Wendy T. Watford

Tumor Progression Locus 2 (Tpl2) Kinase Promotes Chemokine Receptor Expression and Macrophage Migration during Acute Inflammation

Background: The accumulation of activated leukocytes correlates with autoimmunity and is regulated by chemokines. Results: Tpl2-deficient macrophages display impaired chemokine receptor expression and migration under inflammatory conditions. Conclusion: Tpl2 promotes the induction and maintenance of macrophage chemokine receptor expression and cellular migration in vivo. Significance: Tpl2 inhibition may represent a treatment for autoimmune disorders by modulating chemokine receptor expression and preventing local accumulation of activated macrophages. 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.

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/β) 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/β and IFNλ in a cell-type specific manner. Although Tpl2 is important in the regulation of both IFNα/β 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.

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

Background: Tpl2 kinase plays an essential, non-redundant role in activating ERK during TLR signaling. Results: TLRs 2, 4, and 7 directly induce IKKβ-Tpl2-ERK signaling; TLRs 3 and 9 activate ERK indirectly via autocrine ROS signaling. Conclusion: Tpl2-dependent ROS generation drives ERK phosphorylation during TLR 3 and 9 signaling. Significance: The different contributions of Tpl2 to TLR signaling pathways influences early host defense mechanisms. 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 β (IKKβ), 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, IKKβ-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 IKKβ-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.

RGC-32 (Response Gene to Complement 32 Protein) 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-&agr; (tumor necrosis factor-&agr;)–induced monocyte–endothelial cell interaction. Mechanistically, RGC-32 mediated the ICAM-1 and VCAM-1 expression, at least partially, through NF (nuclear factor)-&kgr;B signaling pathway. RGC-32 directly interacted with NF-&kgr;B and facilitated its nuclear translocation and enhanced TNF-&agr;–induced NF-&kgr;B 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-&kgr;B signaling pathway.

A Neoglycoconjugate Containing the Human Milk Sugar LNFPIII Drives Anti-Inflammatory Activation of Antigen Presenting Cells in a CD14 Dependent Pathway.

The milk pentasaccharide LNFPIII has therapeutic action for metabolic and autoimmune diseases and prolongs transplant survival in mice when presented as a neoglycoconjugate. Within LNFPIII is the Lewisx trisaccharide, expressed by many helminth parasites. In humans, LNFPIII is found in human milk and also known as stage-specific embryonic antigen-1. LNFPIII-NGC drives alternative activation of macrophages and dendritic cells via NFκB activation in a TLR4 dependent mechanism. However, the connection between LNFPIII-NGC activation of APCs, TLR4 signaling and subsequent MAP kinase signaling leading to anti-inflammatory activation of APCs remains unknown. In this study we determined that the innate receptor CD14 was essential for LNFPIII-NGC induction of both ERK and NFkB activation in APCs. Induction of ERK activation by LNFPIII-NGC was completely dependent on CD14/TLR4-Ras-Raf1/TPL2-MEK axis in bone marrow derived dendritic cells (BMDCs). In addition, LNFPIII-NGC preferentially induced the production of Th2 “favoring” chemokines CCL22 and matrix metalloprotease protein-9 in a CD14 dependent manner in BMDCs. In contrast, LNFPIII-NGC induces significantly lower levels of Th1 “favoring” chemokines, MIP1α, MIP1β and MIP-2 compared to levels in LPS stimulated cells. Interestingly, NGC of the identical human milk sugar LNnT, minus the alpha 1–3 linked fucose, failed to activate APCs via TLR4/MD2/CD14 receptor complex, suggesting that the alpha 1–3 linked fucose in LNFPIII and not on LNnT, is required for this process. Using specific chemical inhibitors of the MAPK pathway, we found that LNFPIII-NGC induction of CCL22, MMP9 and IL-10 production was dependent on ERK activation. Over all, this study suggests that LNFPIII-NGC utilizes CD14/TLR4-MAPK (ERK) axis in modulating APC activation to produce anti-inflammatory chemokines and cytokines in a manner distinct from that seen for the pro-inflammatory PAMP LPS. These pathways may explain the in vivo therapeutic effect of LNFPIII-NGC treatment for inflammation based diseases.

Tumor progression locus 2 differentially regulates IFNγ and IL-17 production by effector CD4+ T cells in a T cell transfer model of colitis.

Autoimmune diseases are approaching epidemic levels, estimated to affect 5–8% of the population. A number of autoimmune diseases are believed to be driven by autoreactive T cells, specifically by T helper 1 (Th1) cells and T helper 17 (Th17) cells. One molecule gaining interest as a therapeutic target is the serine-threonine kinase, Tpl2, which promotes expression of proinflammatory mediators. We previously demonstrated that Tpl2 regulates Th1 differentiation, secretion of the inflammatory cytokine IFNγ, and host defense against the intracellular parasite Toxoplasma gondii. The goal of this study was to determine whether Tpl2 also regulates Th1 or Th17 differentiation in vivo in a model of colitis associated with mixed Th1/Th17 pathology. In vitro, Tpl2−/− naïve CD4 T cells were significantly impaired in IL-17A secretion under traditional Th17 inducing conditions. Reduced IL-17A secretion correlated with increased expression of FoxP3, a transcription factor known to antagonize RORγt function. In a murine T cell transfer model of colitis, transfer of Tpl2−/− T cells resulted in reduced proportions of CD4 T cells expressing IFNγ, but not IL-17A, compared to that induced by wild type T cells. Further studies revealed that IL-17A differentiation induced by IL-6 and IL-23, cytokines implicated in driving Th17 differentiation in vivo, was unaffected by Tpl2 deficiency. Collectively, these results implicate Tpl2 in TGF-β-induced FoxP3 expression. Additionally, they underscore the contribution of Tpl2 to Th1 immunopathology specifically, which suggests that Tpl2 inhibitors may selectively target Th1-based inflammation.

Estrogen receptor α contributes to T cell–mediated autoimmune inflammation by promoting T cell activation and proliferation

Estrogen receptor α promotes T cell activation and exacerbates autoimmune disease. Estrogen receptor stimulates T cells Sex-linked susceptibility has been described for nearly 75% of all autoimmune disorders. More than any other risk factor discovered, being female confers the greatest risk of developing these diseases. Mohammad et al. identify a direct role for the female sex hormone estrogen in the development of autoimmune T cell responses. Deletion of estrogen receptor α (ERα) in T cells reduced disease burden in a mouse model of colitis. ERα-expressing T cells were more activated after stimulation, proliferated more, and expressed more proinflammatory cytokines than T cells lacking this receptor. Conversely, ERα-deficient T cells were more readily skewed to a regulatory T cell phenotype. Together, these data identify a role for direct sex hormone–dependent activation of T cells in autoimmune responses. It has long been appreciated that most autoimmune disorders are characterized by increased prevalence in females, suggesting a potential role for sex hormones in the etiology of autoimmunity. To study how estrogen receptor α (ERα) contributes to autoimmune diseases, we generated mice in which ERα was deleted specifically in T lymphocytes. We found that ERα deletion in T cells reduced their pathogenic potential in a mouse model of colitis and correlated with transcriptomic changes that affected T cell activation. ERα deletion in T cells contributed to multiple aspects of T cell function, including reducing T cell activation and proliferation and increasing the expression of Foxp3, which encodes a critical transcription factor for the differentiation and function of regulatory T cells. Thus, these data demonstrate that ERα in T cells plays an important role in inflammation and suggest that ERα-targeted immunotherapies could be used to treat autoimmune disorders.

Tpl2 promotes neutrophil trafficking, oxidative burst, and bacterial killing

Tumor progression locus 2 (Tpl2) is a serine/threonine kinase that promotes inflammatory cytokine production by activating the MEK/ERK pathway. Tpl2 has been shown to be important for eliciting the inflammatory properties of macrophages; however, there is relatively little known about the contribution of Tpl2 to neutrophil effector functions. This is an important consideration, as neutrophils provide the first line of defense against infection in the innate immune system. We found that Tpl2 is expressed in both human and murine neutrophils, suggesting a potential function for Tpl2 in this lineage. Despite significantly higher proportions of bone marrow (BM) neutrophils in Tpl2‐deficient (Tpl2−/−) mice compared with wild‐type (WT) mice, Tpl2−/− mice have significantly reduced proportions of circulating neutrophils. Tpl2−/− neutrophils show impaired recruitment to thioglycollate, which was primarily a result of neutrophil‐extrinsic factors in the host. In response to infection, neutrophils secrete inflammatory cytokines and produce reactive oxygen species (ROS), which promote bacterial killing. Tpl2 ablation impaired neutrophil TNF secretion in response to LPS stimulation, superoxide generation in response to the chemotactic peptide fMLP, and killing of the extracellular bacterium, Citrobacter rodentium, despite normal bacterial phagocytosis. These results implicate Tpl2 in the regulation of multiple neutrophil antimicrobial pathways, including inflammatory cytokine secretion and oxidative burst. Furthermore, they indicate that Tpl2 functions early during infection to bolster neutrophil‐mediated innate immunity against extracellular bacteria.

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-1β, 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.

Response by Cui et al to Letter Regarding Article, “RGC-32 (Response Gene to Complement 32) Deficiency Protects Endothelial Cells From Inflammation and Attenuates Atherosclerosis”

We welcome the letter from Rus et al commenting on our recent study showing that RGC-32 (response gene to complement 32) mediates the development of atherosclerosis by facilitating monocyte–endothelial cell (EC) interaction through induction of endothelial intercellular adhesion molecule-1 and vascular cell adhesion molecule-1.1 Our conclusion is based on several observations: (1) RGC-32 is induced in ECs in both human and mouse atherosclerotic lesions; (2) RGC-32 deficiency ( Rgc32 −/−) attenuates the spontaneously-developed and high-fat diet–induced atherosclerosis in Apoe −/− mice; (3) Rgc32 −/− mice transplanted with wild-type bone marrow does not significantly alter the Rgc32 −/− phenotype; (4) Rgc32 −/− inhibits endothelial intercellular adhesion molecule-1 and vascular cell adhesion molecule-1expression and monocyte–EC interaction.1 These data strongly suggest that endothelial RGC-32 plays an essential role in atherosclerosis.nnWe understand the concern in the letter about the differences in RGC-32 expression in human atherosclerotic lesions in our and Dr Vlaicu et al’s2 studies and appreciate the insights that the discrepancy may be the result of the differences in the atherosclerotic lesions from different arterial regions. …