Alois Palmetshofer

Induction of NFATc2 Expression by Interleukin 6 Promotes T Helper Type 2 Differentiation

Interleukin (IL)-6 is produced by professional antigen-presenting cells (APCs) such as B cells, macrophages, and dendritic cells. It has been previously shown that APC-derived IL-6 promotes the differentiation of naive CD4+ T cells into effector T helper type 2 (Th2) cells. Here, we have studied the molecular mechanism for IL-6–mediated Th2 differentiation. During the activation of CD4+ T cells, IL-6 induces the production of IL-4, which promotes the differentiation of these cells into effector Th2 cells. Regulation of IL-4 gene expression by IL-6 is mediated by nuclear factor of activated T cells (NFAT), as inhibition of NFAT prevents IL-6–driven IL-4 production and Th2 differentiation. IL-6 upregulates NFAT transcriptional activity by increasing the levels of NFATc2. The ability of IL-6 to promote Th2 differentiation is impaired in CD4+ T cells that lack NFATc2, demonstrating that NFATc2 is required for regulation of IL-4 gene expression by IL-6. Regulation of NFATc2 expression and NFAT transcriptional activity represents a novel pathway by which IL-6 can modulate gene expression.

NFATc2 and NFATc3 transcription factors play a crucial role in suppression of CD4+ T lymphocytes by CD4+ CD25+ regulatory T cells

The phenotype of NFATc2−/− c3−/− (double knockout [DKO]) mice implies a disturbed regulation of T cell responses, evidenced by massive lymphadenopathy, splenomegaly, and autoaggressive phenomena. The population of CD4+ CD25+ T cells from DKO mice lacks regulatory capacity, except a small subpopulation that highly expresses glucocorticoid-induced tumor necrosis factor receptor family–related gene (GITR) and CD25. However, neither wild-type nor DKO CD4+ CD25+ regulatory T cells (T reg cells) are able to suppress proliferation of DKO CD4+ CD25− T helper cells. Therefore, combined NFATc2/c3 deficiency is compatible with the development of CD4+ CD25+ T reg cells but renders conventional CD4+ T cells unresponsive to suppression, underlining the importance of NFAT proteins for sustaining T cell homeostasis.

Specific and Redundant Roles for NFAT Transcription Factors in the Expression of Mast Cell-Derived Cytokines

By virtue of their ability to express a plethora of biologically highly active mediators, mast cells (MC) are involved in both adaptive and innate immune responses. MC-derived Th2-type cytokines are thought to act as local amplifiers of Th2 reactions, including chronic inflammatory disorders such as allergic asthma, whereas MC-derived TNF-α is a critical initiator of antimicrobial defense. In this study, we demonstrate that the transcription factors NFATc1 and NFATc2 are part of a MC-specific signaling network that regulates the expression of TNF-α and IL-13, whereas NFATc3 is dispensable. Primary murine bone marrow-derived MC from NFATc2−/− mice, activated by either ionomycin or IgE/Ag cross-link, display a strong reduction in the production of these cytokines, compared with bone marrow-derived MC from wild-type mice. Detailed analyses of TNF-α and IL-13 expression using small interfering RNA-mediated knockdown reveals that both NFATc2 and NFATc1 are able to drive the expression of these cytokines, whereas neither degranulation nor the expression of IL-6 depends on NFAT activity. These results support the view that high NFAT activity is necessary for TNF-α and IL-13 promoter induction in MC, irrespective of whether NFATc2 or NFATc1 or a combination of both is present.

NFAT transcription factors in control of peripheral T cell tolerance

The Ca++‐regulated calcineurin/NFAT cascade is one of the crucial signalling pathways that controls adaptive immunity. However, a number of novel experimental data suggest that, in addition to their role in T cell activation, NFATc transcription factors play also a decisive role in the generation of peripheral tolerance against self‐antigens. This function of NFATc factors is mediated by controlling activation‐induced cell death and clonal anergy of T helper cells and the activity of regulatory T cells. The multi‐functional role of NFATc proteins characterize these transcription factors as key regulators of immunological tolerance and, if dysregulated, of development of autoimmune diseases.

Vaccinations with T‐helper type 1 directing adjuvants have different suppressive effects on the development of allergen‐induced T‐helper type 2 responses

Background Allergen‐induced T‐helper type 2 (Th2) responses can be inhibited with Th1 directing vaccines. However, studies comparing the efficacy of the different adjuvants have not been performed in detail.

B Cell Hyperresponsiveness and Expansion of Mature Follicular B Cells but Not of Marginal Zone B Cells in NFATc2/c3 Double-Deficient Mice

Marginal zone (MZ) B cells and peritoneal B-1 cells provide a first defense system of thymus-independent Ab responses against foreign pathogens and therefore share a number of functional properties. Recently, development of B-1a cells was shown to be controlled by the transcription factor NFATc1. We show here that mice deficient for NFATc2 and c3 display a distinct lower representation of MZ B cells, which is correlated with a reduced capturing of trinitrophenyl-Ficoll. In contrast, mature follicular B cells from NFATc2/c3−/− mice are strongly increased in number. NFATc2/c3−/− B cells exhibit a marked increase in BCR-induced intracellular Ca2+ mobilization and proliferation. However, trinitrophenyl-Ficoll-specific IgM and IgG3 responses of NFATc2/c3-deficient mice are intact, and chimeric mice reconstituted with NFATc2/3-deficient B cells show a normal number of MZ B cells and normal BCR responses. These observations suggest that the strongly elevated Th2 cytokine milieu in NFATc2/c3-deficient mice leads to a hyperactivation of mature, follicular B cells, whereas MZ B cells are less responsive to these signals.

Mice Deficient in Nuclear Factor of Activated T-Cell Transcription Factor c2 Mount Increased Th2 Responses after Infection with Nippostrongylus brasiliensis and Decreased Th1 Responses after Mycobacterial Infection

ABSTRACT Infection of nuclear factor of activated T-cell transcription factor c2 (NFATc2)-deficient mice with the helminth Nippostrongylus brasiliensis led to a distinct increase in interleukin-4 (IL-4) and IL-5 protein synthesis by lymph node and spleen cells and to elevated serum immunoglobulin E (IgE) levels in comparison to those seen with infected control mice. While IL-4, IL-5, and IL-13 mRNA expression was also enhanced in lymph node cells from the lungs of infected NFATc2−/− mice, the number of T cells secreting Th2-type lymphokines remained the same in mice infected with N. brasiliensis. In contrast, lymphocytes from NFATc2-deficient mice infected with Mycobacterium bovis BCG secreted less gamma interferon than lymphocytes from infected control mice. These findings indicate that NFATc2 is an activator of Th1 responses and a suppressor of Th2 responses in vivo.

Microtubule associated tumor suppressor 1 deficient mice develop spontaneous heart hypertrophy and SLE-like lymphoproliferative disease

The microtubule associated tumor suppressor gene 1 (MTUS1) is a recently published tumor suppressor gene, which has also been shown to act as an early component in the growth inhibitory signaling cascade of the angiotensin II type 2 receptor (AT2R). In this study we report the generation of MTUS1 knock-out (KO) mice, which develop normally but reveal higher body weights and slightly decreased blood pressure levels. Twenty-eight percent of the studied MTUS1 KO mice also developed heart hypertrophy and 12% developed nephritis, independent of blood pressure levels. Forty-three percent of the MTUS1 KO mice revealed lymphoid hyperplasia affecting spleen (20%), kidney (37%), lung (23%), lymph nodes (17%), and liver (17%) accompanied with leukocytosis, lymphocytosis, and mild anemia. One animal (3%) developed a marginal zone B-cell lymphoma affecting submandibular salivary gland and regional lymph nodes. The symptoms of all mentioned animals are consistent with a B-cell lymphoproliferative disease with features of systemic lupus erythematosus. In addition, body weight of the MTUS1 KO mice was significantly increased and isolated skin fibroblasts showed increased cell proliferation and decreased cell size, compared to wild-type (WT) fibroblasts in response to depleted FCS concentration and lack of growth factors. In conclusion we herein report the first generation of a MTUS1 KO mouse, developing spontaneous heart hypertrophy and increased cell proliferation, confirming once more the anti-proliferative effect of MTUS1, and a SLE-like lymphoproliferative disease suggesting crucial role in regulation of inflammation. These MTUS1 KO mice can therefore serve as a model for further investigations in cardiovascular disease, autoimmune disease and carcinogenesis.

Mice vaccinated with allergen-pulsed myeloid dendritic cells are not protected from developing allergen-induced Th2 responses

Background: Dendritic cells (DC) play a decisive role in the induction of allergen-induced Th1 and Th2 responses. Since the induction of allergen-specific Th1 responses has shown to inhibit allergen-induced Th2-type inflammation, in this study we investigated whether manipulated myeloid-derived DC pulsed with the specific allergen would predominantly induce allergen-specific Th1 responses thereby reducing the development of Th2 responses. Methods: Murine bone marrow (BM)-DC were generated and pulsed with ovalbumin (OVA) and CpG oligodeoxynucleotides (CpG-ODN). Langerhans cells (LC) were also isolated and pulsed in vitro with OVA. Subsequently, mice were vaccinated intravenously with either CpG/OVA-pulsed BM-DC or OVA-pulsed LC, and the protocol to induce OVA-specific Th2 responses using OVA/alum sensitization was initiated. Airway inflammation and OVA-specific serum antibody levels were evaluated 6 days after the intranasal challenge with OVA. Results: The application ofCpG/OVA-pulsed BM-DC was unable to reduce airway eosinophilia and inflammation in OVA/alum-immunized mice. OVA-specific IgG1 or IgE serum levels were also not reduced. The experiments using LC pulsed with OVA yielded similar results. However, mice vaccinated with CpG/OVA-pulsed BM-DC had greatly enhanced levels of serum OVA-specific IgG2a, suggesting the induction of allergen-specific Th1 responses in vivo. Moreover, allergen-induced mast cell degranulation was decreased using this approach. Conclusions: Taken together, our results demonstrated that the vaccination with OVA-pulsed BM-DC matured with CpG-ODN or OVA-pulsed LC did not result in a reduction in allergen-specific Th2 responses in a murine model of severe atopic asthma. Other DC-based vaccination strategies should be evaluated in order to prevent the development of allergic disorders.