inoculation. The dorsal skin developed papillomas and hair follicle tumors (trichoblastomas) as early as 26days after infection. Passive transfer of hyperimmune sera from normal congenic mice immunized with MmuPV1 virus-like particles (VLPs) to T cell-deficient strains of mice prevented infection by virions of experimental mice. This study provides further evidence that T cell deficiency is critical for tumor formation by MmuPV1 infection.

Type I interferon signaling contributes to the bias that Toll-like receptor 4 exhibits for signaling mediated by the adaptor protein TRIF.

Signaling by Toll-like receptor 4 (TLR4) is mediated by either of two adaptor proteins: myeloid differentiation marker 88 (MyD88) or Toll-interleukin-1 (IL-1) receptor (TIR) domain-containing adaptor inducing interferon-ß (TRIF). Whereas MyD88-mediated signaling leads to proinflammatory responses, TRIF-mediated signaling leads to less toxic immunostimulatory responses that are beneficial in boosting vaccine responses. The hypothesis that monophosphorylated lipid A structures act as TRIF-biased agonists of TLR4 offered a potential mechanism to explain their clinical value as vaccine adjuvants, but studies of TRIF-biased agonists have been contradictory. In experiments with mouse dendritic cells, we found that irrespective of the agonist used, TLR4 functioned as a TRIF-biased signaling system through a mechanism that depended on the autocrine and paracrine effects of type I interferons. The TLR4 agonist synthetic lipid A induced expression of TRIF-dependent genes at lower concentrations than were necessary to induce the expression of genes that depend on MyD88-mediated signaling. Blockade of type I interferon signaling selectively decreased the potency of lipid A (increased the concentration required) in inducing the expression of TRIF-dependent genes, thereby eliminating adaptor bias. These data may explain how high-potency TLR4 agonists can act as clinically useful vaccine adjuvants by selectively activating TRIF-dependent signaling events required for immunostimulation, without or only weakly activating potentially harmful MyD88-dependent inflammatory responses.

Adjuvant activity of naturally occurring monophosphoryl lipopolysaccharide preparations from mucosa-associated bacteria.

Natural heterogeneity in the structure of the lipid A portion of lipopolysaccharide (LPS) produces differential effects on the innate immune response. Gram-negative bacterial species produce LPS structures that differ from the classic endotoxic LPS structures. These differences include hypoacylation and hypophosphorylation of the diglucosamine backbone, both differences known to decrease LPS toxicity. The effect of decreased toxicity on the adjuvant properties of many of these LPS structures has not been fully explored. Here we demonstrate that two naturally produced forms of monophosphorylated LPS, from the mucosa-associated bacteria Bacteroides thetaiotaomicron and Prevotella intermedia, function as immunological adjuvants for antigen-specific immune responses. Each form of mucosal LPS increased vaccination-initiated antigen-specific antibody titers in both quantity and quality when given simultaneously with vaccine antigen preparations. Interestingly, adjuvant effects on initial T cell clonal expansion were selective for CD4 T cells. No significant increase in CD8 T cell expansion was detected. MyD88/Toll-like receptor 4 (TLR4) and TRIF/TLR4 signaling pathways showed equally decreased signaling with the LPS forms studied here as with endotoxic LPS or detoxified monophosphorylated lipid A (MPLA). Natural monophosphorylated LPS from mucosa-associated bacteria functions as a weak but effective adjuvant for specific immune responses, with preferential effects on antibody and CD4 T cell responses over CD8 T cell responses.

TRIF is required for TLR4 mediated adjuvant effects on T cell clonal expansion.

Toll like receptor 4 (TLR4) is an important pattern recognition receptor with the ability to drive potent innate immune responses and also to modulate adaptive immune responses needed for long term protection. Activation of TLR4 by its ligands is mediated by engagement of the adapter proteins MyD88 (myeloid differentiation factor 88) and TRIF (Toll-interleukin 1 receptor domain-containing adapter inducing interferon-beta). Previously, we showed that TRIF, but not MyD88, plays an important role in allowing TLR4 agonists to adjuvant early T cell responses. In this study, we investigated the T cell priming events that are regulated specifically by the TRIF signaling branch of TLR4. We found that TRIF deficiency prevented the TLR4 agonist lipid A from enhancing T cell proliferation and survival in an adoptive transfer model of T cell priming. TRIF deficient DC showed defective maturation as evidenced by their failure to upregulate co-stimulatory molecules in response to lipid A stimulation. Importantly, TRIF alone caused CD86 and CD40 upregulation on splenic DC, but both TRIF and MyD88 were required for CD80 upregulation. The impairment of T cell adjuvant effects and defective DC maturation in TRIF (lps/lps) mice after TLR4 stimulation was mainly due to loss of type I IFN production, indicating that type I interferons are central to TLR4's adjuvant effects. These results are useful for the continued development of TLR4 based vaccine adjuvants that avoid inflammatory risks while retaining beneficial immune response.

Effects of Differences in Lipid A Structure on TLR4 Pro-Inflammatory Signaling and Inflammasome Activation.

The vertebrate immune system exists in equilibrium with the microbial world. The innate immune system recognizes pathogen-associated molecular patterns via a family of Toll-like receptors (TLR) that activate cells upon detection of potential pathogens. Because some microbes benefit their hosts, mobilizing the appropriate response, and then controlling that response is critical in the maintenance of health. TLR4 recognizes the various forms of lipid A produced by Gram-negative bacteria. Depending on the structural form of the eliciting lipid A molecule, TLR4 responses range from a highly inflammatory endotoxic response involving inflammasome and other pro-inflammatory mediators, to an inhibitory, protective response. Mounting the correct response against an offending microbe is key to maintaining health when exposed to various bacterial species. Further study of lipid A variants may pave the way to understanding how TLR4 responses are generally able to avoid chronic inflammatory damage.

Strategic nonmyeloablative conditioning: CD154:CD40 costimulatory blockade at primary bone marrow transplantation promotes engraftment for secondary bone marrow transplantation after engraftment failure.

There is an increased risk of failure of engraftment following nonmyeloablative conditioning. Sensitization resulting from failed bone marrow transplantation (BMT) remains a major challenge for secondary BMT. Approaches to allow successful retransplantation would have significant benefits for BMT candidates living with chronic diseases. We used a mouse model to investigate the effect of preparative regimens at primary BMT on outcome for secondary BMT. We found that conditioning with TBI or recipient T cell lymphodepletion at primary BMT did not promote successful secondary BMT. In striking contrast, successful secondary BMT could be achieved in mice conditioned with anti-CD154 costimulatory molecule blockade at first BMT. Blockade of CD154 alone or combined with T cell depletion inhibits generation of the humoral immune response after primary BMT, as evidenced by abrogation of production of anti-donor Abs. The humoral barrier is dominant in sensitization resulting from failed BMT, because almost all CFSE-labeled donor cells were killed at 0.5 and 3 h in sensitized recipients in in vivo cytotoxicity assay, reflecting Ab-mediated cytotoxicity. CD154:CD40 costimulatory blockade used at primary BMT promotes allogeneic engraftment in secondary BMT after engraftment failure at first BMT. The prevention of generation of anti-donor Abs at primary BMT is critical for successful secondary BMT.

Plasmacytoid precursor dendritic cells from NOD mice exhibit impaired function: are they a component of diabetes pathogenesis?

OBJECTIVE: Plasmacytoid precursor dendritic cell facilitating cells (p-preDC FCs) play a critical role in facilitation of syngeneic and allogeneic hematopoietic stem cell (HSC) engraftment. Here, we evaluated the phenotype and function of CD8(+)/TCR(-) FCs from NOD mice. RESEARCH DESIGN AND METHODS: The phenotype of CD8(+)/TCR(-) FCs was analyzed by flow cytometry using sorted FCs from NOD, NOR, or B6 mice. The function of NOD FCs was evaluated by colony-forming cell (CFC) assay in vitro and syngeneic or allogeneic HSC transplantation in vivo. RESULTS: We report for the first time that NOD FCs are functionally impaired. They fail to facilitate engraftment of syngeneic and allogeneic HSCs in vivo and do not enhance HSC clonogenicity in vitro. NOD FCs contain subpopulations similar to those previously described in B6 FCs, including p-preDC, CD19(+), NK1.1(+)DX5(+), and myeloid cells. However, the CD19(+) and NK1.1(+)DX5(+) subpopulations are significantly decreased in number in NOD FCs compared with disease-resistant controls. Removal of the CD19(+) or NK1.1(+)DX5(+) subpopulations from FCs did not significantly affect facilitation. Notably, Flt3 ligand (FL) treatment of NOD donors expanded FC total in peripheral blood and restored facilitating function in vivo. CONCLUSIONS: These data demonstrate that NOD FCs exhibit significantly impaired function that is reversible, since FL restored production of functional FCs in NOD mice and suggest that FL plays an important role in the regulation and development of FC function. FCs may therefore be linked to diabetes pathogenesis and prevention.

Impaired Bcl3 up-regulation leads to enhanced lipopolysaccharide-induced interleukin (IL)-23P19 gene expression in IL-10(-/-) mice.

Genetic and biochemical analyses show that IL-23p19 plays a central role in mediating bacteria-induced colitis in interleukin-10-deficient (IL-10(-/-)) mice. The molecular mechanisms responsible for the dysregulated innate host response leading to enhanced IL-23 gene expression in IL-10(-/-) mice are poorly understood. In this study, we investigated the role of Bcl3 in controlling LPS-induced IL-23p19 gene expression in bone marrow-derived dendritic cells (BMDC) isolated from IL-10(-/-) mice. We report higher IL-23p19 mRNA accumulation and protein secretion in LPS-stimulated BMDC isolated from IL-10(-/-) compared with WT mice. Lipopolysaccharide (LPS)-induced B cell leukemia 3 (Bcl3) expression was strongly impaired (90% decrease) in IL-10(-/-) BMDC compared with WT BMDC. Chromatin immunoprecipitation demonstrated enhanced RelA binding to the IL-23p19 promoter in IL-10(-/-) compared with WT BMDC. Bcl3 overexpression decreased LPS-induced IL-23p19 gene expression in IL-10(-/-) BMDC, which correlated with enhanced NF-kappaB p50 binding and decreased RelA binding to the gene promoter. Conversely, Bcl3 knockdown enhanced LPS-induced IL-23p19 gene expression in WT BMDC. Moreover, LPS-induced IL-23p19 gene expression was significantly enhanced in Bcl3(-/-) BMDC compared with WT BMDC. In conclusion, enhanced LPS-induced IL-23p19 gene expression in IL-10(-/-) mice is due to impaired Bcl3 expression leading to diminished p50 and enhanced RelA recruitment to the IL-23p19 promoter.

Adjuvant induced glucose uptake by activated T cells is not correlated with increased survival.

Authors contributed equally to this manuscript Natural adjuvants, such as bacterial lipopolysaccharide (LPS), activate antigen presenting cells via Toll-like receptors and, indirectly, increase the survival of antigen-activated T cells. The molecular mechanisms leading to increased survival remain poorly defined. Because T cell clonal expansion leads to high energy demands, we hypothesized that increased glucose uptake and/or utilization in adjuvant-activated T cells could be important molecular event(s) that would lead to adjuvant-associated T cell survival advantage. Using a fluorescent analog of 2-deoxyglucose, 2-NBDG, we measured glucose accumulation and rate of uptake in T cells from mice treated with antigen in the absence or presence of LPS. Although adjuvant activated T cells increased the accumulation of 2-NBDG, the rate of uptake was unchanged compared to cells activated with only antigen. Furthermore, glucose transport inhibitors, cytochalasin B or phloretin, decreased the accumulation of glucose in adjuvant-treated T cells, but this decrease did not impair adjuvant-associated survival advantages. Together, these data indicate that increased glucose uptake through glucose transporters is not required for increased survival of activated T cells.