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The BCR-associated protein 31 (BAP31), a transmembrane protein in the endoplasmic reticulum, participates in the regulation of immune cells, such as microglia and T cells, and has potential functions in macrophages that remain to be unexplored. In this study, we designed and bred macrophage-specific BAP31 knockdown mice to detect the polarization and functions of macrophages. The results revealed that M2 macrophage-associated genes were suppressed in mouse bone marrow-derived macrophages of Lyz2 Cre-BAP31 flox/flox mice. Multiple macrophage-associated transcription factors were demonstrated to be able to be regulated by BAP31. Among these factors, C/EBPβ was the most significantly decreased and was regulated by early growth response 2. BAP31 could also affect C/EBPβ via modulating IL-4Rα ubiquitination and proteasome degradation in IL-4-stimulated macrophages. Furthermore, we found that BAP31 affects macrophages functions, including angiogenesis and skin fibrosis, during the wound healing process through IL-4Rα, as confirmed by infection with adeno-associated virus-short hairpin (sh)-IL-4Rα in Lyz2 Cre-BAP31 flox/flox mice. Our findings indicate a novel mechanism of BAP31 in regulating macrophages and provide potential solutions for the prevention and treatment of chronic wounds., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Effector CD8 + T cells are crucial players in adaptive immunity for effective protection against invading pathogens. The regulatory mechanisms underlying CD8 + T cell effector differentiation are incompletely understood. In this study, we defined a critical role of mediator complex subunit 1 (Med1) in controlling effector CD8 + T cell differentiation and survival during acute bacterial infection. Mice with Med1 -deficient CD8 + T cells exhibited significantly impaired expansion with evidently reduced killer cell lectin-like receptor G1 + terminally differentiated and Ly6c + effector cell populations. Moreover, Med1 deficiency led to enhanced cell apoptosis and expression of multiple inhibitory receptors (programmed cell death 1, T cell Ig and mucin domain-containing-3, and T cell immunoreceptor with Ig and ITIM domains). RNA-sequencing analysis revealed that T-bet- and Zeb2-mediated transcriptional programs were impaired in Med1 -deficient CD8 + T cells. Overexpression of T-bet could rescue the differentiation and survival of Med1 -deficient CD8 + effector T cells. Mechanistically, the transcription factor C/EBPβ promoted T-bet expression through interacting with Med1 in effector T cells. Collectively, our findings revealed a novel role of Med1 in regulating effector CD8 + T cell differentiation and survival in response to bacterial infection., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Emergency granulopoiesis, also known as demand-adapted granulopoiesis, is defined as the response of an organism to systemic bacterial infections, and it results in neutrophil mobilization from reservoir pools and increased myelopoiesis in the bone marrow. Indirect and direct initiating mechanisms of emergency granulopoiesis have been hypothesized. However, the detailed mechanism of hyperactive myelopoiesis in the bone marrow, which leads to granulocyte left shift, remains unknown. In this study, we report that TLR4 is expressed on granulo-monocytic progenitors, as well as mobilized human peripheral blood CD34 + cells, which account for 0.2% of monocytes in peripheral blood, and ∼ 10% in bone marrow. LPS, a component of Gram-negative bacteria that results in a systemic bacterial infection, induces the differentiation of peripheral blood CD34 + cells into myelocytes and monocytes in vitro via the TLR4 signaling pathway. Moreover, CD34 + cells directly responded to LPS stimulation by activating the MAPK and NF-κB signaling pathways, and they produced IL-6 that promotes emergency granulopoiesis by phosphorylating C/EBPα and C/EBPβ, and this effect was suppressed by the action of an IL-6 receptor inhibitor. This work supports the finding that TLR is expressed on human hematopoietic stem and progenitor cells, and it provides evidence that human hematopoietic stem and progenitor cells can directly sense pathogens and produce cytokines exerting autocrine and/or paracrine effects, thereby promoting differentiation., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Alcohol use disorders (AUD) increase susceptibility to respiratory infections by 2- to 4-fold in part because of impaired alveolar macrophage (AM) immune function. Alcohol causes AM oxidative stress, diminishing AM phagocytic capacity and clearance of microbes from the alveolar space. Alcohol increases AM NADPH oxidases (Noxes), primary sources of AM oxidative stress, and reduces peroxisome proliferator-activated receptor γ (PPARγ) expression, a critical regulator of AM immune function. To investigate the underlying mechanisms of these alcohol-induced AM derangements, we hypothesized that alcohol stimulates CCAAT/enhancer-binding protein β (C/EBPβ) to suppress Nox-related microRNAs (miRs), thereby enhancing AM Nox expression, oxidative stress, and phagocytic dysfunction. Furthermore, we postulated that pharmacologic PPARγ activation with pioglitazone would inhibit C/EBPβ and attenuate alcohol-induced AM dysfunction. AM isolated from human AUD subjects or otherwise healthy control subjects were examined. Compared with control AM, alcohol activated AM C/EBPβ, decreased Nox1-related miR-1264 and Nox2-related miR-107, and increased Nox1, Nox2, and Nox4 expression and activity. These alcohol-induced AM derangements were abrogated by inhibition of C/EBPβ, overexpression of miR-1264 or miR-107, or pioglitazone treatment. These findings define novel molecular mechanisms of alcohol-induced AM dysfunction mediated by C/EBPβ and Nox-related miRs that are amenable to therapeutic targeting with PPARγ ligands. These results demonstrate that PPARγ ligands provide a novel and rapidly translatable strategy to mitigate susceptibility to respiratory infections and related morbidity in individuals with AUD., (Copyright © 2021 by The American Association of Immunologists, Inc.)

The concept of plasticity of neutrophils is highlighted by studies showing their ability to transdifferentiate into APCs. In this regard, transdifferentiated neutrophils were found at inflammatory sites of autoimmune arthritis (AIA). Exposure of neutrophils to inflammatory stimuli prolongs their survival, thereby favoring the acquisition of pathophysiologically relevant phenotypes and functions. By using microarrays, quantitative RT-PCR, and ELISAs, we showed that long-lived (LL) neutrophils obtained after 48 h of culture in the presence of GM-CSF, TNF, and IL-4 differentially expressed genes related to apoptosis, MHC class II, immune response, and inflammation. The expression of anti-inflammatory genes mainly of peptidase inhibitor families is upregulated in LL neutrophils. Among these, the PI3 gene encoding elafin was the most highly expressed. The de novo production of elafin by LL neutrophils depended on a synergism between GM-CSF and TNF via the activation and cooperativity of C/EBPβ and NF-κB pathways, respectively. Elafin concentrations were higher in synovial fluids (SF) of patients with AIA than in SF of osteoarthritis. SF neutrophils produced more elafin than blood counterparts. These results are discussed with respect to implications of neutrophils in chronic inflammation and the potential influence of elafin in AIA., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Myeloid phagocytes, neutrophils in particular, are easily consumed when they fight against a large number of invading microbes. Hence, they require efficient and constant replenishment from their progenitors via the well-orchestrated emergency myelopoiesis in the hematopoietic organs. The cellular and molecular details of the danger-sensing and warning processes to activate the emergency myelopoiesis are still under debate. In this study, we set up a systemic infection model in zebrafish ( Danio rerio ) larvae via circulative administration of LPS. We focused on the cross-talk of macrophages with myeloid progenitors in the caudal hematopoietic tissue. We revealed that macrophages first detected LPS and sent out the emergency message via il1β The myeloid progenitors, rather than hematopoietic stem and progenitor cells, responded and fulfilled the demand to adapt myeloid expansion through the synergistic cooperation of NF-κB and C/ebpβ. Our study unveiled a critical role of macrophages as the early "whistle blowers" to initiate emergency myelopoiesis., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Mixed-lineage leukemia 1 (MLL1), which exerts its H3K4 methyltransferase activity by interacting with WDR5, ASH2L, and RBBP5, plays a pivotal role in regulating hematopoietic stem cell homeostasis. Disrupting the integrity of MLL1-complex has been reported to be associated with acute leukemia. However, the exact role of MLL1-complex in myeloid cells is unknown. In this study, microarray analysis revealed that the core components of the Mll1-complex, Wdr5, Ash2l, and Mll1, were concurrently downregulated by tumor-secreted factors as well as GM-CSF + IL-6 during the accumulation and activation of murine myeloid-derived suppressor cells (MDSCs). These changes were further validated by quantitative RT-PCR and Western blotting both in vitro and in vivo. The expression levels of WDR5 and ASH2L were also significantly decreased in bone marrow MDSCs of lung cancer patients compared with that of healthy controls. Functionally, ectopic expression of Wdr5, Ash2l, and Mll1 (C terminus) reversed the accumulation and function of GM-CSF + IL-6-induced as well as tumor-cocultured polymorphonuclear MDSCs (PMN-MDSCs) by promoting them to differentiate into mature neutrophil-like cells. Mechanistically, GM-CSF + IL-6-activated Stat3 and Cebpβ synergistically induced the expression of miR-21a, miR-21b, and miR-181b, and thus inhibited the expression of Wdr5, Ash2l, and Mll1 by targeting to their 3' untranslated regions, respectively. Furthermore, knockdown of these microRNAs also suppressed the expansion and function of GM-CSF + IL-6-induced PMN-MDSCs. Taken together, our findings indicate that the Stat3/Cebpβ-miR-21a/b/181b-Mll1-complex axis may play a critical role in PMN-MDSC expansion, activation, and differentiation, and this axis may provide an effectively immunological therapeutic approach for patients with cancer or other immunological diseases., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Pathogen invasion triggers robust antiviral cytokine production via different transcription factor signaling pathways. We have previously demonstrated that major vault protein (MVP) induces type I IFN production during viral infection; however, little is known about the role of MVP in proinflammatory responses. In this study, we found in vitro that expression of MVP, IL-6, and IL-8 was inducible upon dsRNA stimulation or viral infection. Moreover, MVP was essential for the induction of IL-6 and IL-8, as impaired expression of IL-6 and IL-8 in MVP-deficient human PBMCs, human lung epithelial cells (A549), and THP-1 monocytes, as well as in murine splenocytes, peritoneal macrophages, and PBMCs from MVP-knockout (MVP(-/-)) mice, was observed. Upon investigation of the underlying mechanisms, we demonstrated that MVP acted in synergy with AP-1 (c-Fos) and CCAAT/enhancer binding protein (C/EBP)β-liver-enriched transcriptional activating protein to activate the IL6 and IL8 promoters. Introduction of mutations into the AP-1 and C/EBPβ binding sites on the IL6 and IL8 promoters resulted in the loss of synergistic activation with MVP. Furthermore, we found that MVP interacted with both c-Fos and C/EBPβ. The interactions promoted nuclear translocation and recruitment of these transcription factors to IL6 and IL8 promoter regions. In the MVP(-/-) mouse model, significantly decreased expression of early antiviral cytokines resulted in higher viral titer in the lung, higher mortality, and heavier lung damage after infection with lethal influenza A virus. Taken together, our findings help to delineate a novel role of MVP in host proinflammatory response., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Macrophage polarization plays a critical role in tissue homeostasis, disease pathogenesis, and inflammation and its resolution. IL-4-induced macrophage polarization involves induction of STAT6 and Krüppel-like factor 4 (KLF4), which induce each other and promote M2 polarization. However, how these transcription factors implement M2 polarization is not understood. We report that in murine macrophages MCP-1-induced protein (MCPIP), induced by KLF4, inhibits M1 polarization by inhibiting NF-κB activation and implements M2 polarization using both its deubiquitinase and RNase activities that cause sequential induction of reactive oxygen species (ROS), endoplasmic reticulum (ER) stress, and autophagy required for M2 polarization. MCPIP also induces C/EBPβ and PPARγ, which promote M2 polarization. Macrophages from mice with myeloid-targeted overexpression of MCPIP show elevated expression of M2 markers and reduced response to LPS, whereas macrophages from mice with myeloid-specific deletion of MCPIP manifest elevated M1 polarization with enhanced phagocytic activity. Thus, both in vivo and in vitro experiments demonstrate that the transcription factors STAT6 and KLF4 implement IL-4-induced M2 polarization via the dual catalytic activities of MCPIP., (Copyright © 2015 by The American Association of Immunologists, Inc.)

We previously showed that the T cell activation inhibitor, mitochondrial (Tcaim) is highly expressed in grafts of tolerance-developing transplant recipients and that the encoded protein is localized within mitochondria. In this study, we show that CD11c(+) dendritic cells (DCs), as main producers of TCAIM, downregulate Tcaim expression after LPS stimulation or in vivo alloantigen challenge. LPS-stimulated TCAIM-overexpressing bone marrow-derived DC (BMDCs) have a reduced capacity to induce proliferation of and cytokine expression by cocultured allogeneic T cells; this is not due to diminished upregulation of MHC or costimulatory molecules. Transcriptional profiling also revealed normal LPS-mediated upregulation of the majority of genes involved in TLR signaling. However, TCAIM BMDCs did not induce Il2 mRNA expression upon LPS stimulation in comparison with Control-BMDCs. In addition, TCAIM overexpression abolished LPS-mediated Ca(2+) influx and mitochondrial reactive oxygen species formation. Addition of IL-2 to BMDC-T cell cocultures restored the priming capacity of TCAIM BMDCs for cocultured allogeneic CD8(+) T cells. Furthermore, BMDCs of IL-2-deficient mice showed similarly abolished LPS-induced T cell priming as TCAIM-overexpressing wild type BMDCs. Thus, TCAIM interferes with TLR4 signaling in BMDCs and subsequently impairs their T cell priming capacity, which supports its role for tolerance induction., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Multiple sclerosis (MS) is an autoimmune disease of the CNS, and autoreactive CD4(+) T cells are considered important for its pathogenesis. The etiology of MS involves a complex genetic trait and environmental triggers that include viral infections, particularly the EBV. Among the risk alleles that have repeatedly been identified by genome-wide association studies, three are located near the Casitas B-lineage lymphoma proto-oncogene b gene (CBLB). The CBLB protein (CBL-B) is a key regulator of peripheral immune tolerance by limiting T cell activation and expansion and hence T cell-mediated autoimmunity through its ubiquitin E3-ligase activity. In this study, we show that CBL-B expression is reduced in CD4(+) T cells from relapsing-remitting MS (RR-MS) patients during relapse. The MS risk-related single nucleotide polymorphism of CBLB rs12487066 is associated with diminished CBL-B expression levels and alters the effects of type I IFNs on human CD4(+) T cell proliferation. Mechanistically, the CBLB rs12487066 risk allele mediates increased binding of the transcription factor C/EBPβ and reduced CBL-B expression in human CD4(+) T cells. Our data suggest a role of the CBLB rs12487066 variant in the interactions of a genetic risk factor and IFN function during viral infections in MS., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Macrophage accumulation is not only a characteristic hallmark but is also a critical component of pulmonary artery remodeling associated with pulmonary hypertension (PH). However, the cellular and molecular mechanisms that drive vascular macrophage activation and their functional phenotype remain poorly defined. Using multiple levels of in vivo (bovine and rat models of hypoxia-induced PH, together with human tissue samples) and in vitro (primary mouse, rat, and bovine macrophages, human monocytes, and primary human and bovine fibroblasts) approaches, we observed that adventitial fibroblasts derived from hypertensive pulmonary arteries (bovine and human) regulate macrophage activation. These fibroblasts activate macrophages through paracrine IL-6 and STAT3, HIF1, and C/EBPβ signaling to drive expression of genes previously implicated in chronic inflammation, tissue remodeling, and PH. This distinct fibroblast-activated macrophage phenotype was independent of IL-4/IL-13-STAT6 and TLR-MyD88 signaling. We found that genetic STAT3 haplodeficiency in macrophages attenuated macrophage activation, complete STAT3 deficiency increased macrophage activation through compensatory upregulation of STAT1 signaling, and deficiency in C/EBPβ or HIF1 attenuated fibroblast-driven macrophage activation. These findings challenge the current paradigm of IL-4/IL-13-STAT6-mediated alternative macrophage activation as the sole driver of vascular remodeling in PH, and uncover a cross-talk between adventitial fibroblasts and macrophages in which paracrine IL-6-activated STAT3, HIF1α, and C/EBPβ signaling are critical for macrophage activation and polarization. Thus, targeting IL-6 signaling in macrophages by completely inhibiting C/EBPβ or HIF1α or by partially inhibiting STAT3 may hold therapeutic value for treatment of PH and other inflammatory conditions characterized by increased IL-6 and absent IL-4/IL-13 signaling., (Copyright © 2014 by The American Association of Immunologists, Inc.)

LPS induces the expression of NO synthase 2 (nos2) in macrophages. The expression of this molecule is one of the hallmarks of classical activation. In this paper, we describe that trichostatin A (TSA), which inhibits deacetylase activity, blocks LPS-dependent nos2 expression. TSA specifically inhibits LPS-dependent genes of secondary response, which require new protein synthesis for their induction but not those belonging to the primary response, which do not depend on this process. Deacetylase activity acts at the transcriptional level because RNA polymerase II was not bound after LPS stimulus when we added TSA. A link between the global acetylation caused by HDAC inhibitor and gene promoter recruitment of CDK8 was found. This Mediator complex subunit associates with Med 12, Med13, and cyclin C to form a submodule that is a transcriptional negative regulator. We also found that TSA reduces C/EBPβ phosphorylation without affecting its binding to DNA. Taken together, these results shed light on the molecular mechanisms involved in the transcriptional regulation of LPS-treated macrophages and on how TSA targets critical LPS-induced genes, such as nos2 and tnf-α, in inflammatory macrophage response.

Thrombin, a serine protease, is a well-known coagulation factor generated during vascular injury and plays an important role in lung inflammation. We previously showed that the c-Src- and Rac/PI3K/Akt-dependent NF-κB pathways are involved in thrombin-induced IL-8/CXCL8 expression in human lung epithelial cells (A549). In this study, we investigated the role of the MEK kinase (MEKK)1/ERK/p90 ribosomal S6 kinase (RSK)1-dependent C/EBPβ signaling pathway in thrombin-induced IL-8/CXCL8 expression. Thrombin-induced IL-8/CXCL8 release and IL-8/CXCL8-luciferase activity were attenuated by small interfering RNA (siRNA) of C/EBPβ and by cells transfected with the C/EBPβ site mutation of the IL-8/CXCL8 construct. Moreover, thrombin-induced κB-luciferase activity was also inhibited by C/EBPβ siRNA. The thrombin-induced increases in IL-8/CXCL8 release and IL-8/CXCL8-luciferase were also inhibited by MEKK1 siRNA, PD98059 (an MEK inhibitor), U0126 (an ERK inhibitor), and RSK1 siRNA. Treatment of cells with thrombin caused an increase in C/EBPβ phosphorylation at Thr(235), C/EBPβ-luciferase activity, recruitment of C/EBPβ to the IL-8/CXCL8 promoter, and C/EBPβ-specific DNA complex formation. Furthermore, thrombin-mediated C/EBPβ phosphorylation and C/EBPβ-luciferase activity were inhibited by MEKK1 siRNA, PD98059, and RSK1 siRNA. Stimulation of cells with thrombin resulted in an increase in RSK1 phosphorylation at Thr(359)/Ser(363), and this effect was inhibited by MEKK1 siRNA and PD98059. The thrombin-induced increase in ERK activation was inhibited by MEKK1 siRNA. These results imply that thrombin activates the MEKK1/ERK/RSK1 signaling pathway, which in turn initiates C/EBPβ activation, recruitment of C/EBPβ to the IL-8/CXCL8 promoter, and C/EBPβ-specific DNA complex formation, and ultimately induces IL-8/CXCL8 expression and release in lung epithelial cells.

Macrophages and dendritic cells (DC) are distributed throughout the body and play important roles in pathogen detection and tissue homeostasis. In tissues, resident macrophages exhibit distinct phenotypes and activities, yet the transcriptional pathways that specify tissue-specific macrophages are largely unknown. We investigated the functions and origins of two peritoneal macrophage populations in mice: small and large peritoneal macrophages (SPM and LPM, respectively). SPM and LPM differ in their ability to phagocytose apoptotic cells, as well as in the production of cytokines in response to LPS. In steady-state conditions, SPM are sustained by circulating precursors, whereas LPM are maintained independently of hematopoiesis; however, both populations are replenished by bone marrow precursors following radiation injury. Transcription factor analysis revealed that SPM and LPM express abundant CCAAT/enhancer binding protein (C/EBP)-β. Cebpb(-/-) mice exhibit elevated numbers of SPM-like cells but lack functional LPM. Alveolar macrophages are also missing in Cebpb(-/-) mice, although macrophage populations in the spleen, kidney, skin, mesenteric lymph nodes, and liver are normal. Adoptive transfer of SPM into Cebpb(-/-) mice results in SPM differentiation into LPM, yet donor SPM do not generate LPM after transfer into C/EBPβ-sufficient mice, suggesting that endogenous LPM inhibit differentiation by SPM. We conclude that C/EBPβ plays an intrinsic, tissue-restricted role in the generation of resident macrophages.

Neutrophils influence innate and adaptative immunity by generating numerous cytokines and chemokines whose regulation largely depends on transcriptional activators such as NF-κB and C/EBP factors. In this study, we describe the critical involvement of CREB transcription factors (CREB1 and activating transcription factor-1) in this functional response as well as relevant upstream signaling components. Neutrophil stimulation with LPS or TNF led to the phosphorylation, DNA binding activity, and chemokine promoter association of CREB1 and activating transcription factor-1. These responses occurred downstream of the p38-MSK1 signaling axis, as did the phosphorylation and promoter association of another bZIP factor, C/EBPβ. Conversely, inhibition of RSK1 failed to alter the phosphorylation of either CREB1 or C/EBPβ in neutrophils. From a more functional standpoint, the inhibition of p38 MAPK or MSK1 interfered with cytokine generation in neutrophils. Likewise, overexpression of a dominant-negative CREB1 mutant (K-CREB) or of a point mutant (S133A) resulted in a decreased ability of human neutrophil-like PLB-985 cells to generate inflammatory cytokines (CXCL8, CCL3, CCL4, and TNF-α). Collectively, our data show the involvement of CREB1 in neutrophil cytokine production, the key role of its S133 residue, important upstream signaling events, and the parallel activation of another bZIP factor. These are all potential molecular targets that could be exploited in the context of several chronic inflammatory diseases that prominently feature neutrophils and their products.

IL-10 is important in the resistance response of BALB/c mice to experimental Pseudomonas aeruginosa corneal infection. However, the cellular mechanisms by which this anti-inflammatory cytokine is regulated remain unknown. Because the mammalian target of rapamycin (mTOR) regulates IL-10 in other disease models, the present study tested its role in bacterial keratitis. After infection, corneas of rapamycin versus control-treated BALB/c mice showed worsened disease, and real-time RT-PCR confirmed that mTOR mRNA levels were significantly decreased. Rapamycin treatment also increased clinical score, polymorphonuclear neutrophil (PMN) infiltration (determined by myeloperoxidase assay), and bacterial load, but it diminished PMN bactericidal activity. Inhibition of mTOR also led to elevated mRNA and protein levels of IL-12p40, matrix metalloproteinase 9, and inducible NO synthase, whereas mRNA and protein levels of IL-10, its regulator/effector STAT-3, and suppressor of cytokine signaling 3 (a proinflammatory cytokine regulator) were decreased. Furthermore, mTOR inhibition reduced levels of proapoptotic caspase-3 and increased levels of B cell lymphoma-2 (antiapoptotic), indicative of delayed apoptosis. mTOR inhibition also altered genes related to TLR signaling, including elevation of TLR4, TLR5, and IL-1R1, with decreases in IL-1R-associated kinase 1 and an inhibitor of NF-κB, NF-κB inhibitor-like 1. Rapamycin treatment also increased levels of IFN-γ and CCAAT/enhancer binding protein, β, a gene that regulates expression of preprotachykinin-A (the precursor of substance P). Collectively, these data, as well as a rescue experiment using rIL-10 together with rapamycin, which decreased PMN in cornea, provide concrete evidence that mTOR regulates IL-10 in P. aeruginosa-induced bacterial keratitis and is critical to balancing pro- and anti-inflammatory events, resulting in better disease outcome.

The C/EBP β is a basic leucine zipper transcription factor that regulates a variety of biological processes, including metabolism, cell proliferation and differentiation, and immune response. Recent findings show that C/EBP β-induced inflammatory responses mediate kainic acid-triggered excitotoxic brain injury. In this article, we show that protein kinase C ζ enhances K-ras expression and subsequently activates the Raf/MEK/ERK1/2 pathway in the hippocampus of domoic acid (DA)-treated mice, which promotes C/EBP β expression and induces inflammatory responses. Elevated production of TNF-α impairs mitochondrial function and increases the levels of reactive oxygen species by IκB kinase β/NF-κB signaling. The aforementioned inflammation and oxidative stress lead to memory deficits in DA-treated mice. However, troxerutin inhibits cyclin-dependent kinase 1 expression, enhances type 1 protein phosphatase α dephosphorylation, and abolishes MEK/ERK1/2/C/EBP β activation, which subsequently reverses the memory impairment observed in the DA-treated mice. Thus, troxerutin is recommended as a potential candidate for the prevention and therapeutic treatment of cognitive deficits resulting from excitotoxic brain damage and other brain disorders.

Comparison of the inflammatory cytokine profile in bone marrow-derived macrophages (BMDMs) from normal and Src homology domain 2-containing tyrosine phosphatase-1 (SHP-1)-deficient Motheaten (me/me) mice revealed a dramatic suppression of IL-6 transcript and protein in me/me BMDMs after LPS stimulation. Interfering with SHP-1 expression using antisense SHP-1 oligonucleotides led to a significant downregulation of IL-6 in normal BMDMs. Conversely, reconstitution of me/me BMDMs with the SHP-1 gene using adenoviral vectors restored IL-6 production. Expression of only SHP-1 Src homology region 2 domains in normal BMDMs inhibited IL-6 production, confirming that IL-6 regulation depends on SHP-1 phosphatase activity. We further demonstrated that loss of SHP-1 function affects proper phosphorylation of Erk1/2 MAPKs and, to a lesser degree, of NF-κB downstream of TLR4 in BMDMs. Inefficient phosphorylation of Erk1/2 MAPKs abrogated the activation of C/EBPβ transcription factor, which was reversed on restoration of SHP-1 function and led to a concomitant enhancement of IL-6 production. We demonstrate that IL-6 production is regulated by a complex network of signaling pathways that include SHP-1-dependent activation of Erk1/2-C/EBPβ and NF-κB, in addition to SHP-1-independent IκB pathway through the activation of protein tyrosine kinases downstream of TLR4. Taken together, these results revealed for the first time, to our knowledge, a positive and critical role of SHP-1 in IL-6 regulation and dependence of Erk1/2-C/EBPβ pathway in addition to that of IκB on SHP-1 activity required for IL-6 induction after LPS stimulation.

CXCL8 is a neutrophil and mast cell chemoattractant that is involved in regulating inflammatory cell influx in asthma. Here, we investigated the transcriptional mechanism involved in CXCL8 induction by TNF-alpha in cultured human airway smooth muscle (HASM) cells and compared these in cells from nonasthmatic and asthmatic individuals. Transfection studies with mutated CXCL8 promoter constructs identified NF-kappaB, activating protein-1, and CAAT/enhancer binding protein (C/EBP)beta as key transcription factors, and binding of these three transcription factors to the CXCL8 promoter after TNF-alpha stimulation was confirmed by chromatin immunoprecipitation analysis. Cells derived from asthmatic individuals produced significantly higher levels of CXCL8 than nonasthmatic cells both basally and following 24 h of stimulation with TNF-alpha (p < 0.001). Furthermore, chromatin immunoprecipitation studies detected increased binding of NF-kappaB p65 and RNA polymerase II to the CXCL8 promoter of asthmatic HASM cells both in the presence and absence of TNF-alpha stimulation. This was not due to either an increased activation or phosphorylation of NF-kappaB per se or to an increase in its translocation to the nucleus. Increased binding of C/EBPbeta to the CXCL8 promoter of unstimulated cells was also detected in the asthmatic HASM cells. Collectively these studies show that HASM cells from asthmatic individuals have increased CXCL8 production due to the presence of a transcription complex on the CXCL8 promoter, which contains NF-kappaB, C/EBPbeta, and RNA polymerase II. This is the first description of an abnormality in transcription factor binding altering chemokine expression in airway structural cells in asthma.

A growing number of neutrophil-derived cytokines have proven to be crucial to various inflammatory and immune processes in vivo. Whereas C/EBP (CCAAT/enhancer-binding protein) transcription factors are important for neutrophil differentiation from myeloid precursors, we report herein that they also regulate cytokine production in mature neutrophils. All known C/EBP proteins but C/EBPgamma are expressed in neutrophils; most isoforms localize to the nucleus, except for C/EBPalpha, which is cytoplasmic. Neutrophil stimulation does not alter the overall levels, cellular distribution, or turnover of C/EBP proteins; it also does not further induce the constitutive DNA-binding activity detected in nuclear extracts, consisting of C/EBPbeta and C/EBPepsilon. However, nuclear C/EBPbeta is rapidly phosphorylated upon cell stimulation, suggesting that it can activate cytokine promoters. Indeed, the transactivation of an IL-8 promoter-luciferase construct in a human neutrophil-like cell line was impaired when its C/EBP or NF-kappaB sites were mutated. Overexpression of a C/EBP repressor also impeded IL-8 promoter transactivation, as well as the generation of IL-8, Mip-1alpha, and Mip-1beta in this cellular model, whereas TNF-alpha generation was mostly unaffected. Finally, overexpression of a C/EBPbeta mutant (T235A) as well as chromatin immunoprecipitation assays unveiled an important role for this residue in cytokine induction. This is the first demonstration that C/EBP factors are important regulators of cytokine expression in human neutrophils.

Expression of the acute phase protein C-reactive protein (CRP) is tightly regulated in hepatocytes. Although very little CRP mRNA is transcribed normally, inflammatory stimuli are followed by a dramatic increase in mRNA synthesis and accumulation. IL-6 and IL-1beta are believed to be the major cytokines responsible for induction of CRP and other acute phase proteins. Our previous studies, using transient transfection and EMSA experiments, implicated involvement of the transcription factors C/EBPbeta, STAT3, Rel p50, and c-Rel in CRP induction. In the current study we used chromatin immunoprecipitation assays to determine the kinetics of transcription factor occupancy of these transcription factors on the endogenous CRP promoter. All of these transcription factors were found bound to the endogenous CRP promoter in the absence of cytokines, but cytokine treatment markedly increased binding of only C/EBPbeta. In addition, c-Rel and TATA box-binding protein (TBP) appeared to occupy the promoter in parallel in the presence of cytokines. In the absence of cytokines, CRP mRNA accumulation was not measurable but began to increase by 3 h after exposure of cells to IL-1beta plus IL-6, peaking at 12 h with secondary peaks at 18 and 24 h. The secondary peaks in mRNA expression paralleled the pattern of binding of c-Rel and TBP to the CRP promoter. We conclude that the CRP promoter has a low level of transcription factor occupancy in the absence of cytokines and induction occurs with binding of C/EBP, and that c-Rel and TBP are important for modulating CRP expression.

TLR2 signaling by Mycobacterium tuberculosis 19-kDa lipoprotein (LpqH) inhibits IFN-gamma-induced expression of CIITA by macrophages. Microarray analysis, quantitative RT-PCR, and Western blots showed that LpqH induced C/EBPbeta and C/EBPdelta in kinetic correlation with inhibition of CIITA expression. Of the C/EBPbeta isoforms, liver inhibitory protein (LIP) was notably induced and liver-activating protein was increased by LpqH. Putative C/EBP binding sites were identified in CIITA promoters I and IV (pI and pIV). LpqH induced binding of C/EBPbeta (LIP and liver-activating protein) to biotinylated oligodeoxynucleotide containing the pI or pIV binding sites, and chromatin immunoprecipitation showed that LpqH induced binding of C/EBPbeta and C/EBPdelta to endogenous CIITA pI and pIV. Constitutive expression of C/EBPbeta LIP inhibited IFN-gamma-induced CIITA expression in transfected cells. In summary, LpqH induced expression of C/EBPbeta and C/EBPdelta, and their binding to CIITA pI and pIV, in correlation with inhibition of IFN-gamma-induced expression of CIITA in macrophages, suggesting a role for C/EBP as a novel regulator of CIITA expression.

Although it is widely accepted that glucocorticoids (GC) are a mainstay of the treatment of diseases characterized by airway inflammation, little is known about the effects of GC on local innate immunity. In this article, we report that respiratory epithelial cells manifested a local "acute phase response" after stimulation with TLR activation and TNF-alpha and that GC spared or enhanced the epithelial expression of molecules that are involved in host defense, including complement, collectins, and other antimicrobial proteins. As expected, GC inhibited the expression of molecules responsible for inflammation such as cytokines (IFNbeta and GM-CSF) and chemokines (RANTES and IL-8). Studies using Western blotting, EMSA, and functional analysis indicated that the selective effects of GC are mediated through activation of the transcription factor C/EBP. Knockdown of C/EBPbeta by small interfering RNA blocked the enhancement by GC of host defense molecule expression but had no effect on inflammatory gene expression. These results suggest that GC spare or enhance local innate host defense responses in addition to exerting anti-inflammatory actions. It is possible that the known ability of GC to reduce the exacerbation of diseases in which infectious organisms serve as triggering factors (e.g., asthma, allergic bronchopulmonary aspergillosis, and chronic obstructive pulmonary disease) may result in part from enhanced innate immune responses in airway mucosa.

Regulation of basal and cytokine (IL-6 and IL-1beta)-induced expression of C-reactive protein (CRP) in human hepatoma Hep3B cells occurs during transcription. A critical transcriptional regulatory element on the CRP promoter is a C/EBP binding site overlapping a NF-kappaB p50 binding site. In response to IL-6, C/EBPbeta and p50 occupy the C/EBP-p50 site on the CRP promoter. The aim of this study was to identify the transcription factors occupying the C/EBP-p50 site in the absence of C/EBPbeta. Accordingly, we treated Hep3B nuclear extract with a C/EBP-binding consensus oligonucleotide to generate an extract lacking active C/EBPbeta. Such treated nuclei contain only C/EBPzeta (also known as CHOP10 and GADD153) because the C/EBP-binding consensus oligonucleotide binds to all C/EBP family proteins except C/EBPzeta. EMSA using this extract revealed formation of a C/EBPzeta-containing complex at the C/EBP-p50 site on the CRP promoter. This complex also contained RBP-Jkappa, a transcription factor known to interact with kappaB sites. RBP-Jkappa was required for the formation of C/EBPzeta-containing complex. The RBP-Jkappa-dependent C/EBPzeta-containing complexes were formed at the C/EBP-p50 site on the CRP promoter in the nuclei of primary human hepatocytes also. In luciferase transactivation assays, overexpressed C/EBPzeta abolished both C/EBPbeta-induced and (IL-6 + IL-1beta)-induced CRP promoter-driven luciferase expression. These results indicate that under basal conditions, C/EBPzeta occupies the C/EBP site, an action that requires RBP-Jkappa. Under induced conditions, C/EBPzeta is replaced by C/EBPbeta and p50. We conclude that the switch between C/EBPbeta and C/EBPzeta participates in regulating CRP transcription. This process uses a novel phenomenon, that is, the incorporation of RBP-Jkappa into C/EBPzeta complexes solely to support the binding of C/EBPzeta to the C/EBP site.

TNF is a major mediator of inflammation, immunity, and apoptosis. Pre-exposure to TNF reduces sensitivity to restimulation, a phenomenon known as tolerance, considered as protective in sepsis, but also as a paradigm for immunoparalysis. Earlier experiments in TNF-tolerant cells display inhibition of NF-kappaB-dependent IL-8 gene expression at the transcriptional level with potential involvement of C/EBPbeta. In this study, we have shown that a kappaB motive was sufficient to mediate transcriptional inhibition under TNF tolerance conditions in monocytic cells. Furthermore, in tolerant cells, TNF-induced NF-kappaB p65 phosphorylation was markedly decreased, which was accompanied by the formation of C/EBPbeta-p65 complexes. Remarkably, in C/EBPbeta(-/-) cells incubated under the conditions of TNF tolerance, neither impairment of transcription nor inhibition of p65 phosphorylation was observed. Finally, we showed that C/EBPbeta overexpression reduced p65-mediated transactivation and that association of C/EBPbeta with p65 specifically prevented p65 phosphorylation. Our data demonstrate that C/EBPbeta is an essential signaling component for inhibition of NF-kappaB-mediated transcription in TNF-tolerant cells and suggest that this is caused by blockade of p65 phosphorylation. These results define a new molecular mechanism responsible for TNF tolerance in monocytic cells that may contribute to the unresponsiveness seen in patients with sepsis.

The transcription factor C/EBPbeta transactivates the IL-4 gene in murine T lymphocytes and facilitates Th2 cell responses. In this study, we demonstrate that C/EBPbeta also acts as a repressor of T cell proliferation. By binding to the c-myc promoter(s), C/EBPbeta represses c-Myc expression and, therefore, arrests T cells in the G1 phase of the cell cycle. For C/EBPbeta-mediated repression, the integrity of its N-terminal transactivation domain is essential whereas the central regulatory domain is dispensable. This central regulatory domain is sumoylated in vivo which leads to an alteration of the activity of C/EBPbeta. Whereas sumoylation does not affect the C/EBPbeta-mediated activation of the IL-4 gene, it relieves its repressive effect on c-Myc expression and T cell proliferation. Similar to several other transcription factors, sumoylation redistributes nuclear C/EBPbeta and targets it to pericentric heterochromatin. These results suggest an important role of sumoylation in adjusting the finely tuned balance between proliferation and differentiation in peripheral T cells which is controlled by C/EBPbeta.

IFN-gamma-activated transcriptional element (GATE)-binding factor 1 (GBF1) was identified as a transactivator that induces gene expression through GATE, a novel IFN-inducible element. Although it can induce gene expression, it is an extremely weak DNA-binding protein on its own. GATE also binds another transcription factor, C/EBP-beta. Therefore, we explored whether GBF1 physically interacts with C/EBP-beta to induce IFN-gamma-regulated transcription. In response to IFN-gamma, C/EBP-beta undergoes phosphorylation at a critical ERK1/2 phosphorylation motif. Mutational inactivation of this motif and/or interference with the ERK1/2 activation prevented the IFN-gamma-induced interactions between GBF1 and C/EBP-beta. A 37-aa long peptide derived from the GBF1 protein can associate with C/EBP-beta in an IFN-inducible manner. These results identify a converging point for two transactivators that exert their effects through a single response element. Together, our studies identify a novel regulatory mechanism that controls IFN-induced transcription.

The role of IL-1beta in inflammation is amply documented, but its ability to inhibit myofibroblast differentiation and, in particular, the suppression of alpha-smooth muscle actin (alpha-SMA) gene expression is less well understood. Because IL-1beta can induce C/EBPbeta expression, the role of C/EBPbeta isoforms in IL-1beta regulation of alpha-SMA gene expression was investigated in rat lung myofibroblasts. The results showed that IL-1beta inhibited alpha-SMA expression in a dose-dependent manner, which was associated with stimulation of the expression of both C/EBPbeta isoforms, liver-enriched activating protein (LAP) and liver-enriched inhibitory protein (LIP). However, a greater increase in LIP relative to LAP expression resulted in a reduced LAP/LIP ratio after IL-1beta treatment. Transfection with an LAP-expressing plasmid stimulated, whereas an LIP-expressing plasmid inhibited, alpha-SMA expression. Cells from C/EBPbeta-deficient mice had reduced levels of alpha-SMA expression and promoter activity, which failed to respond to IL-1beta treatment. Sequence analysis identified the presence of a C/EBPbeta consensus binding sequence in the alpha-SMA promoter, which, when mutated, resulted in diminished promoter activity and abolished its responsiveness to IL-1beta treatment. EMSA revealed binding of C/EBPbeta to this C/EBPbeta consensus binding sequence from the alpha-SMA promoter. Finally, IL-1beta enhanced the expression of eukaryotic initiation factor 4E, a stimulator of LIP expression, which may account for a mechanism by which IL-1beta could alter the LAP/LIP ratio. These data taken together suggest that C/EBPbeta isoforms regulate alpha-SMA gene expression, and that its inhibition by IL-1beta was due to preferential stimulation of LIP expression.

Previous work indicates that treatment of human T cells with PGE(2) results in an increase of HIV-1 long terminal repeat (LTR) transcriptional activity. The noticed PGE(2)-mediated activation of virus gene activity required the participation of specific intracellular second messengers such as calcium and two transcription factors, i.e., NF-kappaB and CREB. We report in this work that the nuclear transcription factor CCAAT/enhancer binding protein (C/EBP) is also important for PGE(2)-dependent up-regulation of HIV-1 LTR-driven gene activity. The implication of C/EBP was shown by using a trans-dominant negative inhibitor of C/EBP (i.e., liver-enriched transcriptional inhibitory protein) and several molecular constructs carrying site-directed mutations in the C/EBP binding sites located within the HIV-1 LTR. Mutated HIV-1 LTR constructs also revealed the involvement of the two most proximal C/EBP binding sites. Data from cotransfection experiments with vectors coding for dominant negative mutants and gel mobility shift assays indicated that PGE(2)-mediated induction of HIV-1 LTR activity results from a cooperative interaction between C/EBPbeta and CREB, two members of the basic leucine zipper family of transcription factors. Altogether these findings indicate that treatment of human T cells with PGE(2) induces HIV-1 LTR activity through a complex interplay between C/EBPbeta and CREB. Such a combinatorial regulation may represent a mechanism that permits a fine regulation of HIV-1 expression by PGE(2) in human T cells.

Appropriate regulation of IL-12 expression is critical for cell-mediated immune responses. In the present study, we have analyzed the regulation of IL-12 p40 promoter activity in primary human monocytes in vivo. Accordingly, we analyzed the p40 promoter by in vivo footprinting in resting and activated primary human blood CD14(+) monocytes. Interestingly, footprints at binding sites for trans-activating proteins such as C/EBP, NF-kappaB, and ETS were only found upon stimulation with LPS and IFN-gamma. In contrast, a footprint over a purine-rich sequence at -155, termed GA-12 (GATA sequence in the IL-12 promoter), was observed in resting, but not activated, cells. Further characterization of this site revealed specific complex formation at a protected GATA core motif in unstimulated primary monocytes and RAW264.7 macrophages. Mutagenesis within the GA-12 sequence caused a significant up-regulation of inducible IL-12 p40 promoter activity in both transient and stable transfection systems, suggesting a repressor function of this site. Furthermore, binding activity of the GA-12 binding protein GAP-12 was increased by treatment with two potent inhibitors of IL-12 expression, IL-4 and PGE(2). Finally, we observed that IL-4-mediated repression of IL-12 p40 promoter activity is critically dependent on an intact GA-12 sequence. In summary, our data underline the complex regulation of the human IL-12 p40 promoter and identify GA-12 as a potent, novel repressor element that mediates IL-4-dependent suppression of inducible promoter activity in monocytes. Regulation of GAP-12 binding may thus modulate IL-12 p40 gene expression.

C/EBPbeta is a member of a family of leucine zipper transcription factors that are involved in regulating the expression of several cytokines, including IL-1, IL-6, IL-8, TNF, and macrophage-inflammatory protein-1alpha. We identified multiple C/EBPbeta binding sites within the gene for CCR5, suggesting that C/EBPbeta may be involved in its regulation. Transient transfection experiments in both myeloid and lymphoid cells showed an increase in CCR5 promoter-driven green fluorescent protein production in the presence of C/EBPbeta. Deletion analysis identified two C/EBPbeta-responsive regions in the CCR5 gene, one in the promoter region and one at the 3' part of the intron. We provide evidence that, in myeloid cells (U937), C/EBPbeta independently activates CCR5 expression through sites located either in the promoter region or in the intron of the CCR5 gene. In contrast, in lymphoid cells (Jurkat) the presence of the intronic cis-regulatory regions is required for C/EBPbeta-mediated activation. In agreement with the functional data, EMSA demonstrated that in both myeloid and lymphoid cells C/EBPbeta binds specifically to sites present in the intron, whereas interaction with the sites located in the promoter was cell type specific and was detected only in myeloid cells. Analysis of C/EBPbeta in primary PBMCs obtained from HIV-1-infected individuals revealed a significant increase in C/EBPbeta expression. The enhanced C/EBPbeta activity correlated with a higher frequency of circulating CCR5(+) lymphocytes in AIDS patients and with a decline in CD4 lymphocyte numbers. Taken together, these results suggest that C/EBPbeta is an important regulator of CCR5 expression and may play a relevant role in the pathogenesis of HIV disease.

We have previously found that overexpression of the Rel protein p50 stimulated C-reactive protein (CRP) expression in Hep 3B cells and that p50 could bind to a nonconsensus kappaB site overlapping the CCAAT/enhancer binding protein (C/EBP) binding site centered at position -53 on the CRP promoter. Accordingly, we employed EMSA to investigate possible cooperation between p50 and C/EBP proteins using an oligonucleotide probe (-63/-41) derived from the CRP promoter and containing both C/EBP and p50 binding sites. Abs to p50, but not to p65, decreased formation of C/EBPbeta-containing complexes in nuclei of IL-6-treated cells, indicating that ternary complexes containing C/EBPbeta and p50 are formed on the CRP promoter. Depletion of free Rel proteins by pretreatment of nuclear extracts with a kappaB consensus oligonucleotide markedly decreased formation of C/EBP complexes, indicating that Rel proteins are required for formation of such complexes. Overexpression of p50 in transient cotransfection studies using the proximal CRP promoter (-125/+9) linked to a luciferase reporter caused a 3-fold increase of luciferase activity, while C/EBPbeta overexpression caused an 18-fold increase; simultaneous overexpression of both transcription factors increased luciferase activity approximately 600-fold. Mutation of either the C/EBP binding site or the p50 binding site drastically reduced the effects of overexpressed transcription factors. Taken together, our findings indicate that binding of Rel p50 to the nonconsensus kappaB site enhances and stabilizes binding of C/EBPbeta to the CRP promoter and that binding of both C/EBPbeta and p50 to their overlapping cognate sites is required for induction of CRP expression by IL-6.