The transcription factor T-bet is induced by multiple pathways and prevents an endogenous Th2 cell program during Th1 cell responses.

T-bet is a critical transcription factor for T helper 1 (Th1) cell differentiation. To study the regulation and functions of T-bet, we developed a T-bet-ZsGreen reporter mouse strain. We determined that interleukin-12 (IL-12) and interferon-γ (IFN-γ) were redundant in inducing T-bet in mice infected with Toxoplasma gondii and that T-bet did not contribute to its own expression when induced by IL-12 and IFN-γ. By contrast, T-bet and the transcription factor Stat4 were critical for IFN-γ production whereas IFN-γ signaling was dispensable for inducing IFN-γ. Loss of T-bet resulted in activation of an endogenous program driving Th2 cell differentiation in cells expressing T-bet-ZsGreen. Genome-wide analyses indicated that T-bet directly induced many Th1 cell-related genes but indirectly suppressed Th2 cell-related genes. Our study revealed redundancy and synergy among several Th1 cell-inducing pathways in regulating the expression of T-bet and IFN-γ, and a critical role of T-bet in suppressing an endogenous Th2 cell-associated program.

Islet antigen-specific Th17 cells can induce TNF-α-dependent autoimmune diabetes.

Type 1 diabetes (T1D) results from autoimmune destruction of pancreatic ß-cells. Although Th1 cells are key orchestrators of T1D, the function(s) of the more recently identified Th17 subset are unclear due to inherent plasticity. In this study, we analyzed Th17 cells for stability and diabetogenicity in NOD mice. We found that like Th1 cells, Th17 are a distinct population throughout the prediabetic phase. At diabetes onset, there were marked increases in IL-17-producing Th17 cells and IFN-γ-producing Th1 cells in the pancreas as well as in the serum levels of these cytokines, indicating that these proinflammatory mediators serve as biomarkers of advanced autoimmunity. Although naturally occurring Th17 cells in diabetic mice did not contribute to diabetes development in transfer models, islet-specific Th17 cells were diabetogenic independently of IL-17 and displayed inflammation-induced Th17-to-Th1 reprogramming that could be elicited by Th1 cells. However, an inability to generate Th1 cells because of Stat4, Ifngr, and Ifng deficiencies did not prevent diabetes. Instead, TNF-α could mediate diabetes in response to either Th17 cells or Th1 cells. The results identify a previously unknown mechanism by which Th17 cells can contribute to T1D. Our studies also suggest that when developing interventions for T1D, it will be potentially advantageous to focus on mechanisms common to effector T cells rather than on the signature cytokines of various subsets.

STAT4 is critical for immunity but not for antileishmanial activity of antimonials in experimental visceral leishmaniasis.

We and others have previously shown that IL-12 is indispensable for immunity and is required for the optimal antiparasitic activity of antimonials in experimental visceral leishmaniasis caused by Leishmania donovani. Here we investigated the role of STAT4 in immunity against L. donovani using STAT4 knockout mice and also determined the effect of STAT4 deficiency in response to antimonial therapy. Upon infection with L. donovani, stat4⁻/⁻ BALB/c and C57BL/6 mice showed enhanced susceptibility to Leishmania during late time points of infection which was associated with a marked reduction in Th1 responses and hepatic immunopathology. Interestingly, these defects in Th1 responses in stat4⁻/⁻ did not impair the antimonial chemotherapy as both stat4⁻/⁻ and WT mice showed comparable levels of parasite clearance from the liver and spleen. These findings highlight the role of STAT4 in immunity to L. donovani infection and also provide evidence that STAT4 is dispensable for antimonial-based chemotherapy.

STAT4 controls GM-CSF production by both Th1 and Th17 cells during EAE.

BACKGROUND: In experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, mice genetically deficient in the transcription factor signal transducer and activator of transcription 4 (STAT4) are resistant to disease. In contrast, deletion or inhibition of the Th1-associated cytokines IL-12 or IFNγ which act upstream and downstream of STAT4, respectively, does not ameliorate disease. These discordant findings imply that STAT4 may act in a non-canonical role during EAE. Recently, STAT4 has been shown to regulate GM-CSF production by CD4 T cells and this cytokine is necessary for the induction of EAE. However, it is not known if STAT4 controls GM-CSF production by both Th1 and Th17 effector CD4 T cells. METHODS: This study utilized the MOG(35-55) peptide immunization model of EAE. Intracellular cytokine staining and novel mixed bone marrow chimeric mice were used to study the CD4 T cell-intrinsic role of STAT4 during disease. STAT4 chromatin-immunoprecipitation (ChIP-PCR) experiments were performed to show STAT4 directly interacts with the Csf2 gene loci. RESULTS: Herein, we demonstrate that STAT4 controls CD4 T cell-intrinsic GM-CSF production by both Th1 and Th17 CD4 T cells during EAE as well as in vitro. Importantly, we show that STAT4 interacts with the Csf2 locus in MOG(35-55)-activated effector CD4 T cells demonstrating direct modulation of GM-CSF. CONCLUSIONS: Overall, these studies illustrate a previously unrecognized role of STAT4 to regulate GM-CSF production by not only Th1 cells, but also Th17 effector CD4 T cell subsets during EAE pathogenesis. Critically, these data highlight for the first time that STAT4 is able to modulate the effector profile of Th17 CD4 T cell subsets, which redefines our current understanding of STAT4 as a Th1-centric factor.

STAT4 deficiency fails to induce lung Th2 or Th17 immunity following primary or secondary respiratory syncytial virus (RSV) challenge but enhances the lung RSV-specific CD8+ T cell immune response to secondary challenge.

UNLABELLED: Immune-mediated lung injury is a hallmark of lower respiratory tract illness caused by respiratory syncytial virus (RSV). STAT4 plays a critical role in CD4+ Th1 lineage differentiation and gamma interferon (IFN-γ) protein expression by CD4+ T cells. As CD4+ Th1 differentiation is associated with negative regulation of CD4+ Th2 and Th17 differentiation, we hypothesized that RSV infection of STAT4-/- mice would result in enhanced lung Th2 and Th17 inflammation and impaired lung Th1 inflammation compared to wild-type (WT) mice. We performed primary and secondary RSV challenges in WT and STAT4-/- mice and used STAT1-/- mice as a positive control for the development of RSV-specific lung Th2 and Th17 inflammation during primary challenge. Primary RSV challenge of STAT4-/- mice resulted in decreased T-bet and IFN-γ expression levels in CD4+ T cells compared to those of WT mice. Lung Th2 and Th17 inflammation did not develop in primary RSV-challenged STAT4-/- mice. Decreased IFN-γ expression by NK cells, CD4+ T cells, and CD8+ T cells was associated with attenuated weight loss and enhanced viral clearance with primary challenge in STAT4-/- mice compared to WT mice. Following secondary challenge, WT and STAT4-/- mice also did not develop lung Th2 or Th17 inflammation. In contrast to primary challenge, secondary RSV challenge of STAT4-/- mice resulted in enhanced weight loss, an increased lung IFN-γ expression level, and an increased lung RSV-specific CD8+ T cell response compared to those of WT mice. These data demonstrate that STAT4 regulates the RSV-specific CD8+ T cell response to secondary infection but does not independently regulate lung Th2 or Th17 immune responses to RSV challenge. IMPORTANCE: STAT4 is a protein critical for both innate and adaptive immune responses to viral infection. Our results show that STAT4 regulates the immune response to primary and secondary challenge with RSV but does not restrain RSV-induced lung Th2 or Th17 immune responses. These findings suggest that STAT4 expression may influence lung immunity and severity of illness following primary and secondary RSV infections.

Opposing roles of STAT4 and Dnmt3a in Th1 gene regulation.

The STAT transcription factor STAT4 is a critical regulator of Th1 differentiation and inflammatory disease. Yet, how STAT4 regulates gene expression is still unclear. In this report, we define a STAT4-dependent sequence of events including histone H3 lysine 4 methylation, Jmjd3 association with STAT4 target loci, and a Jmjd3-dependent decrease in histone H3 lysine 27 trimethylation and DNA methyltransferase (Dnmt) 3a association with STAT4 target loci. Dnmt3a has an obligate role in repressing Th1 gene expression, and in Th1 cultures deficient in both STAT4 and Dnmt3a, there is recovery in the expression of a subset of Th1 genes that is sufficient to increase IFN-γ production. Moreover, although STAT4-deficient mice are protected from the development of experimental autoimmune encephalomyelitis, mice deficient in STAT4 and conditionally deficient in Dnmt3a in T cells develop paralysis. Th1 genes that are derepressed in the absence of Dnmt3a have greater induction after the ectopic expression of the Th1-associated transcription factors T-bet and Hlx1. Together, these data demonstrate that STAT4 and Dnmt3a play opposing roles in regulating Th1 gene expression, and that one mechanism for STAT4-dependent gene programming is in establishing a derepressed genetic state susceptible to transactivation by additional fate-determining transcription factors.

STAT4 deficiency protects against neointima formation following arterial injury in mice.

Signal transducer and activator of transcription 4 (STAT4) has been associated with susceptibility to autoimmune diseases. Intriguingly, we previously reported that STAT4 might play a critical role in vascular smooth muscle cell (VSMC) proliferation. The present study therefore investigated the impact of STAT4 on VSMC migration, apoptosis and neointimal hyperplasia postinjury, as well as the underlying mechanisms. Guide-wire injury was associated with development of intimal neointima, STAT4 and phosphorylated STAT4 (p-STAT4) expressions were apparently up-regulated in the injured arteries. Neointima was greatly blocked in STAT4 knockout (KO) mice compared with wild type (WT) mice. A marked loss of inflammatory cells was identified in the vasculature postinjury in STAT4 KO mice. VSMC apoptosis was enhanced in the vasculature postinjury in STAT4 KO mice compared with WT mice. Cultured primary STAT4 KO VSMCs displayed reduced migration in comparison with WT controls. Mechanically, the deletion of STAT4 potently decreased the level of MCP-1, and its downstream targets MMP1 and MMP2. The effect of STAT4 on VSMC apoptosis was mainly mediated by the activation of the mitochondrial apoptotic pathway, as manifested by increased cytochrome c release and the activation of caspase-3. STAT4 therefore represents a promising molecular target to limit restenosis after artery intervention.

Twist1 regulates Ifng expression in Th1 cells by interfering with Runx3 function.

A transcription factor network that includes STAT4, T-bet, and Runx3 promotes the differentiation of Th1 cells and inflammatory immune responses. How additional transcription factors regulate the function of Th1 cells has not been defined. In this study we show that the negative regulatory factor Twist1 decreases expression of T-bet, Runx3, and IL-12Rß2 as it inhibits IFN-γ production. Ectopic expression of Runx3, but not T-bet or IL-12Rß2, compensates for the effects of Twist1 on IFN-γ production, and Twist1 regulation of Ifng depends on complex formation with Runx3. Twist1 decreases Runx3 and T-bet binding at the Ifng locus, and it decreases chromatin looping within the Ifng locus. These data define an IL-12/STAT4-induced negative regulatory loop that impacts multiple components of the Th1 transcriptional network and provide further insight into regulation of Th1 differentiation.

High basal STAT4 balanced by STAT1 induction to control type 1 interferon effects in natural killer cells.

The best-characterized type 1 interferon (IFN) signaling pathway depends on signal transducer and activator of transcription 1 (STAT1) and STAT2. The cytokines can, however, conditionally activate all STATs. Regulation of their access to particular signaling pathways is poorly understood. STAT4 is important for IFN-gamma induction, and NK cells are major producers of this cytokine. We report that NK cells have high basal STAT4 levels and sensitivity to type 1 IFN-mediated STAT4 activation for IFN-gamma production. Increases in STAT1, driven during viral infection by either type 1 IFN or IFN-gamma, are associated with decreased STAT4 access. Both STAT1 and STAT2 are important for antiviral defense, but STAT1 has a unique role in protecting against sustained NK cell IFN-gamma production and resulting disease. The regulation occurs with an NK cell type 1 IFN receptor switch from a STAT4 to a STAT1 association. Thus, a fundamental characteristic of NK cells is high STAT4 bound to the type 1 IFN receptor. The conditions of infection result in STAT1 induction with displacement of STAT4. These studies elucidate the critical role of STAT4 levels in predisposing selection of specific signaling pathways, define the biological importance of regulation within particular cell lineages, and provide mechanistic insights for how this is accomplished in vivo.

Helper T cell IL-2 production is limited by negative feedback and STAT-dependent cytokine signals.

Although required for many fundamental immune processes, ranging from self-tolerance to pathogen immunity, interleukin (IL)-2 production is transient, and the mechanisms underlying this brevity remain unclear. These studies reveal that helper T cell IL-2 production is limited by a classic negative feedback loop that functions autonomously or in collaboration with other common gamma chain (IL-4 and IL-7) and IL-6/IL-12 family cytokines (IL-12 and IL-27). Consistent with this model for cytokine-dependent regulation, they also demonstrate that the inhibitory effect can be mediated by several signal transducer and activator of transcription (STAT) family transcription factors, namely STAT5, STAT4, and STAT6. Collectively, these findings establish that IL-2 production is limited by a network of autocrine and paracrine signals that are readily available during acute inflammatory responses and, thus, provide a cellular and molecular basis for its transient pattern of expression.

Acquired STAT4 deficiency as a consequence of cancer chemotherapy.

Signal Transducer and Activator of Transcription 4 (STAT4) is a transcription factor that is activated by IL-12 signaling and promotes Th1-cell differentiation and IFN-γ production. Defective IFN-γ production because of STAT4 mRNA and protein deficiency occurs after autologous stem cell transplantation for lymphoma. In the present study, we investigated the mechanisms of STAT4 deficiency in lymphoma patients. The tumor-bearing state is not responsible, because STAT4 levels were not significantly different in PBMCs obtained from healthy control subjects compared with those from lymphoma patients before treatment. STAT4 protein levels were significantly decreased in PBMCs and T cells obtained from lymphoma patients after standard-dose chemotherapy. Furthermore, treatment of control PBMC cultures or a natural killer cell line with chemotherapy drugs in vitro also resulted in reduced STAT4 protein and diminished, IL-12-induced IFN-γ production. Translation of STAT4 protein was not impaired in chemotherapy-treated cells, whereas the STAT4 protein half-life was significantly reduced. Chemotherapy drugs promoted the ubiquitination and proteasomal degradation of STAT4. Treatment with the proteasome inhibitor bortezomib reversed chemotherapy-induced STAT4 deficiency and defective IFN-γ production. We conclude that acquired STAT4 deficiency in lymphoma patients is a consequence of treatment with chemotherapy, results that have important implications for the design of optimal immunotherapy for lymphoma.

Targeting transcription factor Stat4 uncovers a role for interleukin-18 in the pathogenesis of severe lupus nephritis in mice.

Polymorphisms in the transcription factor Stat4 gene have been implicated as risk factors for systemic lupus erythematosus. Although some polymorphisms have a strong association with autoantibodies and nephritis, their impact on pathophysiology is still unknown. To explore this further we used signal transducers and activators of transcription 4 (Stat4) knockout MRL/MpJ-Fas(lpr)/Fas(lpr) (MRL-Fas(lpr)) mice and found that they did not differ in survival or renal function from Stat4-intact MRL-Fas(lpr) mice. Circulating interleukin (IL)-18 levels, however, were elevated in Stat4-deficient compared to Stat4-intact mice, suggesting that this interleukin might contribute to the progression of lupus nephritis independent of Stat4. In a second approach, Stat4 antisense or missense oligonucleotides or vehicle were given to MRL-Fas(lpr) mice with advanced nephritis. Each of these treatments temporarily ameliorated disease, although IL-18 was increased in each setting. Based on these findings, studies using gene transfer to overexpress IL-18 in MRL-Fas(lpr) and IL-12p40/IL-23 knockout MRL-Fas(lpr) mice reveal a critical role for IL-18 in mediating disease. Thus, the Stat4 and IL-12 (an activator of Stat4)-independent factor, IL-18, can drive autoimmune lupus nephritis in MRL-Fas(lpr) mice. Temporarily blocking Stat4 during advanced nephritis ameliorates disease, suggesting a time-dependent compensatory proinflammatory mechanism.

Impaired development of human Th1 cells in patients with deficient expression of STAT4.

IL-12 activates STAT4, which is a critical regulator of inflammation and T helper type I (Th1) lineage development in murine systems. The requirement for STAT4 in the generation of human Th1 cells has not been examined thoroughly. Compared with control Th1 cultures, expression of the Th1 genes IFNgamma, IL-12Rbeta2, and TNFalpha is greatly reduced in Th1 cultures of CD4 T cells isolated from lymphoma patients after autologous stem cell transplantation who have acquired STAT4 deficiency. Moreover, IL-4 and IL-5 production is increased in patient Th1 cultures though there are no defects in the development of Th2 cells. Reconstitution of STAT4 in patient T cells allowed recovery of IFNgamma and IL-12Rbeta2 expression, whereas ectopic expression of IL-12Rbeta2 did not rescue STAT4 expression, and increased IFNgamma production only to levels intermediate between control and patient samples. These results demonstrate that, as in murine systems, STAT4 is required for optimal human Th1 lineage development.

Attenuated expression of tenascin-C in ovalbumin-challenged STAT4-/- mice.

BACKGROUND: Asthma leads to structural changes in the airways, including the modification of extracellular matrix proteins such as tenascin-C. The role of tenascin-C is unclear, but it might act as an early initiator of airway wall remodelling, as its expression is increased in the mouse and human airways during allergic inflammation. In this study, we examined whether Th1 or Th2 cells are important regulators of tenascin-C in experimental allergic asthma utilizing mice with impaired Th1 (STAT4-/-) or Th2 (STAT6-/-) immunity. METHODS: Balb/c wildtype (WT), STAT4-/- and STAT6-/- mice were sensitized with intraperitoneally injected ovalbumin (OVA) followed by OVA or PBS airway challenge. Airway hyperreactivity (AHR) was measured and samples were collected. Real time PCR and immunohistochemistry were used to study cytokines and differences in the expression of tenascin-C. Tenascin-C expression was measured in human fibroblasts after treatment with TNF-α and IFN-γ in vitro. RESULTS: OVA-challenged WT mice showed allergic inflammation and AHR in the airways along with increased expression of TNF-α, IFN-γ, IL-4 and tenascin-C in the lungs. OVA-challenged STAT4-/- mice exhibited elevated AHR and pulmonary eosinophilia. The mRNA expression of TNF-α and IFN-γ was low, but the expression of IL-4 was significantly elevated in these mice. OVA-challenged STAT6-/- mice had neither AHR nor pulmonary eosinophilia, but had increased expression of mRNA for TNF-α, IFN-γ and IL-4. The expression of tenascin-C in the lungs of OVA-challenged STAT4-/- mice was weaker than in those of OVA-challenged WT and STAT6-/- mice suggesting that TNF-α and IFN-γ may regulate tenascin-C expression in vivo. The stimulation of human fibroblasts with TNF-α and IFN-γ induced the expression of tenascin-C confirming our in vivo findings. CONCLUSIONS: Expression of tenascin-C is significantly attenuated in the airways of STAT4-/- mice, which may be due to the impaired secretion of TNF-α and IFN-γ in these mice.

CD4+ T-cell-mediated anti-tumor immunity can be uncoupled from autoimmunity via the STAT4/STAT6 signaling axis.

Previous reports have suggested that autoimmune sequelae may be an unavoidable consequence of successful immunization against tumor-associated antigens, which are typically non-mutated self-antigens. Using a melanoma model, we demonstrated that CD4(+) T-cell-mediated anti-tumor immunity and autoimmunity could be separated by modulating the STAT4/STAT6 signaling axis. Our results have revealed an unexpected dichotomy in the effector phase following cancer vaccination where anti-tumor immunity is mediated via a STAT6 and IL-4-dependent pathway, whereas autoimmune pathology is mediated via STAT4 through a mechanism that relies partially on IFN-gamma. Our results offer a possibility to elicit specific anti-tumor responses without triggering unwanted tissue autoimmune diseases.

Involvement of STAT4 in IgG subtype switching and ocular HSV-1 replication in mice.

PURPOSE: To assess the relative impact of elevated T-helper 2 (T(H)2)- and reduced T-Helper 1 (T(H)1)-dependent immune responses on ocular herpes simplex virus type 1 (HSV-1) infection. METHODS: Signal transducer and activator of transcription protein 4 knockout mice (BALB/c-STAT4(-/-)) and wild-type BALB/c control mice were immunized with avirulent HSV-1 strain KOS or were mock-immunized. Three weeks after the third immunization, neutralizing antibody titers were determined by plaque reduction assays. Following ocular infection with virulent HSV-1 strain McKrae, viral replication in the eye, blepharitis, corneal scarring (CS), survival, and immunoglobulin (Ig) isotypes in sera were determined. RESULTS: Vaccinated STAT4(-/-) and BALB/c mice contained significant and similar neutralizing antibody titers and were completely protected against HSV-1-induced death and CS. In contrast to vaccinated STAT4(-/-) mice, mock-vaccinated STAT4(-/-) mice had higher ocular HSV-1 titers than mock-vaccinated BALB/c mice on days 2-3 post-ocular infection. There were also significant differences in the levels of IgG2a, IgG2b, and IgG3 in the sera of STAT4(-/-) mice when compared to the control BALB/c mice. CONCLUSIONS: These results suggest that the absence of T(H)1 cytokine responses did alter protection against viral replication and IgG isotypes but not eye disease or survival.

STAT4 isoforms differentially regulate Th1 cytokine production and the severity of inflammatory bowel disease.

STAT4, a critical regulator of inflammation in vivo, can be expressed as two alternative splice forms, a full-length STAT4alpha, and a STAT4beta isoform lacking a C-terminal transactivation domain. Each isoform is sufficient to program Th1 development through both common and distinct subsets of target genes. However, the ability of these isoforms to mediate inflammation in vivo has not been examined. Using a model of colitis that develops following transfer of CD4(+) CD45RB(high) T cells expressing either the STAT4alpha or STAT4beta isoform into SCID mice, we determined that although both isoforms mediate inflammation and weight loss, STAT4beta promotes greater colonic inflammation and tissue destruction. This correlates with STAT4 isoform-dependent expression of TNF-alpha and GM-CSF in vitro and in vivo, but not Th1 expression of IFN-gamma or Th17 expression of IL-17, which were similar in STAT4alpha- and STAT4beta-expressing T cells. Thus, higher expression of a subset of inflammatory cytokines from STAT4beta-expressing T cells correlates with the ability of STAT4beta-expressing T cells to mediate more severe inflammatory disease.

Immune Suppression Mediated by STAT4 Deficiency Promotes Lymphatic Metastasis in HNSCC.

Head and neck squamous cell carcinoma (HNSCC) is a prevalent form of cancer with 5-years survival rates around 57%, and metastasis is a leading cause of mortality. Host-derived immunological factors that affect HNSCC tumor development and metastasis are not completely understood. We investigated the role of host-derived signal transducer and activator of transcription 4 (STAT4) during experimental HNSCC using an aggressive and metastatic HNSCC cell line, LY2, which was orthotopically injected into the buccal sulcus of wild type (WT) and STAT4 deficient (Stat4-/-) BALB/c mice. Necropsies performed at terminal sacrifice revealed that Stat4-/- mice displayed comparable primary tumor growth to the WT mice. However, the rate and extent of lymph node and lung metastasis among Stat4-/- mice was significantly higher. Downstream analyses performed on primary tumors, draining lymph nodes, spleens and bone marrow revealed significant upregulation of lymphocytic immunosuppressive biomarkers as well as an accumulation of granulocytic MDSC subpopulations in draining lymph nodes of metastatic Stat4-/- mice. Further, we observed a significant decrease in TH1, TH17, and cytotoxic activity in tumor bearing Stat4-/- compared to WT mice. Our results demonstrate that STAT4 mediates resistance to HNSCC metastasis, and activation of STAT4 could potentially mitigate lymphatic metastasis in HNSCC patients.

Signal transducer and activator of transcription 4 (STAT4), but not IL-12 contributes to Pseudomonas aeruginosa-induced lung inflammation in mice.

Pseudomonas aeruginosa is a major opportunistic pathogen in immune-compromised individuals and cystic fibrosis patients. This organism stimulates a complex inflammatory response in the lung, including production of various cytokines and chemokines. The specific contribution of these mediators in the host defense against this bacterium has yet to be fully characterized. Interleukin-12 (IL-12) is commonly known as a master regulator of innate and adaptive immunity. IL-12 induces its biological effects through its associated intracellular signaling molecule, the signal transducer and activator of transcription 4 (STAT4). To examine a specific role of IL-12 and STAT4 in P. aeruginosa lung infection in mice, STAT4-deficient (STAT4-/-) and IL-12 p40-deficient (IL-12 p40-/-) mice were infected with P. aeruginosa intranasally. Interestingly, STAT4-/- mice, but not IL-12 p40-/- mice after 24h infection showed impaired production of the pro-inflammatory cytokines tumor necrosis factor, interleukin-1beta, and macrophage-inflammatory protein-2. However, neither STAT4 nor IL-12 p40 deficiency significantly affected INFgamma production or bacterial clearance compared to wild-type mice. Similarly, neutrophil recruitment was not affected in the STAT4-/- and IL-12 p40-/- mice. These results suggest that STAT4 contributes to P. aeruginosa-induced inflammation, but it is not essential for bacterial clearance. Although IL-12 is essential for the host defense against various pathogens, this cytokine is likely not a major player in the host response to P. aeruginosa lung infection.

Contribution of IL-12R mediated feedback loop to Th1 cell differentiation.

T helper 1 (Th1) cell fate is induced by overlapping signaling pathways, whose kinetic principles and regulatory motifs are largely unknown. We identified a simple positive feedback loop in the STAT4 signaling pathway, whereby activation by IL-12 leads to the increased expression in IL-12 receptor. A computational analysis shows that this feedback loop synergizes with the one mediated by the IFN-gamma secreted by differentiating cells, when the induction of Th1 cell fate is weak. Positive feedback loops are often utilized to enhance phenotypic differentiation. This effect was confirmed by experiments showing that stochastic fluctuations in the expression of IL-12 receptor gene were amplified, leading to two discrete levels of expression in a cell population.