Chien-Kuo Lee

What does Stat3 do

The Stat protein family was discovered in the course of studies of signaling specificity from IFN receptors (1). The initial finding of a family of related proteins, each activated by a different cytokine receptor, suggested that these proteins would fulfill the requirements predicted for carriers of intracellular signaling information capable of retaining the specificity inherent in cytokine-receptor interactions (2). While studies in cell culture systems belied some of this early promise, the initial findings with genetically manipulated mice, especially using gene-targeting approaches, generally suggested a high degree of specificity for the various Stat proteins in individual signaling pathways. As reviewed elsewhere in this Perspective series, the analysis of mice lacking one or more Stat genes has shown relatively discrete phenotypes, assigning each Stat protein to a relatively specific pathway. Not so with Stat3. Unlike all other members of the Stat gene family, ablation of Stat3 leads to embryonic lethality (3). This finding, along with evidence of its activation by a wide variety of cytokines, growth factors, and other stimuli (4, 5), implied that Stat3 might be more generally deployed than its relatives and has led to the suggestion that it might represent a primordial Stat protein. Recent data, especially from the analysis of conditional loss of Stat3 protein in adult tissues, confirm that Stat3 participates in a wide variety of physiological processes and even directs seemingly contradictory responses.

Stat5 Is Essential for the Myelo- and Lymphoproliferative Disease Induced by TEL/JAK2

STAT5 is activated in a broad spectrum of human hematologic malignancies. We addressed whether STAT5 activation is necessary for the myelo- and lymphoproliferative disease induced by TEL/JAK2 using a genetic approach. Whereas mice transplanted with bone marrow transduced with retrovirus expressing TEL/JAK2 develop a rapidly fatal myelo- and lymphoproliferative syndrome, reconstitution with bone marrow derived from Stat5ab-deficient mice expressing TEL/JAK2 did not induce disease. Disease induction in the Stat5a/b-deficient background was rescued with a bicistronic retrovirus encoding TEL/JAK2 and Stat5a. Furthermore, myeloproliferative disease was induced by reconstitution with bone marrow cells expressing a constitutively active mutant, Stat5a, or a single Stat5a target, murine oncostatin M (mOSM). These data define a critical role for Stat5a/b and mOSM in the pathogenesis of TEL/JAK2 disease.

CLEC5A is critical for dengue-virus-induced lethal disease

Dengue haemorrhagic fever and dengue shock syndrome, the most severe responses to dengue virus (DV) infection, are characterized by plasma leakage (due to increased vascular permeability) and low platelet counts. CLEC5A (C-type lectin domain family 5, member A; also known as myeloid DAP12-associating lectin (MDL-1)) contains a C-type lectin-like fold similar to the natural-killer T-cell C-type lectin domains and associates with a 12-kDa DNAX-activating protein (DAP12) on myeloid cells. Here we show that CLEC5A interacts with the dengue virion directly and thereby brings about DAP12 phosphorylation. The CLEC5A–DV interaction does not result in viral entry but stimulates the release of proinflammatory cytokines. Blockade of CLEC5A–DV interaction suppresses the secretion of proinflammatory cytokines without affecting the release of interferon-α, supporting the notion that CLEC5A acts as a signalling receptor for proinflammatory cytokine release. Moreover, anti-CLEC5A monoclonal antibodies inhibit DV-induced plasma leakage, as well as subcutaneous and vital-organ haemorrhaging, and reduce the mortality of DV infection by about 50% in STAT1-deficient mice. Our observation that blockade of CLEC5A-mediated signalling attenuates the production of proinflammatory cytokines by macrophages infected with DV (either alone or complexed with an enhancing antibody) offers a promising strategy for alleviating tissue damage and increasing the survival of patients suffering from dengue haemorrhagic fever and dengue shock syndrome, and possibly even other virus-induced inflammatory diseases.

Type I IFN Modulates Innate and Specific Antiviral Immunity

IFNs protect from virus infection by inducing an antiviral state and by modulating the immune response. Using mice deficient in multiple aspects of IFN signaling, we found that type I and type II IFN play distinct although complementing roles in the resolution of influenza viral disease. Both types of IFN influenced the profile of cytokines produced by T lymphocytes, with a significant bias toward Th2 differentiation occurring in the absence of responsiveness to either IFN. However, although a Th1 bias produced through inhibition of Th2 differentiation by IFN-γ was not required to resolve infection, loss of type I IFN responsiveness led to exacerbated disease pathology characterized by granulocytic pulmonary inflammatory infiltrates. Responsiveness to type I IFN did not influence the generation of virus-specific cytotoxic lymphocytes or the rate of viral clearance, but induction of IL-10 and IL-15 in infected lungs through a type I IFN-dependent pathway correlated with a protective response to virus. Combined loss of both IFN pathways led to a severely polarized proinflammatory immune response and exacerbated disease. These results reveal an unexpected role for type I IFN in coordinating the host response to viral infection and controlling inflammation in the absence of a direct effect on virus replication.

STAT3 is a negative regulator of granulopoiesis but is not required for G-CSF-dependent differentiation

STAT3 has been described as an essential component of G-CSF-driven cell proliferation and granulopoiesis. This notion was tested by conditional gene ablation in transgenic mice. Contrary to expectation, granulocytes developed from STAT3 null bone marrow progenitors, and STAT3 null neutrophils displayed mature effector functions. Rather than a deficit in granulopoiesis, mice lacking STAT3 in their hematopoietic progenitors developed neutrophilia, and bone marrow cells were hyperresponsive to G-CSF stimulation. These studies provide direct evidence for STAT3-independent granulopoiesis and suggest that STAT3 directs a negative feedback loop necessary for controlling neutrophil numbers, possibly through induced expression of the signaling inhibitor, SOCS3.

Distinct Requirements for IFNs and STAT1 in NK Cell Function

NK cell functions were examined in mice with a targeted mutation of the STAT1 gene, an essential mediator of IFN signaling. Mice deficient in STAT1 displayed impaired basal NK cytolytic activity in vitro and were unable to reject transplanted tumors in vivo, despite the presence of normal numbers of NK cells. IL-12 enhanced NK-mediated cytolysis, but poly(I:C) did not, and a similar phenotype occurred in mice lacking IFNα receptors. Molecules involved in activation and lytic function of NK cells (granzyme A, granzyme B, perforin, DAP10, and DAP12) were expressed at comparable levels in both wild-type and STAT1−/− mice, and serine esterase activity necessary for CTL function was normal, showing that the lytic machinery was intact. NK cells with normal cytolytic activity could be derived from STAT1−/− bone marrow progenitors in response to IL-15 in vitro, and enhanced NK lytic activity and normal levels of IFN-γ were produced in response to IL-12 treatment in vivo. Despite these normal responses to cytokines, STAT1−/− mice could not reject the NK-sensitive tumor RMA-S, even following IL-12 treatment in vivo. Whereas in vitro NK cytolysis was also reduced in mice lacking both type I and type II IFN receptors, these mice resisted tumor challenge. These results demonstrate that both IFN-α and IFN-γ are required to maintain NK cell function and define a STAT1-dependent but partially IFN-independent pathway required for NK-mediated antitumor activity.

STAT1 Affects Lymphocyte Survival and Proliferation Partially Independent of Its Role Downstream of IFN-γ

Lymphocytes derived from mice deficient in STAT1 showed reduced apoptosis and enhanced proliferation in vitro. To understand the involvement of STAT1 in the observed reduction in apoptosis, we examined the levels of caspase and bcl-2 family genes that are involved in cell survival and/or apoptosis. The levels of caspase 1 and 11, two enzymes involved in both cytokine protein processing and induction of apoptosis, were reduced in STAT1−/− cells compared with wild-type. However, the levels of bcl-2 genes were comparable in both mice. STAT1−/− cells also displayed an enhanced proliferation following TCR stimulation. This hyperproliferation could not be ascribed completely to the loss of IFN-γ-mediated antiproliferation. First, similar phenotypes were also observed in fibroblasts and pre-B cells derived from STAT1−/− mice, which do not produce IFN-γ. Second, comparisons with cells lacking the gene for IFN-γ or with cells treated with neutralizing Abs to IFN-γ only partially mimicked the STAT1−/− phenotype. Interestingly, the kinetics of degradation of p27kip1, a CDK inhibitor, following TCR ligation were faster, and, concomitantly, the up-regulation of CDK2 kinase activity and protein levels were increased in stimulated T cells of STAT1−/− mice relative to those of wild-type mice. Furthermore, STAT1−/− animals were more susceptible to carcinogen-induced thymic tumors, a possible consequence of altered T cell growth and/or survival. These results demonstrate an essential role for STAT1 for lymphocyte survival and proliferation that is only partially dependent on IFN-γ signaling.

Tissue-specific Positive Feedback Requirements for Production of Type I Interferon following Virus Infection

Type I interferon (IFN) is synthesized by most nucleated cells following viral infection. Robust IFN production in cell culture requires positive feedback expression of inducible signaling components, such as the transcription factor IRF7. However, the role of positive feedback and IRF7 in vivo may be more complex. We found that IFN produced locally in the respiratory tract of influenza virus-infected mice displayed characteristics of positive feedback, including Stat1-dependent induction of IRF7 and IFN gene expression. IRF7 expression was similarly stimulus-dependent in most tissues. However, lymphoid tissue constitutively expressed high levels of IRF7 in the absence of induction or positive feedback, and this expression was largely confined to plasmacytoid dendritic cells (DC). These cells rapidly produced large quantities of multiple IFNα species following viral infection without positive feedback, whereas other hematopoietic cells, including other DC subtypes, expressed little IRF7 and were poor IFN producers in the absence of positive feedback. These data reveal a dual mechanism for the regulation of IFN production by differential expression of IRF7, involving positive feedback at local sites of infection combined with robust systemic production by IRF7-expressing plasmacytoid DC.

Regulation of Interferon-α Responsiveness by the Duration of Janus Kinase Activity

Daudi B lymphoblastoid cells are highly sensitive to the anti-growth and anti-viral effects of interferon (IFN). Unlike many cell lines, these cells show prolonged transcription of IFN-stimulated genes following treatment with IFN-α. This prolonged response correlated with the continued presence of the activated transcription factor, IFN-stimulated gene factor 3 (ISGF3). Pulse-chase labeling experiments indicated that the half-life of the phosphorylation of signal transducers and activators of transcription (Stat)1 and Stat2 was short (<2 h) although the turnover of the proteins themselves was slow (>24 h), indicative of a constitutive phosphatase activity. The administration of protein-tyrosine kinase inhibitors at any time point during IFN stimulation led to rapid inhibition of the response, indicating that tyrosine kinase activity was continuously required. Catalytic activity of Jak1 and Tyk2 kinases remained elevated for prolonged periods following stimulation. Continuous presence of IFN-α was necessary for maintaining prolonged activation of ISGF3 and of Janus kinases, an activity that was blocked by antibodies to IFN-α or by cycloheximide. Conditioned medium of IFN-α-stimulated cells was capable of stimulating STAT activation in naive cells. Taken together, these results suggest that the response to IFN-α is controlled by the duration of stimulated Janus kinase activity over the background of constitutive dephosphorylation and that this response can be sustained by autocrine secretion of IFN-α.

STAT3 Negatively Regulates Type I IFN-Mediated Antiviral Response

Type I IFNs are crucial cytokines of innate immunity for combating viral infections. Signaling through type I IFN receptors triggers the activation of STAT proteins, including STAT1, STAT2, and STAT3. Although an essential role of STAT1 and STAT2 for type I IFN-induced antiviral response has been well established by studies of gene-targeted mice and human mutations, the role of STAT3 for this response remains unclear. Using gain-of-function and loss-of-function approaches, we demonstrated that STAT3 negatively regulates type I IFN-mediated response. STAT3 knockdown or knockout cells displayed enhanced gene expression and antiviral activity in response to IFN-α/β. Restoration of STAT3 to STAT3KO cells resulted in attenuation of the response. Upon viral infection, increased type I IFN production in STAT3KO cells resulted in enhanced STAT activation and ISG expression. One mechanism for the enhanced IFN production and response in the absence of STAT3 might operate through an MDA5-dependent manner. STAT3 also appeared to suppress IFN response directly in a manner dependent on its N-terminal domain and independent of its function as a transcriptional factor. Taken together, these results define STAT3 as a negative regulator of type I IFN response and provide a therapeutic target for viral infections.