Short-Chain Fatty Acid Decreases the Expression of CEBPB to Inhibit miR-145-Mediated DUSP6 and Thus Further Suppresses Intestinal Inflammation.

Intestinal inflammation is a common disease which can further lead to inflammatory bowel disease and even intestinal cancer. The increasing focus has come to the role of short-chain fatty acid (SCFA) in various bowel diseases. Hence, this study was designed to explore the specific role of SCFA in intestinal inflammation. In vivo and in vitro models of intestinal inflammation were constructed by lipopolysaccharide (LPS) injection in mice and LPS treatment on intestinal epithelial cells. A possible regulatory mechanism involving SCFA, CCAAT enhancer-binding protein beta (CEBPB), microRNA-145 (miR-145), and dual-specificity phosphatase 6 (DUSP6) in intestinal inflammation was verified by ChIP assay and dual-luciferase reporter gene assay. To evaluate the effects of SCFA on LPS-treated intestinal epithelial cells, the expression of relevant genes and inflammatory factors (IL-6, TNF-α, and IL-1ß) were determined. Last, the role of SCFA in vivo was explored through the scoring of disease activity index (DAI) and observation of colonic histology of LPS-treated mice. SCFA decreased the CEBPB expression in mouse colon tissues and small intestine epithelial cells induced by LPS. Furthermore, CEBPB could bind to the miR-145 promoter to inhibit its expression, thereby promoting the expression of DUSP6. In addition, SCFA improved the DAI, colonic histology, and the expression of serum inflammatory factors in LPS-treated mice and cells, noting that SCFA alleviated intestinal inflammation in vitro and in vivo. To sum up, SCFA inhibited DUSP6 by upregulating miR-145 through CEBPB repression and thus prevented the development of intestinal inflammation.

DHEA Inhibits Leukocyte Recruitment through Regulation of the Integrin Antagonist DEL-1.

Leukocytes are rapidly recruited to sites of inflammation via interactions with the vascular endothelium. The steroid hormone dehydroepiandrosterone (DHEA) exerts anti-inflammatory properties; however, the underlying mechanisms are poorly understood. In this study, we show that an anti-inflammatory mechanism of DHEA involves the regulation of developmental endothelial locus 1 (DEL-1) expression. DEL-1 is a secreted homeostatic factor that inhibits ß2-integrin-dependent leukocyte adhesion, and the subsequent leukocyte recruitment and its expression is downregulated upon inflammation. Similarly, DHEA inhibited leukocyte adhesion to the endothelium in venules of the inflamed mouse cremaster muscle. Importantly, in a model of lung inflammation, DHEA limited neutrophil recruitment in a DEL-1-dependent manner. Mechanistically, DHEA counteracted the inhibitory effect of inflammation on DEL-1 expression. Indeed, whereas TNF reduced DEL-1 expression and secretion in endothelial cells by diminishing C/EBPß binding to the DEL-1 gene promoter, DHEA counteracted the inhibitory effect of TNF via activation of tropomyosin receptor kinase A (TRKA) and downstream PI3K/AKT signaling that restored C/EBPß binding to the DEL-1 promoter. In conclusion, DHEA restrains neutrophil recruitment by reversing inflammation-induced downregulation of DEL-1 expression. Therefore, the anti-inflammatory DHEA/DEL-1 axis could be harnessed therapeutically in the context of inflammatory diseases.

Infection and RNA-seq analysis of a zebrafish tlr2 mutant shows a broad function of this toll-like receptor in transcriptional and metabolic control and defense to Mycobacterium marinum infection.

BACKGROUND: The function of Toll-like receptor 2 (TLR2) in host defense against pathogens, especially Mycobacterium tuberculosis (Mtb) is poorly understood. To investigate the role of TLR2 during mycobacterial infection, we analyzed the response of tlr2 zebrafish mutant larvae to infection with Mycobacterium marinum (Mm), a close relative to Mtb, as a model for tuberculosis. We measured infection phenotypes and transcriptome responses using RNA deep sequencing in mutant and control larvae. RESULTS: tlr2 mutant embryos at 2 dpf do not show differences in numbers of macrophages and neutrophils compared to control embryos. However, we found substantial changes in gene expression in these mutants, particularly in metabolic pathways, when compared with the heterozygote tlr2+/- control. At 4 days after Mm infection, the total bacterial burden and the presence of extracellular bacteria were higher in tlr2-/- larvae than in tlr2+/-, or tlr2+/+ larvae, whereas granuloma numbers were reduced, showing a function of Tlr2 in zebrafish host defense. RNAseq analysis of infected tlr2-/- versus tlr2+/- shows that the number of up-regulated and down-regulated genes in response to infection was greatly diminished in tlr2 mutants by at least 2 fold and 10 fold, respectively. Analysis of the transcriptome data and qPCR validation shows that Mm infection of tlr2 mutants leads to decreased mRNA levels of genes involved in inflammation and immune responses, including il1b, tnfb, cxcl11aa/ac, fosl1a, and cebpb. Furthermore, RNAseq analyses revealed that the expression of genes for Maf family transcription factors, vitamin D receptors, and Dicps proteins is altered in tlr2 mutants with or without infection. In addition, the data indicate a function of Tlr2 in the control of induction of cytokines and chemokines, such as the CXCR3-CXCL11 signaling axis. CONCLUSION: The transcriptome and infection burden analyses show a function of Tlr2 as a protective factor against mycobacteria. Transcriptome analysis revealed tlr2-specific pathways involved in Mm infection, which are related to responses to Mtb infection in human macrophages. Considering its dominant function in control of transcriptional processes that govern defense responses and metabolism, the TLR2 protein can be expected to be also of importance for other infectious diseases and interactions with the microbiome.

MAPK and NF-κB signalling pathways regulate the expression of miRNA, let-7f in human endocervical epithelial cells.

MicroRNAs (miRNAs) mediate post-transcriptional gene suppression and are a critical component of the complex regulatory networks in epithelial immune responses. Transcription of miRNA genes in epithelial cells can be elaborately controlled through Toll-like receptors (TLRs), and associated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) pathways, leading to nuclear transcription factor associated-transactivation and transrepression of miRNAs. MiRNA, let-7f is involved in the regulation of innate immune responses post TLR3 stimulation in human endocervical cells (End1/E6E7) and decreased let-7f is associated with poor immune activation. Thus, expression of let-7f is under strict control. However, the mechanism by which let-7f is regulated in these cells is not known. Therefore, in the present study, we have investigated the role of MAPK and NF-κB in the transcription of let-7f. We report that signalling of TLR3, results in activation of multiple signalling pathways including MAPK/ERK, JNK, p38, and NF-κB. Of these MAPK/ p38 and JNK directly influence the expression of let-7f in End1/E6E7 cells. Inhibition of ERK and NF-κB up regulates the expression of let-7f and its transcription factor, C/EBPß. In conclusion, we have identified a system through which TLR3 mediated immune response is regulated by C/EBPß and let-7f through the temporal activation of MAPK and NF-κB in human endocervical cells.

Mouse adenovirus type 1 infection of adipose tissue.

Human adenovirus (HAdV) type 36 seropositivity has been linked to obesity in humans. That link is supported by a small number of studies using HAdV-36 infection of animals that are not natural hosts for HAdVs. In this study, we infected mice with mouse adenovirus type 1 (MAV-1), a mouse pathogen, to determine whether MAV-1 infected adipose tissue and was associated with adipose tissue inflammation and obesity. We detected MAV-1 in adipose tissue during acute MAV-1 infection, but we did not detect virus-induced increases in adipose tissue cytokine expression or histological evidence of adipose tissue inflammation during acute infection. MAV-1 did not persist in adipose tissue at later times, and we did not detect long-term adipose inflammation, increased adipose tissue mass, or body weight in infected mice. Our data indicate that MAV-1 is not associated with obesity in infected mice.

Impairment of Hematopoietic Precursor Cell Activation during the Granulopoietic Response to Bacteremia in Mice with Chronic-Plus-Binge Alcohol Administration.

Alcohol abuse impairs immune defense. To study the effect of chronic-plus-binge alcohol exposure on the granulopoietic response, acute alcohol intoxication (intraperitoneal injection of 5 g alcohol/kg body weight) was introduced to mice chronically fed on the Lieber-DeCarli low-fat liquid alcohol diet for 5 weeks. Bacteremia was induced by intravenous injection of Escherichia coli Bacteremia caused a remarkable increase in marrow lin- c-kit+ Sca-1+ cells. Activation of cell proliferation supported the increase in marrow lin- c-kit+ Sca-1+ cells. Alcohol administration inhibited this activation of lin- c-kit+ Sca-1+ cells. The bone marrow of pair-fed control mice receiving intraperitoneal saline stored a large number of mature granulocytes expressing a high level of Gr1 (Gr1hi cells). The proportion of Gr1hi cells and the total number of Gr1+ cells were markedly reduced in the bone marrow, along with an increase in the ratio of Gr1+ granulocytes in peripheral white blood cells following bacteremia. E. coli infection stimulated proliferation of granulopoietic precursor cells, resulting in a marked increase in the ratio of immature Gr1lo cells in the bone marrow. Alcohol administration itself triggered marrow release of Gr1+ cells, resulting in reduction of the marrow granulocyte reserve with an elevation of granulocytes in the circulation. Alcohol also impaired activation of granulopoietic precursor proliferation following bacteremia. Alcohol disrupted lipopolysaccharide (LPS)-TLR4-ERK1/2-cyclin D1 signaling and inhibited upregulation of Sca-1 and C/EBPß expression by lineage-negative marrow cells in response to bacteremia. These results indicate that chronic-plus-binge alcohol exposure inhibits the granulopoietic response by disrupting key cell signaling for hematopoietic precursor cell activation and commitment to granulocyte lineage development.

MicroRNA 26a (miR-26a)/KLF4 and CREB-C/EBPß regulate innate immune signaling, the polarization of macrophages and the trafficking of Mycobacterium tuberculosis to lysosomes during infection.

For efficient clearance of Mycobacterium tuberculosis (Mtb), macrophages tilt towards M1 polarization leading to the activation of transcription factors associated with the production of antibacterial effector molecules such as nitric oxide (NO) and proinflammatory cytokines such as interleukin 1 ß (IL-1ß) and tumor necrosis factor α (TNF-α). At the same time, resolution of inflammation is associated with M2 polarization with increased production of arginase and cytokines such as IL-10. The transcriptional and post-transcriptional mechanisms that govern the balance between M1 and M2 polarization, and bacteria-containing processes such as autophagy and trafficking of Mtb to lysosomes, are incompletely understood. Here we report for the first time, that the transcription factor KLF4 is targeted by microRNA-26a (miR-26a). During Mtb infection, downregulation of miR-26a (observed both ex vivo and in vivo) facilitates upregulation of KLF4 which in turn favors increased arginase and decreased iNOS activity. We further demonstrate that KLF4 prevents trafficking of Mtb to lysosomes. The CREB-C/EBPß signaling axis also favors M2 polarization. Downregulation of miR-26a and upregulation of C/ebpbeta were observed both in infected macrophages as well as in infected mice. Knockdown of C/ebpbeta repressed the expression of selected M2 markers such as Il10 and Irf4 in infected macrophages. The importance of these pathways is substantiated by observations that expression of miR-26a mimic or knockdown of Klf4 or Creb or C/ebpbeta, attenuated the survival of Mtb in macrophages. Taken together, our results attribute crucial roles for the miR-26a/KLF4 and CREB-C/EBPßsignaling pathways in regulating the survival of Mtb in macrophages. These studies expand our understanding of how Mtb hijacks host signaling pathways to survive in macrophages, and open up new exploratory avenues for host-targeted interventions.

p300 and C/EBPß-regulated IKKß expression are involved in thrombin-induced IL-8/CXCL8 expression in human lung epithelial cells.

Asthma and chronic obstructive pulmonary disease (COPD) are common chronic lung inflammatory diseases. Thrombin and interleukin (IL)-8/C-X-C chemokine ligand 8 (CXCL8) play critical roles in lung inflammation. Our previous study showed that c-Src-dependent IκB kinase (IKK)/IκBα/nuclear factor (NF)-κB and mitogen-activated protein kinase kinase kinase 1 (MEKK1)/extracellular signal-regulated kinase (ERK)/ribosomal S6 protein kinase (RSK)-dependent CAAT/enhancer-binding protein ß (C/EBPß) activation are involved in thrombin-induced IL-8/CXCL8 expression in human lung epithelial cells. In this study, we aimed to investigate the roles of p300 and C/EBPß-reliant IKKß expression in thrombin-induced IL-8/CXCL8 expression. Thrombin-induced increases in IL-8/CXCL8-luciferase activity and IL-8/CXCL8 release were inhibited by p300 small interfering (siRNA). Thrombin-caused histone H3 acetylation was attenuated by p300 siRNA. Stimulation of cells with thrombin for 12h resulted in increases in IKKß expression and phosphorylation in human lung epithelial cells. However, thrombin did not affect p65 expression. Moreover, 12h of thrombin stimulation produced increases in IKKß expression and phosphorylation, and IκBα phosphorylation, which were inhibited by C/EBPß siRNA. Finally, treatment of cells with thrombin caused increases in p300 and C/EBPß complex formation, p65 and C/EBPß complex formation, and recruitment of p300, p65, and C/EBPß to the IL-8/CXCL8 promoter. These results imply that p300-dependent histone H3 acetylation and C/EBPß-regulated IKKß expression contribute to thrombin-induced IL-8/CXCL8 expression in human lung epithelial cells. Results of this study will help clarify C/EBPß signaling pathways involved in thrombin-induced IL-8/CXCL8 expression in human lung epithelial cells.

2-Methoxyestradiol protects against IgG immune complex-induced acute lung injury by blocking NF-κB and CCAAT/enhancer-binding protein ß activities.

Increasing evidences indicate that 2-Methoxyestradiol (2ME2) plays an essential role in protecting against inflammatory responses. However, its effect on IgG immune complex (IC)-induced acute lung injury (ALI) remains enigmatic. In the study, by using i.p. administration of 2ME2, we evaluated its influence on IgG IC-induced pulmonary injury in mice. We found that during IgG IC-induced ALI, mice treated by 2ME2 displayed a substantial decrease in vascular permeability and neutrophil influx (represented by myeloperoxidase activity) when compared with their counterparts receiving vehicle treatment. Furthermore, 2ME2 treatment significantly decreased pro-inflammatory mediator production and inflammatory cell, especially neutrophil accumulation in bronchoalveolar lavage fluids (BALFs) upon IgG IC stimulation. In vitro, IgG IC-triggered inflammatory mediator production was markedly down-regulated by 2ME2 in macrophages. Moreover, we verified that the activation of the transcription factors, NF-κB and CCAAT/enhancer-binding protein (C/EBP) ß, were inhibited by 2ME2 in IgG IC-challenged macrophages. We demonstrated that alleviation of NF-κB-dependent transcription might be associated with reduced phosphorylation of NF-κB p65, and reduction of C/EBP activation was directly linked to its expression. In addition, we discovered that IgG IC-stimulated phosphorylation of both p38 MAPK and ERK1/2 was alleviated by 2ME2. These data indicated a novel strategy for blockade of IgG IC-induced inflammatory activities.

Inhibition of IFN-γ-Induced Nitric Oxide Dependent Antimycobacterial Activity by miR-155 and C/EBPß.

miR-155 (microRNA-155) is an important non-coding RNA in regulating host crucial biological regulators. However, its regulatory function in mycobacterium infection remains unclear. Our study demonstrates that miR-155 expression is significantly increased in macrophages after Mycobacterium marinum (M.m) infection. Transfection with anti-miR-155 enhances nitric oxide (NO) synthesis and decreases the mycobacterium burden, and vice versa, in interferon γ (IFN-γ) activated macrophages. More importantly, miR-155 can directly bind to the 3'UTR of CCAAT/enhancer binding protein ß (C/EBPß), a positive transcriptional regulator of nitric oxide synthase (NOS2), and regulate C/EBPß expression negatively. Knockdown of C/EBPß inhibit the production of nitric oxide synthase and promoted mycobacterium survival. Collectively, these data suggest that M.m-induced upregulation of miR-155 downregulated the expression of C/EBPß, thus decreasing the production of NO and promoting mycobacterium survival, which may provide an insight into the function of miRNA in subverting the host innate immune response by using mycobacterium for its own profit. Understanding how miRNAs partly regulate microbicidal mechanisms may represent an attractive way to control tuberculosis infectious.

Antagonistic effects of IL-17 and D-resolvins on endothelial Del-1 expression through a GSK-3ß-C/EBPß pathway.

Del-1 is an endothelial cell-secreted anti-inflammatory protein. In humans and mice, Del-1 expression is inversely related to that of IL-17, which inhibits Del-1 through hitherto unidentified mechanism(s). Here we show that IL-17 downregulates human endothelial cell expression of Del-1 by targeting a critical transcription factor, C/EBPß. Specifically, IL-17 causes GSK-3ß-dependent phosphorylation of C/EBPß, which is associated with diminished C/EBPß binding to the Del-1 promoter and suppressed Del-1 expression. This inhibitory action of IL-17 can be reversed at the GSK-3ß level by PI3K/Akt signalling induced by D-resolvins. The biological relevance of this regulatory network is confirmed in a mouse model of inflammatory periodontitis. Intriguingly, resolvin-D1 (RvD1) confers protection against IL-17-driven periodontal bone loss in a Del-1-dependent manner, indicating an RvD1-Del-1 axis against IL-17-induced pathological inflammation. The dissection of signalling pathways regulating Del-1 expression provides potential targets to treat inflammatory diseases associated with diminished Del-1 expression, such as periodontitis and multiple sclerosis.

Helios, and not FoxP3, is the marker of activated Tregs expressing GARP/LAP.

Regulatory T cells (Tregs) are key players of immune regulation/dysregulation both in physiological and pathophysiological settings. Despite significant advances in understanding Treg function, there is still a pressing need to define reliable and specific markers that can distinguish different Treg subpopulations. Herein we show for the first time that markers of activated Tregs [latency associated peptide (LAP) and glycoprotein A repetitions predominant (GARP, or LRRC32)] are expressed on CD4+FoxP3- T cells expressing Helios (FoxP3-Helios+) in the steady state. Following TCR activation, GARP/LAP are up-regulated on CD4+Helios+ T cells regardless of FoxP3 expression (FoxP3+/-Helios+). We show that CD4+GARP+/-LAP+ Tregs make IL-10 immunosuppressive cytokine but not IFN-γ effector cytokine. Further characterization of FoxP3/Helios subpopulations showed that FoxP3+Helios+ Tregs proliferate in vitro significantly less than FoxP3+Helios- Tregs upon TCR stimulation. Unlike FoxP3+Helios- Tregs, FoxP3+Helios+ Tregs secrete IL-10 but not IFN-γ or IL-2, confirming they are bona fide Tregs with immunosuppressive characteristics. Taken together, Helios, and not FoxP3, is the marker of activated Tregs expressing GARP/LAP, and FoxP3+Helios+ Tregs have more suppressive characteristics, compared with FoxP3+Helios- Tregs. Our work implies that therapeutic modalities for treating autoimmune and inflammatory diseases, allergies and graft rejection should be designed to induce and/or expand FoxP3+Helios+ Tregs, while therapies against cancers or infectious diseases should avoid such expansion/induction.

COX-2 gene promoter DNA methylation status in eutopic and ectopic endometrium of Egyptian women with endometriosis.

The pathophysiology of COX-2 expression in endometriosis is a matter of debate. The aim was to investigate the role of DNA methylation of the NF-IL6 site within the promoter of COX-2 gene in the pathogenesis of endometriosis. The endometrial tissues (ectopic and eutopic) were collected from 60 women with endometriosis and 30 women without endometriosis (control group). The methylation status of COX-2 was examined by methylation-specific PCR. Quantitative real-time PCR (RT-PCR) was performed to measure COX-2 mRNA levels in endometrial tissues. We found significantly higher levels of COX-2 in ectopic endometriotic tissue compared with eutopic tissue. Also, we found that the frequencies of methylation status of the NF-IL6 site within the COX-2 promoter in the eutopic and ectopic endometrial tissues of endometriosis groups were significantly decreased in comparison to controls (P=0.002, P=0.000 respectively). Our study demonstrated that DNA hypomethylation of the NF-IL6 site within the promoter of COX-2 gene could be a key mechanism for its elevated expression in the eutopic and ectopic tissues of endometriosis.

Endogenous prostaglandin E2 potentiates anti-inflammatory phenotype of macrophage through the CREB-C/EBP-ß cascade.

Macrophages have important functions in tissue homeostasis, but the exact mechanisms regarding wide spectrum of macrophage phenotype remain unresolved. In this study, we report that mouse bone marrow derived naïve macrophages produce prostaglandin E2 (PGE2 ) endogenously, resulting in anti-inflammatory gene expression upon differentiation induced by macrophage colony stimulating factor (M-CSF). Cyclooxygenase (COX) inhibition by indomethacin reduced endogenous PGE2 production of macrophages and subsequently reduced arg1, IL10 and Mrc1, YmI and FizzI gene expressions. Of note, PGE2 phosphorylates CREB via EP2 and EP4 receptor ligation, thereby transcriptionally increasing C/EBP-ß expression in BALB/c bone marrow derived macrophages. Activated CREB directly binds to the CREB-responsive element of the C/EBP-ß promoter, such that PGE2 ultimately reinforces arg1, IL10 and Mrc1 gene expression. Cyclic AMP activator forskolin also phosphorylated CREB and induced the C/EBP-ß cascade, but this was completely blocked by the PKA inhibitor, H89. Consequently, M-CSF grown macrophages inhibited T-cell proliferation but the inhibition ability was reduced when the COX is inhibited by indomethacin or macrophage C/EBP-ß expression was decreased by siRNA transduction. Our results collectively describe the molecular basis for homeostatic macrophage differentiation by endogenous PGE2 .

Degradation of polyomavirus JC T-antigen by stress involves the LIP isoform of C/EBP.

Endoplasmic reticulum (ER) stress is caused by the accumulation of misfolded or unfolded proteins in the lumen of the endoplasmic reticulum. CCAAT/enhancer binding proteins are one of the cellular proteins whose expression is upregulated during ER stress. Previously, we have identified C/EBPbeta isoforms, especially LIP, as a negative regulator of polyomavirus JC (JCV), the causative agent of the demyelinating disease progressive multifocal leukoencephalopathy (PML). Here, we show that the induction of ER stress by thapsigargin increase the expression of endogenous LIP and the degradation of JCV T-antigen in a JCV-transgenic mouse tumor cell line. Our results also revealed that overexpression of LIP significantly reduced the level of T-Ag and this effect is reversed upon siRNA-mediated silencing of LIP. Immunoprecipitation/Western blot experiments indicated that LIP interacts with T-antigen directly. Treatment of cells that overexpress LIP with MG115, a proteasome inhibitor, partially rescued LIP-mediated degradation of T-antigen. Our observations point to a role of LIP in ER stress regulation of T-antigen stability and may open a new avenue to study host-virus interaction during ER stress.

Dendritic cell-mediated survival signals in Eµ-Myc B-cell lymphoma depend on the transcription factor C/EBPß.

The capacity of dendritic cells (DCs) to regulate tumour-specific adaptive immune responses depends on their proper differentiation and homing status. Whereas DC-associated tumour-promoting functions are linked to T-cell tolerance and formation of an inflammatory milieu, DC-mediated direct effects on tumour growth have remained unexplored. Here we show that deletion of DCs substantially delays progression of Myc-driven lymphomas. Lymphoma-exposed DCs upregulate immunomodulatory cytokines, growth factors and the CCAAT/enhancer-binding protein ß (C/EBPß). Moreover, Eµ-Myc lymphomas induce the preferential translation of the LAP/LAP* isoforms of C/EBPß. C/EBPß(-/-) DCs are unresponsive to lymphoma-associated cytokine changes and in contrast to wild-type DCs, they are unable to mediate enhanced Eµ-Myc lymphoma cell survival. Antigen-specific T-cell proliferation in lymphoma-bearing mice is impaired; however, this immune suppression is reverted by the DC-restricted deletion of C/EBPß. Thus, we show that C/EBPß-controlled DC functions are critical steps for the creation of a lymphoma growth-promoting and -immunosuppressive niche.

Can the suppressive activity of myeloid-derived suppressor cells be "chop"ped?

Myeloid-derived suppressor cells (MDSCs) are one of the major negative regulators of immune responses. In this issue of Immunity, Thevenot et al. (2014) showed that in tumors, the suppressive activity of MDSCs is regulated by transcription factor Chop.

The stress-response sensor chop regulates the function and accumulation of myeloid-derived suppressor cells in tumors.

Adaptation of malignant cells to the hostile milieu present in tumors is an important determinant of their survival and growth. However, the interaction between tumor-linked stress and antitumor immunity remains poorly characterized. Here, we show the critical role of the cellular stress sensor C/EBP-homologous protein (Chop) in the accumulation and immune inhibitory activity of tumor-infiltrating myeloid-derived suppressor cells (MDSCs). MDSCs lacking Chop had decreased immune-regulatory functions and showed the ability to prime T cell function and induce antitumor responses. Chop expression in MDSCs was induced by tumor-linked reactive oxygen and nitrogen species and regulated by the activating-transcription factor-4. Chop-deficient MDSCs displayed reduced signaling through CCAAT/enhancer-binding protein-ß, leading to a decreased production of interleukin-6 (IL-6) and low expression of phospho-STAT3. IL-6 overexpression restored immune-suppressive activity of Chop-deficient MDSCs. These findings suggest the role of Chop in tumor-induced tolerance and the therapeutic potential of targeting Chop in MDSCs for cancer immunotherapy.

TSC1 controls macrophage polarization to prevent inflammatory disease.

Macrophages acquire distinct phenotypes during tissue stress and inflammatory responses, but the mechanisms that regulate the macrophage polarization are poorly defined. Here we show that tuberous sclerosis complex 1 (TSC1) is a critical regulator of M1 and M2 phenotypes of macrophages. Mice with myeloid-specific deletion of TSC1 exhibit enhanced M1 response and spontaneously develop M1-related inflammatory disorders. However, TSC1-deficient mice are highly resistant to M2-polarized allergic asthma. Inhibition of the mammalian target of rapamycin (mTOR) fails to reverse the hypersensitive M1 response of TSC1-deficient macrophages, but efficiently rescues the defective M2 polarization. Deletion of mTOR also fails to reverse the enhanced inflammatory response of TSC1-deficient macrophages. Molecular studies indicate that TSC1 inhibits M1 polarization by suppressing the Ras GTPase-Raf1-MEK-ERK pathway in mTOR-independent manner, whereas TSC1 promotes M2 properties by mTOR-dependent CCAAT/enhancer-binding protein-ß pathways. Overall, these findings define a key role for TSC1 in orchestrating macrophage polarization via mTOR-dependent and independent pathways.

Differential control of Mincle-dependent cord factor recognition and macrophage responses by the transcription factors C/EBPß and HIF1α.

Trehalose-6,6-dimycolate (TDM), the mycobacterial cord factor, and its synthetic analog Trehalose-6,6-dibehenate (TDB) bind to the C-type lectin receptors macrophage-inducible C-type lectin (Mincle) and Mcl to activate macrophages. Genetically, the transcriptional response to TDB/TDM has been defined to require FcRγ-Syk-Card9 signaling. However, TDB/TDM-triggered kinase activation has not been studied well, and it is largely unknown which transcriptional regulators bring about inflammatory gene expression. In this article, we report that TDB/TDM caused only weak Syk-phosphorylation in resting macrophages, consistent with low basal Mincle expression. However, LPS-priming caused MYD88-dependent upregulation of Mincle, resulting in enhanced TDB/TDM-induced kinase activation and more rapid inflammatory gene expression. TLR-induced Mincle expression partially circumvented the requirement for Mcl in the response to TDB/TDM. To dissect transcriptional responses to TDB/TDM, we mined microarray data and identified early growth response (Egr) family transcription factors as direct Mincle target genes, whereas upregulation of Cebpb and Hif1a required new protein synthesis. Macrophages and dendritic cells lacking C/EBPß showed nearly complete abrogation of TDB/TDM responsiveness, but also failed to upregulate Mincle. Retroviral rescue of Mincle expression in Cebpb-deficient cells restored induction of Egr1, but not of G-CSF. This pattern of C/EBPß dependence was also observed after stimulation with the Dectin-1 ligand Curdlan. Inducible expression of hypoxia-inducible factor 1α (HIF1α) also required C/EBPß. In turn, HIF1α was not required for Mincle expression, kinase activation, and Egr1 or Csf3 expression, but critically contributed to NO production. Taken together, we identify C/EBPß as central hub in Mincle expression and inflammatory gene induction, whereas HIF1α controls Nos2 expression. C/EBPß also connects TLR signals to cord factor responsiveness through MYD88-dependent upregulation of Mincle.