Thomas Welte

STAT3 deletion during hematopoiesis causes Crohn's disease-like pathogenesis and lethality : a critical role of STAT3 in innate immunity

Signal transducer and activator of transcription 3 (STAT3) is a key transcriptional mediator for many cytokines and is essential for normal embryonic development. We have generated a unique strain of mice with tissue-specific disruption of STAT3 in bone marrow cells during hematopoiesis. This specific STAT3 deletion causes death of these mice within 4–6 weeks after birth with Crohns disease-like pathogenesis in both the small and large intestine, including segmental inflammatory cell infiltration, ulceration, bowel wall thickening, and granuloma formation. Deletion of STAT3 causes significantly increased cell autonomous proliferation of cells of the myeloid lineage, both in vivo and in vitro. Most importantly, Stat3 deletion during hematopoiesis causes overly pseudoactivated innate immune responses. Although inflammatory cytokines, including tumor necrosis factor α and IFN-γ, are overly produced in these mice, the NAPDH oxidase activity, which is involved in antimicrobial and innate immune responses, is inhibited. The signaling responses to lipopolysaccharide are changed in the absence of STAT3, leading to enhanced NF-κB activation. Our results suggest a model in which STAT3 has critical roles in the development and regulation of innate immunity, and deletion of STAT3 during hematopoiesis results in abnormalities in myeloid cells and causes Crohns disease-like pathogenesis.

STAT3 Is Required for Flt3L-Dependent Dendritic Cell Differentiation

The signals that control decisions of progenitor commitment involve the interplay of both cytokines and transcription factors. Flt3L has emerged as a potential regulator of dendritic cell (DC) development, but regulation of HSC commitment to the DC lineage remains poorly understood. Our central finding is the identification of STAT3 activation as a checkpoint of Flt3L-regulated DC development. Deletion of STAT3 caused profound deficiency in the DC compartment and abrogated Flt3L effects on DC development. DC derivation by Flt3L revealed a normal HSC pool, a 2- to 3-fold accumulation of CLP/CMP, but absence of common DC precursors as well as their DC progeny in STAT3-deficient mice. The formation of CMP and CLP represents the first decisive lineage commitment step, and in this regard we provide evidence that commitments of CLP/CMP to the DC lineage strictly depend on the interplay of both Flt3L and STAT3 activation.

The Osteogenic Niche Promotes Early-Stage Bone Colonization of Disseminated Breast Cancer Cells

Breast cancer bone micrometastases can remain asymptomatic for years before progressing into overt lesions. The biology of this process, including the microenvironment niche and supporting pathways, is unclear. We find that bone micrometastases predominantly reside in a niche that exhibits features of osteogenesis. Niche interactions are mediated by heterotypic adherens junctions (hAJs) involving cancer-derived E-cadherin and osteogenic N-cadherin, the disruption of which abolishes niche-conferred advantages. We elucidate that hAJ activates the mTOR pathway in cancer cells, which drives the progression from single cells to micrometastases. Human data set analyses support the roles of AJ and the mTOR pathway in bone colonization. Our study illuminates the initiation of bone colonization, and provides potential therapeutic targets to block progression toward osteolytic metastases.

Dominance of IL-12 Over IL-4 in γδ T Cell Differentiation Leads to Default Production of IFN-γ: Failure to Down-Regulate IL-12 Receptor β2-Chain Expression

γδ T cells secrete Th1- and Th2-like cytokines that help mediate innate and acquired immunity. We have addressed the mechanism whereby murine γδ T cells acquire the capacity to differentially produce such cytokines. Splenic γδ T cells could be polarized into IFN-γ- or IL-4-secreting cells in vitro; however, in contrast to CD4+ αβ T cells, γδ T cells predominantly produced IFN-γ, even in the presence of IL-4, a finding independent of genetic background. Like CD4+ Th1 cells, IFN-γ-producing cells expressed the IL-12 receptor β2-chain after activation in the presence of IL-12; however, unlike Th2 cells, IL-4-primed γδ T cells also expressed this receptor, even in the absence of IFN-γ and despite the presence of the transcription factor GATA-3. IL-12 also induced IL-4-primed γδ T cells to proliferate and to translocate Stat3/Stat4, indicating signaling through the IL-12 receptor. These molecular events can account for the predominant production of IFN-γ by γδ T cells in the presence of IL-12, despite the availability of IL-4. Early and predominant production of IFN-γ by γδ T cells likely is critical for the roles that these cells play in protection against intracellular pathogens and in tumor immunity.

Toll-like receptor 7-induced immune response to cutaneous West Nile virus infection

The Toll-like receptor (TLR) 7 response represents a vital host-defence mechanism in a murine model of systemic West Nile virus (WNV) infection. Here, we investigated the role of the TLR7-induced immune response following cutaneous WNV infection. We found that there was no difference in susceptibility to WNV encephalitis between wild-type and TLR7(-/-) mice upon intradermal injection or infected mosquito feeding. Viral load analysis revealed similar levels of WNV RNA in the peripheral tissues and brains of these two groups of mice following intradermal infection. There was a higher level of cytokines in the blood of wild-type mice at early stages of infection; however, this difference was diminished in the blood and brains at later stages. Langerhans cells (LCs) are permissive to WNV infection and migrate from the skin to draining lymph nodes upon intradermal challenge. Our data showed that WNV infection of TLR7(-/-) keratinocytes was significantly higher than that of wild-type keratinocytes. Infection of wild-type keratinocytes induced higher levels of alpha interferon and interleukin-1beta (IL-1beta), IL-6 and IL-12, which might promote LC migration from the skin. Co-culture of naïve LCs of wild-type mice with WNV-infected wild-type keratinocytes resulted in the production of more IL-6 and IL-12 than with TLR7(-/-) keratinocytes or by cultured LCs alone. Moreover, LCs in the epidermis were reduced in wild-type mice, but not in TLR7(-/-) mice, following intradermal WNV infection. Overall, our results suggest that the TLR7 response following cutaneous infection promotes LC migration from the skin, which might compromise its protective effect in systemic infection.

γδ T Cells Facilitate Adaptive Immunity against West Nile Virus Infection in Mice

West Nile (WN) virus causes fatal meningoencephalitis in laboratory mice, and γδ T cells are involved in the protective immune response against viral challenge. We have now examined whether γδ T cells contribute to the development of adaptive immune responses that help control WN virus infection. Approximately 15% of TCRδ−/− mice survived primary infection with WN virus compared with 80–85% of the wild-type mice. These mice were more susceptible to secondary challenge with WN virus than the wild-type mice that survived primary challenge with the virus. Depletion of γδ T cells in wild-type mice that survived the primary infection, however, does not affect host susceptibility during secondary challenge with WN virus. Furthermore, γδ T cells do not influence the development of Ab responses during primary and at the early stages of secondary infection with WN virus. Adoptive transfer of CD8+ T cells from wild-type mice that survived primary infection with WN virus to naive mice afforded partial protection from lethal infection. In contrast, transfer of CD8+ T cells from TCRδ−/− mice that survived primary challenge with WN virus failed to alter infection in naive mice. This difference in survival correlated with the numeric and functional reduction of CD8 memory T cells in these mice. These data demonstrate that γδ T cells directly link innate and adaptive immunity during WN virus infection.

Mutual regulation of tumour vessel normalization and immunostimulatory reprogramming

Blockade of angiogenesis can retard tumour growth, but may also paradoxically increase metastasis. This paradox may be resolved by vessel normalization, which involves increased pericyte coverage, improved tumour vessel perfusion, reduced vascular permeability, and consequently mitigated hypoxia. Although these processes alter tumour progression, their regulation is poorly understood. Here we show that type 1 T helper (TH1) cells play a crucial role in vessel normalization. Bioinformatic analyses revealed that gene expression features related to vessel normalization correlate with immunostimulatory pathways, especially T lymphocyte infiltration or activity. To delineate the causal relationship, we used various mouse models with vessel normalization or T lymphocyte deficiencies. Although disruption of vessel normalization reduced T lymphocyte infiltration as expected, reciprocal depletion or inactivation of CD4+ T lymphocytes decreased vessel normalization, indicating a mutually regulatory loop. In addition, activation of CD4+ T lymphocytes by immune checkpoint blockade increased vessel normalization. TH1 cells that secrete interferon-γ are a major population of cells associated with vessel normalization. Patient-derived xenograft tumours growing in immunodeficient mice exhibited enhanced hypoxia compared to the original tumours in immunocompetent humans, and hypoxia was reduced by adoptive TH1 transfer. Our findings elucidate an unexpected role of TH1 cells in vasculature and immune reprogramming. TH1 cells may be a marker and a determinant of both immune checkpoint blockade and anti-angiogenesis efficacy.

MECHANISM OF INTERACTION BETWEEN THE GLUCOCORTICOID RECEPTOR AND STAT5 : ROLE OF DNA-BINDING

The functional interaction between the glucocorticoid receptor (GR) and the signal transducer and activator of transcription-5 (Stat5) was investigated by studying the synergistic activation of beta-cascin gene transcription by prolactin and glucocorticoids. The synergism was shown to be mediated by a complex hormone response region with multiple binding sites for Stat5, the glucocorticoid receptor, and CCAAT/enhancer binding proteins (C/EBP). HC11 mammary epithelial cells, which contain physiological levels of GR and Stat5, and COS-7 cells overexpressing GR and Stat5 were employed. In both cell types intact binding sites for Stat5 and the GR were a prerequisite for the synergism, whereas C/EBP sites were only required in HC11 cells. Interestingly, the GR sites employed for the synergism were nonclassical, half palindromic sites, which did not function in the absence of activated Stat5 to mediate the action of the GR on transcription. The interaction of GR and Stat5 triggered by the unusual configuration of binding sites appears to represent a novel mechanism by which these two distinct types of transcription factors cooperate. The mode of interaction provides an efficient means to restrict gene expression to conditions where both Stat5 and the GR are activated.

Glucocorticoid receptor binding sites in the promoter region of milk protein genes

The action of glucocorticoids on the induction of the two milk protein genes beta-casein and whey acidic protein (WAP) is delayed and appears to be indirect. The response requires a co-stimulation of cells with prolactin, is restricted to mammary epithelial cells and is dependent on cis-acting sequences localized in the promoter region of the two genes. We have searched for glucocorticoid receptor (GR) binding sites in these hormone response regions. In vitro DNaseI footprinting experiments were performed with a purified preparation of the GR from rat liver. The sequences between -231 and -7 and between -250 and -79 of mouse WAP and rat beta-casein gene promoter, respectively, were found to contain multiple sites which were protected from the attack of DNaseI by the GR preparation. The sites comprise sequence motifs related to the delayed secondary glucocorticoid response elements described (Chan et al., J. Biol. Chem. 266, 22,634-22,644, 1991), which represent half sites of classical GR binding sites. In the WAP gene promoter, the motifs are arranged head to tail with irregular spacing. The GR binding sites are in close proximity or even overlap with the bindings sites for candidate factors involved in mammary cell specific gene expression. The results suggest a direct co-operation between GR and mammary cell specific transcription factors in mediating the expression of milk protein genes.

Comparative study on the phosphotyrosine motifs of different cytokine receptors involved in STAT5 activation

Several cytokines and growth factors activate transcription of their target genes via the JAK/STAT signalling pathway. It has been shown that the interaction between SH2 domains of STAT factors and receptor phosphotyrosine residues plays an essential role in the specific recruitment of STATs. For STAT5, however, the importance of receptor tyrosines is still controversial. Using a chimeric receptor system in COS‐7 cells, we studied the activation of STAT5 through the interleukin‐6 signal transducer gp130. In contrast to previous reports, we did not detect gp130‐mediated STAT5 activation. However, STAT5 activation was achieved when tyrosine motifs of other cytokine receptors were fused to the membrane‐proximal part of gp130. The comparison of the relative potency of different tyrosine motifs revealed that hydrophobic amino acids, preferentially leucine, in positions +1 and +3, and an aspartate residue in position ‐1 or ‐2 with respect to the tyrosine are likely to be required for efficient STAT5 recruitment. In summary, we show here for the first time that phosphotyrosine motifs can confer the ability to activate STAT5 to a heterologous receptor.