CtpB Facilitates Mycobacterium tuberculosis Growth in Copper-Limited Niches.

Copper is required for aerobic respiration by Mycobacterium tuberculosis and its human host, but this essential element is toxic in abundance. Copper nutritional immunity refers to host processes that modulate levels of free copper to alternately starve and intoxicate invading microbes. Bacteria engulfed by macrophages are initially contained within copper-limited phagosomes, which fuse with ATP7A vesicles that pump in toxic levels of copper. In this report, we examine how CtpB, a P-type ATPase in M. tuberculosis, aids in response to nutritional immunity. In vitro, the induced expression of ctpB in copper-replete medium inhibited mycobacterial growth, while deletion of the gene impaired growth only in copper-starved medium and within copper-limited host cells, suggesting a role for CtpB in copper acquisition or export to the copper-dependent respiration supercomplex. Unexpectedly, the absence of ctpB resulted in hypervirulence in the DBA/2 mouse infection model. As ctpB null strains exhibit diminished growth only in copper-starved conditions, reduced copper transport may have enabled the mutant to acquire a "Goldilocks" amount of the metal during transit through copper-intoxicating environments within this model system. This work reveals CtpB as a component of the M. tuberculosis toolkit to counter host nutritional immunity and underscores the importance of elucidating copper-uptake mechanisms in pathogenic mycobacteria.

The immunity-related GTPase Irgm1 promotes the expansion of activated CD4+ T cell populations by preventing interferon-gamma-induced cell death.

Mice deficient in the interferon-gamma (IFN-gamma)-inducible, immunity-related GTPase Irgm1 have defective host resistance to a variety of intracellular pathogens. This greater susceptibility to infection is associated with impaired IFN-gamma-dependent macrophage microbicidal activity in vitro. Here we show that Irgm1 also regulated the survival of mature effector CD4(+) T lymphocytes by protecting them from IFN-gamma-induced autophagic cell death. Mice deficient in both IFN-gamma and Irgm1 were 'rescued' from the lymphocyte depletion and greater mortality that occurs in mice singly deficient in Irgm1 after mycobacterial infection. Our studies identify a feedback mechanism in the T helper type 1 response that limits the detrimental effects of IFN-gamma on effector T lymphocyte survival while promoting the antimicrobial functions of IFN-gamma.

Diverse targets of the transcription factor STAT3 contribute to T cell pathogenicity and homeostasis.

STAT3, an essential transcription factor with pleiotropic functions, plays critical roles in the pathogenesis of autoimmunity. Despite recent data linking STAT3 with inflammatory bowel disease, exactly how it contributes to chronic intestinal inflammation is not known. Using a T cell transfer model of colitis, we found that STAT3 expression in T cells was essential for the induction of both colitis and systemic inflammation. STAT3 was critical in modulating the balance of T helper 17 (Th17) and regulatory T (Treg) cells, as well as in promoting CD4(+) T cell proliferation. We used chromatin immunoprecipitation and massive parallel sequencing (ChIP-Seq) to define the genome-wide targets of STAT3 in CD4(+) T cells. We found that STAT3 bound to multiple genes involved in Th17 cell differentiation, cell activation, proliferation, and survival, regulating both expression and epigenetic modifications. Thus, STAT3 orchestrates multiple critical aspects of T cell function in inflammation and homeostasis.

Discrete roles of STAT4 and STAT6 transcription factors in tuning epigenetic modifications and transcription during T helper cell differentiation.

Signal transducer and activator of transcription 4 (STAT4) and STAT6 are key factors in the specification of helper T cells; however, their direct roles in driving differentiation are not well understood. Using chromatin immunoprecipitation and massive parallel sequencing, we quantitated the full complement of STAT-bound genes, concurrently assessing global STAT-dependent epigenetic modifications and gene transcription by using cells from cognate STAT-deficient mice. STAT4 and STAT6 each bound over 4000 genes with distinct binding motifs. Both played critical roles in maintaining chromatin configuration and transcription of a core subset of genes through the combination of different epigenetic patterns. Globally, STAT4 had a more dominant role in promoting active epigenetic marks, whereas STAT6 had a more prominent role in antagonizing repressive marks. Clusters of genes negatively regulated by STATs were also identified, highlighting previously unappreciated repressive roles of STATs. Therefore, STAT4 and STAT6 play wide regulatory roles in T helper cell specification.

RGC-32 (Response Gene to Complement 32) Deficiency Protects Endothelial Cells From Inflammation and Attenuates Atherosclerosis.

OBJECTIVE: The objective of this study is to determine the role and underlying mechanisms of RGC-32 (response gene to complement 32 protein) in atherogenesis. APPROACH AND RESULTS: RGC-32 was mainly expressed in endothelial cells of atherosclerotic lesions in both ApoE-/- (apolipoprotein E deficient) mice and human patients. Rgc-32 deficiency (Rgc32-/-) attenuated the high-fat diet-induced and spontaneously developed atherosclerotic lesions in ApoE-/- mice without affecting serum cholesterol concentration. Rgc32-/- seemed to decrease the macrophage content without altering collagen and smooth muscle contents or lesional macrophage proliferation in the lesions. Transplantation of WT (wild type) mouse bone marrow to lethally irradiated Rgc32-/- mice did not alter Rgc32-/--caused reduction of lesion formation and macrophage accumulation, suggesting that RGC-32 in resident vascular cells, but not the macrophages, plays a critical role in the atherogenesis. Of importance, Rgc32-/- decreased the expression of ICAM-1 (intercellular adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule-1) in endothelial cells both in vivo and in vitro, resulting in a decrease in TNF-α (tumor necrosis factor-α)-induced monocyte-endothelial cell interaction. Mechanistically, RGC-32 mediated the ICAM-1 and VCAM-1 expression, at least partially, through NF (nuclear factor)-κB signaling pathway. RGC-32 directly interacted with NF-κB and facilitated its nuclear translocation and enhanced TNF-α-induced NF-κB binding to ICAM-1 and VCAM-1 promoters. CONCLUSIONS: RGC-32 mediates atherogenesis by facilitating monocyte-endothelial cell interaction via the induction of endothelial ICAM-1 and VCAM-1 expression, at least partially, through NF-κB signaling pathway.

Lyn kinase controls basophil GATA-3 transcription factor expression and induction of Th2 cell differentiation.

T helper 1 (Th1)-Th2 cell balance is key to host defense and its dysregulation has pathophysiological consequences. Basophils are important in Th2 cell differentiation. However, the factors controlling the onset and extent of basophil-mediated Th2 cell differentiation are unknown. Here, we demonstrate that Lyn kinase dampened basophil expression of the transcription factor GATA-3 and the initiation and extent of Th2 cell differentiation. Lyn-deficient mice had a marked basophilia, a constitutive Th2 cell skewing that was exacerbated upon in vivo challenge of basophils, produced antibodies to a normally inert antigen, and failed to appropriately respond to a Th1 cell-inducing pathogen. The Th2 cell skewing was dependent on basophils, immunoglobulin E, and interleukin-4, but was independent of mast cells. Our findings demonstrate that basophil-expressed Lyn kinase exerts regulatory control on Th2 cell differentiation and function.

Global mapping of H3K4me3 and H3K27me3 reveals specificity and plasticity in lineage fate determination of differentiating CD4+ T cells.

Multipotential naive CD4(+) T cells differentiate into distinct lineages including T helper 1 (Th1), Th2, Th17, and inducible T regulatory (iTreg) cells. The remarkable diversity of CD4(+) T cells begs the question whether the observed changes reflect terminal differentiation with heritable epigenetic modifications or plasticity in T cell responses. We generated genome-wide histone H3 lysine 4 (H3K4) and lysine 27 (H3K27) trimethylation maps in naive, Th1, Th2, Th17, iTreg, and natural Treg (nTreg) cells. We found that although modifications of signature-cytokine genes (Ifng, Il4, and Il17) partially conform to the expectation of lineage commitment, genes encoding transcription factors like Tbx21 exhibit a broad spectrum of epigenetic states, consistent with our demonstration of T-bet and interferon-gamma induction in nTreg cells. Our data suggest an epigenetic mechanism underlying the specificity and plasticity of effector and regulatory T cells and also provide a framework for understanding complexity of CD4(+) T helper cell differentiation.

Inflammasome-mediated disease animal models reveal roles for innate but not adaptive immunity.

NLRP3 nucleates the inflammasome, a protein complex responsible for cleavage of prointerleukin-1beta (IL-1beta) to its active form. Mutations in the NLRP3 gene cause the autoinflammatory disease spectrum cryopyrin-associated periodic syndromes (CAPS). The central role of IL-1beta in CAPS is supported by the response to IL-1-targeted therapy. We developed two Nlrp3 mutant knockin mouse strains to model CAPS to examine the role of other inflammatory mediators and adaptive immune responses in an innate immune-driven disease. These mice had systemic inflammation and poor growth, similar to some human CAPS patients, and demonstrated early mortality, primarily mediated by myeloid cells. Mating these mutant mice to various gene mutant backgrounds showed that the mouse disease phenotype required an intact inflammasome, was only partially dependent on IL-1beta, and was independent of T cells. These data suggest that CAPS are true inflammasome-mediated diseases and provide insight for more common inflammatory disorders.

Tumor Progression Locus 2 (Tpl2) Activates the Mammalian Target of Rapamycin (mTOR) Pathway, Inhibits Forkhead Box P3 (FoxP3) Expression, and Limits Regulatory T Cell (Treg) Immunosuppressive Functions.

The serine/threonine kinase tumor progression locus 2 (Tpl2, also known as Map3k8/Cot) is a potent inflammatory mediator that drives the production of TNFα, IL-1ß, and IFNγ. We previously demonstrated that Tpl2 regulates T cell receptor (TCR) signaling and modulates T helper cell differentiation. However, very little is known about how Tpl2 modulates the development of regulatory T cells (Tregs). Tregs are a specialized subset of T cells that express FoxP3 and possess immunosuppressive properties to limit excess inflammation. Because of the documented role of Tpl2 in promoting inflammation, we hypothesized that Tpl2 antagonizes Treg development and immunosuppressive function. Here we demonstrate that Tpl2 constrains the development of inducible Tregs. Tpl2(-/-) naïve CD4(+) T cells preferentially develop into FoxP3(+) inducible Tregs in vitro as well as in vivo in a murine model of ovalbumin (OVA)-induced systemic tolerance. Treg biasing of Tpl2(-/-) T cells depended on TCR signal strength and corresponded with reduced activation of the mammalian target of rapamycin (mTOR) pathway. Importantly, Tpl2(-/-) Tregs have basally increased expression of FoxP3 and immunosuppressive molecules, IL-10 and cytotoxic T lymphocyte-associated protein 4 (CTLA-4). Furthermore, they were more immunosuppressive in vivo in a T cell transfer model of colitis, as evidenced by reduced effector T cell accumulation, systemic production of inflammatory cytokines, and colonic inflammation. These results demonstrate that Tpl2 promotes inflammation in part by constraining FoxP3 expression and Treg immunosuppressive functions. Overall, these findings suggest that Tpl2 inhibition could be used to preferentially drive Treg induction and thereby limit inflammation in a variety of autoimmune diseases.

Tpl2 Promotes Innate Cell Recruitment and Effector T Cell Differentiation To Limit Citrobacter rodentium Burden and Dissemination.

Tumor progression locus 2 (Tpl2) is a serine-threonine kinase that regulates Th1 differentiation, secretion of the inflammatory cytokine gamma interferon (IFN-γ), and host defense against the intracellular pathogens Toxoplasma gondii, Listeria monocytogenes, and Mycobacterium tuberculosis However, relatively little is known about the contribution of Tpl2 to Th17 differentiation and immune cell function during infection with an extracellular pathogen. The goal of this study was to determine whether Tpl2 influences the immune response generated to the extracellular bacterium Citrobacter rodentium, which induces a mixed Th1 and Th17 response. During peak infection with C. rodentium, Tpl2-/- mice experienced greater bacterial burdens with evidence of dissemination to the liver and spleen but ultimately cleared the bacteria within 3 weeks postinfection, similar to the findings for wild-type mice. Tpl2-/- mice also recruited fewer neutrophils and monocytes to the colon during peak infection, which correlated with increased bacterial burdens. In mixed bone marrow chimeras, Tpl2 was shown to play a T cell-intrinsic role in promoting both IFN-γ and interleukin-17A production during infection with C. rodentium However, upon CD4 T cell transfer into Rag-/- mice, Tpl2-/- CD4 T cells were as protective as wild-type CD4 T cells against the dissemination of bacteria and mortality. These data indicate that the enhanced bacterial burdens in Tpl2-/- mice are not caused primarily by impairments in CD4 T cell function but result from defects in innate immune cell recruitment and function.

Tumor Progression Locus 2 Promotes Induction of IFNλ, Interferon Stimulated Genes and Antigen-Specific CD8+ T Cell Responses and Protects against Influenza Virus.

Mitogen-activated protein kinase (MAP) cascades are important in antiviral immunity through their regulation of interferon (IFN) production as well as virus replication. Although the serine-threonine MAP kinase tumor progression locus 2 (Tpl2/MAP3K8) has been implicated as a key regulator of Type I (IFNα/ß) and Type II (IFNγ) IFNs, remarkably little is known about how Tpl2 might contribute to host defense against viruses. Herein, we investigated the role of Tpl2 in antiviral immune responses against influenza virus. We demonstrate that Tpl2 is an integral component of multiple virus sensing pathways, differentially regulating the induction of IFNα/ß and IFNλ in a cell-type specific manner. Although Tpl2 is important in the regulation of both IFNα/ß and IFNλ, only IFNλ required Tpl2 for its induction during influenza virus infection both in vitro and in vivo. Further studies revealed an unanticipated function for Tpl2 in transducing Type I IFN signals and promoting expression of interferon-stimulated genes (ISGs). Importantly, Tpl2 signaling in nonhematopoietic cells is necessary to limit early virus replication. In addition to early innate alterations, impaired expansion of virus-specific CD8+ T cells accompanied delayed viral clearance in Tpl2-/- mice at late time points. Consistent with its critical role in facilitating both innate and adaptive antiviral responses, Tpl2 is required for restricting morbidity and mortality associated with influenza virus infection. Collectively, these findings establish an essential role for Tpl2 in antiviral host defense mechanisms.

Proprotein convertase FURIN constrains Th2 differentiation and is critical for host resistance against Toxoplasma gondii.

The proprotein convertase subtilisin/kexin enzymes proteolytically convert immature proproteins into bioactive molecules, and thereby they serve as key regulators of cellular homeostasis. The archetype proprotein convertase subtilisin/kexin, FURIN, is a direct target gene of the IL-12/STAT4 pathway and it is upregulated in Th1 cells. We have previously demonstrated that FURIN expression in T cells critically regulates the maintenance of peripheral immune tolerance and the functional maturation of pro-TGF-ß1 in vivo, but FURIN's role in cell-mediated immunity and Th polarization has remained elusive. In this article, we show that T cell-expressed FURIN is essential for host resistance against a prototypic Th1 pathogen, Toxoplasma gondii, and for the generation of pathogen-specific Th1 lymphocytes, including Th1-IL-10 cells. FURIN-deficient Th cells instead show elevated expression of IL-4R subunit α on cell surface, sensitized IL-4/STAT6 signaling, and a propensity to polarize toward the Th2 phenotype. By exploring FURIN-interacting proteins in Jurkat T cells with Strep-Tag purification and mass spectrometry, we further identify an association with a cytoskeleton modifying Ras-related C3 botulinum toxin substrate/dedicator of cytokinesis 2 protein complex and unravel that FURIN promotes F-actin polymerization, which has previously been shown to downregulate IL-4R subunit α cell surface expression and promote Th1 responses. In conclusion, our results demonstrate that in addition to peripheral immune tolerance, T cell-expressed FURIN is also a central regulator of cell-mediated immunity and Th1/2 cell balance.

Generation of pathogenic T(H)17 cells in the absence of TGF-ß signalling.

CD4(+) T-helper cells that selectively produce interleukin (IL)-17 (T(H)17), are critical for host defence and autoimmunity. Although crucial for T(H)17 cells in vivo, IL-23 has been thought to be incapable of driving initial differentiation. Rather, IL-6 and transforming growth factor (TGF)-ß1 have been proposed to be the factors responsible for initiating specification. Here we show that T(H)17 differentiation can occur in the absence of TGF-ß signalling. Neither IL-6 nor IL-23 alone efficiently generated T(H)17 cells; however, these cytokines in combination with IL-1ß effectively induced IL-17 production in naive precursors, independently of TGF-ß. Epigenetic modification of the Il17a, Il17f and Rorc promoters proceeded without TGF-ß1, allowing the generation of cells that co-expressed RORγt (encoded by Rorc) and T-bet. T-bet(+)RORγt(+) T(H)17 cells are generated in vivo during experimental allergic encephalomyelitis, and adoptively transferred T(H)17 cells generated with IL-23 without TGF-ß1 were pathogenic in this disease model. These data indicate an alternative mode for T(H)17 differentiation. Consistent with genetic data linking IL23R with autoimmunity, our findings re-emphasize the importance of IL-23 and therefore may have therapeutic implications.

Tumor progression locus 2-dependent oxidative burst drives phosphorylation of extracellular signal-regulated kinase during TLR3 and 9 signaling.

Signal transduction via NFκB and MAP kinase cascades is a universal response initiated upon pathogen recognition by Toll-like receptors (TLRs). How activation of these divergent signaling pathways is integrated to dictate distinct immune responses to diverse pathogens is still incompletely understood. Herein, contrary to current perception, we demonstrate that a signaling pathway defined by the inhibitor of κB kinase ß (IKKß), MAP3 kinase tumor progression locus 2 (Tpl2/MAP3K8), and MAP kinase ERK is differentially activated by TLRs. TLRs 2, 4, and 7 directly activate this inflammatory axis, inducing immediate ERK phosphorylation and early TNFα secretion. In addition to TLR adaptor proteins, IKKß-Tpl2-ERK activation by TLR4 is regulated by the TLR4 co-receptor CD14 and the tyrosine kinase Syk. Signals from TLRs 3 and 9 do not initiate early activation of IKKß-Tpl2-ERK pathway but instead induce delayed, NADPH-oxidase-dependent ERK phosphorylation and TNFα secretion via autocrine reactive oxygen species signaling. Unexpectedly, Tpl2 is an essential regulator of ROS production during TLR signaling. Overall, our study reveals distinct mechanisms activating a common inflammatory signaling cascade and delineates differences in MyD88-dependent signaling between endosomal TLRs 7 and 9. These findings further confirm the importance of Tpl2 in innate host defense mechanisms and also enhance our understanding of how the immune system tailors pathogen-specific gene expression patterns.

Tumor progression locus 2 (Tpl2) kinase promotes chemokine receptor expression and macrophage migration during acute inflammation.

In autoimmune diseases, the accumulation of activated leukocytes correlates with inflammation and disease progression, and, therefore, the disruption of leukocyte trafficking is an active area of research. The serine/threonine protein kinase Tpl2 (MAP3K8) regulates leukocyte inflammatory responses and is also being investigated for therapeutic inhibition during autoimmunity. Here we addressed the contribution of Tpl2 to the regulation of macrophage chemokine receptor expression and migration in vivo using a mouse model of Tpl2 ablation. LPS stimulation of bone marrow-derived macrophages induced early CCR1 chemokine receptor expression but repressed CCR2 and CCR5 expression. Notably, early induction of CCR1 expression by LPS was dependent upon a signaling pathway involving Tpl2, PI3K, and ERK. On the contrary, Tpl2 was required to maintain the basal expression of CCR2 and CCR5 as well as to stabilize CCR5 mRNA expression. Consistent with impairments in chemokine receptor expression, tpl2(-/-) macrophages were defective in trafficking to the peritoneal cavity following thioglycollate-induced inflammation. Overall, this study demonstrates a Tpl2-dependent mechanism for macrophage expression of select chemokine receptors and provides further insight into how Tpl2 inhibition may be used therapeutically to disrupt inflammatory networks in vivo.

Regulating type 1 IFN effects in CD8 T cells during viral infections: changing STAT4 and STAT1 expression for function.

Type 1 IFNs can conditionally activate all of the signal transducers and activators of transcription molecules (STATs), including STAT4. The best-characterized signaling pathways use STAT1, however, and type 1 IFN inhibition of cell proliferation is STAT1 dependent. We report that type 1 IFNs can basally stimulate STAT1- and STAT4-dependent effects in CD8 T cells, but that CD8 T cells responding to infections of mice with lymphocytic choriomenigitis virus have elevated STAT4 and lower STAT1 expression with significant consequences for modifying the effects of type 1 IFN exposure. The phenotype was associated with preferential type 1 IFN activation of STAT4 compared with STAT1. Stimulation through the TCR induced elevated STAT4 expression, and STAT4 was required for peak expansion of antigen-specific CD8 T cells, low STAT1 levels, and resistance to type 1 IFN-mediated inhibition of proliferation. Thus, a mechanism is discovered for regulating the consequences of type 1 IFN exposure in CD8 T cells, with STAT4 acting as a key molecule in driving optimal antigen-specific responses and overcoming STAT1-dependent inhibition of proliferation.

T-cell-expressed proprotein convertase furin is essential for maintenance of peripheral immune tolerance.

Furin is one of seven proprotein convertase family members that promote proteolytic maturation of proproteins. It is induced in activated T cells and is reported to process a variety of substrates including the anti-inflammatory cytokine transforming growth factor (TGF)-beta1 (refs 2-4), but the non-redundant functions of furin versus other proprotein convertases in T cells are unclear. Here we show that conditional deletion of furin in T cells allowed for normal T-cell development but impaired the function of regulatory and effector T cells, which produced less TGF-beta1. Furin-deficient T regulatory (Treg) cells were less protective in a T-cell transfer colitis model and failed to induce Foxp3 in normal T cells. Additionally, furin-deficient effector cells were inherently over-active and were resistant to suppressive activity of wild-type Treg cells. Thus, our results indicate that furin is indispensable in maintaining peripheral tolerance, which is due, at least in part, to its non-redundant, essential function in regulating TGF-beta1 production. Targeting furin has emerged as a strategy in malignant and infectious disease. Our results suggest that inhibiting furin might activate immune responses, but may result in a breakdown in peripheral tolerance.

Attenuated retinoic acid signaling is among the early responses in mouse uterus approaching embryo attachment.

The uterus is transiently receptive for embryo implantation. It remains to be understood why the uterus does not reject a semi-allogeneic embryo (to the biological mother) or an allogeneic embryo (to a surrogate) for implantation. To gain insights, we examined uterine early response genes approaching embryo attachment on day 3 post coitum (D3) at 22 hours when blue dye reaction, an indication of embryo attachment, had not manifested in mice. C57BL/6 pseudo-pregnant (control) and pregnant mouse uteri were collected on D3 at 22 hours for microarray analysis. The self-assembling-manifold (SAM) algorithm identified 21,858 unique probesets. Principal component analysis indicated a clear separation between the pseudo-pregnant and pregnant groups. There were 106 upregulated and five downregulated protein-coding genes in the pregnant uterus with fold change (fc) >1.5 and q value <5%. Gene ontology (GO) analysis of the 106 upregulated genes revealed 38 significant GO biological process (GOBP) terms (P <0.05), and 32 (84%) of them were associated with immune responses, with a dominant natural killer (NK) cell activation signature. Among the top eight upregulated protein-coding genes, Cyp26a1 inactivates retinoic acid (RA) while Lrat promotes vitamin A storage, both of which are expected to attenuate RA bioavailability; Atp6v0d2 and Gjb2 play roles in ion transport and transmembrane transport; Gzmb, Gzmc, and Il2rb are involved in immune responses; and Tdo2 is important for kynurenine pathway. Most of these genes or their related pathways have functions in immune regulations. RA signaling has been implicated in immune tolerance and immune homeostasis, and uterine NK cells have been implicated in immunotolerance at the maternal-fetal interface in the placenta. The mechanisms of immune responses approaching embryo attachment remain to be elucidated. The coordinated effects of the early response genes may hold the keys to the question of why the uterus does not reject an implanting embryo.