The transcription factor C/EBPß orchestrates dendritic cell maturation and functionality under homeostatic and malignant conditions.

Dendritic cell (DC) maturation is a prerequisite for the induction of adaptive immune responses against pathogens and cancer. Transcription factor (TF) networks control differential aspects of early DC progenitor versus late-stage DC cell fate decisions. Here, we identified the TF C/EBPß as a key regulator for DC maturation and immunogenic functionality under homeostatic and lymphoma-transformed conditions. Upon cell-specific deletion of C/EBPß in CD11c+MHCIIhi DCs, gene expression profiles of splenic C/EBPß-/- DCs showed a down-regulation of E2F cell cycle target genes and associated proliferation signaling pathways, whereas maturation signatures were enriched. Total splenic DC cell numbers were modestly increased but differentiation into cDC1 and cDC2 subsets were unaltered. The splenic CD11c+MHCIIhiCD64+ DC compartment was also increased, suggesting that C/EBPß deficiency favors the expansion of monocytic-derived DCs. Expression of C/EBPß could be mimicked in LAP/LAP* isoform knockin DCs, whereas the short isoform LIP supported a differentiation program similar to deletion of the full-length TF. In accordance with E2F1 being a negative regulator of DC maturation, C/EBPß-/- bone marrow-derived DCs matured much faster enabling them to activate and polarize T cells stronger. In contrast to a homeostatic condition, lymphoma-exposed DCs exhibited an up-regulation of the E2F transcriptional pathways and an impaired maturation. Pharmacological blockade of C/EBPß/mTOR signaling in human DCs abrogated their protumorigenic function in primary B cell lymphoma cocultures. Thus, C/EBPß plays a unique role in DC maturation and immunostimulatory functionality and emerges as a key factor of the tumor microenvironment that promotes lymphomagenesis.

Function and characterization of the promoter region of perilipin 1 (PLIN1): Roles of E2F1, PLAG1, C/EBPß, and SMAD3 in bovine adipocytes.

Perilipin 1 (PLIN1) protein, also known as lipid droplet-associated protein, is encoded by the PLIN1 gene and is able to anchor itself to the membranes of lipid droplets. The phosphorylation of PLIN1 is critical for the mobilization of fat in adipose tissue and plays an important role in regulating lipolysis and lipid storage in adipocytes. However, research on the synthesis and lipid metabolism of lipid droplets by PLIN1 in bovine adipocytes is limited. In the present study, we found that bovine PLIN1 was highly expressed in subcutaneous adipose tissue. The highest level of PLIN1 mRNA expression in bovine adipocytes was observed on day 6 of differentiation. Moreover, the cytoplasmic subcellular localization of PLIN1 was observed in bovine preadipocytes. To elucidate the molecular mechanism of bovine PLIN1 transcriptional regulation, we cloned eight fragments containing the 5' regulatory region of the PLIN1 gene. The results showed that the -209/-17 bp region of the bovine PLIN1 gene was the core promoter region. Based on the transcriptional activities of the promoter vector fragments, the luciferase activity of the mutated fragment, the siRNA interference, and the results of the electrophoretic mobility shift assay (EMSA), we identified the binding sites of E2F transcription factor 1 (E2F1), pleiomorphic adenoma gene 1 (PLAG1), CCAAT enhancer binding protein beta (C/EBPß), and SMAD family member 3 (SMAD3) as the transcriptional activators or repressors of the core promoter region. Further experiments confirmed that the knockdown of the PLIN1 gene affected the ability of these transcription factors to regulate the lipid metabolism in bovine adipocytes. In conclusion, our results reveal a potential mechanism for the transcriptional regulation of PLIN1 in bovine adipocytes.

Regulation of senescence and the SASP by the transcription factor C/EBPß.

Oncogene-induced senescence (OIS) serves as an important barrier to tumor progression in cells that have acquired activating mutations in RAS and other oncogenes. Senescent cells also produce a secretome known as the senescence-associated secretory phenotype (SASP) that includes pro-inflammatory cytokines and chemokines. SASP factors reinforce and propagate the senescence program and identify senescent cells to the immune system for clearance. The OIS program is executed by several transcriptional effectors that include p53, RB, NF-κB and C/EBPß. In this review, we summarize the critical role of C/EBPß in regulating OIS and the SASP. Post-translational modifications induced by oncogenic RAS signaling control C/EBPß activity and dimerization, and these alterations switch C/EBPß to a pro-senescence form during OIS. In addition, C/EBPß is regulated by a unique 3'UTR-mediated mechanism that restrains its activity in tumor cells to facilitate senescence bypass and suppression of the SASP.

Long Non-coding RNAs: Regulators of the Activity of Myeloid-Derived Suppressor Cells.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous cell population with potent immunosuppressive functions. They play major roles in cancer and many of the pathologic conditions associated with inflammation. Long non-coding RNAs (lncRNAs) are untranslated functional RNA molecules. The lncRNAs are involved in the control of a wide variety of cellular processes and are dysregulated in different diseases. They can participate in the modulation of immune function and activity of inflammatory cells, including MDSCs. This mini review focuses on the emerging role of lncRNAs in MDSC activity. We summarize how lncRNAs modulate the generation, recruitment, and immunosuppressive functions of MDSCs and the underlying mechanisms.

CCAAT/Enhancer Binding Protein ß-Mediated MMP3 Upregulation Promotes Esophageal Squamous Cell Cancer Invasion In Vitro and Is Associated with Metastasis in Human Patients.

Aims: Metastasis is a significant obstacle to curing esophageal squamous cell carcinoma (ESCC). The CCAAT/enhancer binding protein ß (C/EBPß) and matrix metalloproteinase 3 (MMP3) are thought to play key roles in cancer invasion and metastasis. In this study, we aimed to detect whether C/EBPß-mediated tumor invasion was dependent on MMP3. In addition, we determined whether C/EBPß upregulation was associated with MMP3 levels and metastatic status in patients with ESCC. Materials and Methods: A total of 126 patients with ESCC were recruited for this study. The mRNA and protein levels of C/EBPß and MMP3 in ESCC cell lines and specimens from ESCC patient were determined by reverse transcription-polymerase chain reaction and western blot, respectively. Tumor cell invasion was analyzed using an in vitro Matrigel Invasion Assay. The correlation between C/EBPß and MMP3 expression was determined by Pearson's correlation analysis. Results: Both mRNA and protein levels of MMP3 were upregulated by C/EBPß overexpression and downregulated by C/EBPß siRNA in KYSE150 cell cultures. The promotion of ESCC cell invasion through C/EBPß was inhibited by MMP3 siRNA. The level of C/EBPß was correlated with MMP3 and metastatic status in patients with ESCC. Conclusions: C/EBPß upregulation promoted tumor cell invasion in an MMP3-dependent manner in vitro and was associated with metastatic status in ESCC.

CCAAT/Enhancer Binding Protein ß inhibits myogenic differentiation via ID3.

Myogenesis is regulated by the coordinated expression of muscle regulatory factors, a family of transcription factors that includes MYOD, MYF5, myogenin and MRF4. Muscle regulatory factors are basic helix-loop-helix transcription factors that heterodimerize with E proteins to bind the regulatory regions of target genes. Their activity can be inhibited by members of the Inhibitor of DNA binding and differentiation (ID) family, which bind E-proteins with high affinity, thereby preventing muscle regulatory factor-dependent transcriptional responses. CCAAT/Enhancer Binding protein beta (C/EBPß) is a transcription factor expressed in myogenic precursor cells that acts to inhibit myogenic differentiation, though the mechanism remains poorly understood. We identify Id3 as a novel C/EBPß target gene that inhibits myogenic differentiation. Overexpression of C/EBPß stimulates Id3 mRNA and protein expression, and is required for C/EBPß-mediated inhibition of myogenic differentiation. Misexpression of C/EBPß in myogenic precursors, such as in models of cancer cachexia, prevents the differentiation of myogenic precursors and we show that loss of Id3 rescues differentiation under these conditions, suggesting that the stimulation of Id3 expression by C/EBPß is an important mechanism by which C/EBPß inhibits myogenic differentiation.

[Differentiation and proliferation of hematopoietic stem cells are regulated by C/EBPß, a transcription factor required for emergency granulopoiesis].

Under stress conditions such as infection, inflammation, and hematopoietic recovery following chemotherapy or transplantation, the hematopoietic system is required to meet the increasing demands, especially from myeloid cells. Therefore, an understanding of the molecular mechanism underlying stress hematopoiesis is clinically imperative. We previously showed that C/EBPß, which is a transcription factor required for emergency granulopoiesis, plays a pivotal role at the level of hematopoietic stem/progenitor cells under stress conditions. Upon exposure to stress, the C/EBPß protein is upregulated in the hematopoietic stem cells. A close examination of C/EBPß knockout mice revealed that C/EBPß regulates the proliferation and differentiation of hematopoietic stem cells at the cost of the self-renewing activity. Further elucidation of the functions and regulation of C/EBPß in hematopoietic stem cells will facilitate an understanding of stress hematopoiesis.

[Functional diversity of disorder-specific macrophages].

Recent research has revealed that macrophages and monocytes comprise various subtypes. Previously, we demonstrated that JMJD3 is vital for macrophage differentiation in response to allergic stimuli. Moreover, we substantiated that Trib1 controls the differentiation of tissue-resident macrophages in peripheral organs, such as adipose tissue. This study aims to elucidate that Ceacam1+Msr1+Ly6C-F4/80-Mac1+ monocytes are essential for the development of fibrosis. Remarkably, these cell types harbor bilobed-like nucleus and some granules in the cytoplasm. Thus, we named these as cell segregated-nucleus-containing atypical monocytes (SatM). The results revealed that NFIL6 is critical for the differentiation of SatM, and the lack of this protein causes a complete deficiency of SatM. Furthermore, the development of fibrosis was prevented in NFIL6-/- chimeric mice and the adoptive transfer of SatM into NFIL6-/- chimeric mice resulted in fibrosis. Thus, macrophage and monocytes comprised multiple subtypes with functional diversity.

The expression of VEGF, myoglobin and CRP2 proteins regulating endometrial remodeling in the porcine endometrial tissues during follicular and luteal phase.

Endometrial remodeling is important for successful embryo development and implantation in pigs. Therefore, this study investigated change of proteins regulating endometrial remodeling on follicular and luteal phase in porcine endometrial tissues. The endometrial tissue samples were collected from porcine uterus during follicular and luteal phase, vascular endothelial growth factor (VEGF), myoglobin and cysteine-rich protein 2 (CRP2) proteins were expressed by immnofluorescence, immunoblotting, and determined by 2-DE and MALDI-TOF/MS. We found that VEGF, myoglobin and CRP2 were strongly localized in endometrial tissues during luteal phase, but not follicular phase. The protein levels of VEGF, myoglobin and CRP2 in endometrial tissues were higher than luteal phase (P < 0.05). These results may provide understanding of intrauterine environment during estrous cycle in pigs, and will be used in animal reproduction for developing specific biomarkers in the future.

The recurrent architecture of tumour initiation, progression and drug sensitivity.

Recent studies across multiple tumour types are starting to reveal a recurrent regulatory architecture in which genomic alterations cluster upstream of functional master regulator (MR) proteins, the aberrant activity of which is both necessary and sufficient to maintain tumour cell state. These proteins form small, hyperconnected and autoregulated modules (termed tumour checkpoints) that are increasingly emerging as optimal biomarkers and therapeutic targets. Crucially, as their activity is mostly dysregulated in a post-translational manner, rather than by mutations in their corresponding genes or by differential expression, the identification of MR proteins by conventional methods is challenging. In this Opinion article, we discuss novel methods for the systematic analysis of MR proteins and of the modular regulatory architecture they implement, including their use as a valuable reductionist framework to study the genetic heterogeneity of human disease and to drive key translational applications.

Epigenetic and Transcriptional Regulation of IRAK-M Expression in Macrophages.

During macrophage activation, expression of IL-1R-associated kinase (IRAK)-M is induced to suppress TLR-mediated responses and is a hallmark of endotoxin tolerance. Endotoxin tolerance requires tight regulation of genes occurring at the transcriptional and epigenetic levels. To identify novel regulators of IRAK-M, we used RAW 264.7 macrophages and performed a targeted RNA interference screen of genes encoding chromatin-modifying enzymes, signaling molecules, and transcription factors involved in macrophage activation. Among these, the transcription factor CCAAT/enhancer binding protein (C/EBP)ß, known to be involved in macrophage inactivation, was necessary for the induction of IRAK-M expression. Chromatin immunoprecipitation showed that C/EBPß was recruited to the IRAK-M promoter following LPS stimulation and was indispensable for IRAK-M transcriptional activation. Among histone 3-modifying enzymes, our screen showed that knockdown of the histone 3 lysine 27 (H3K27) methyltransferase and part of the polycomb recessive complex 2, enhancer of Zeste 2, resulted in IRAK-M overexpression. In contrast, knockdown of the H3K27 demethylase ubiquitously transcribed tetratricopeptide repeat X chromosome suppressed the induction of IRAK-M in response to LPS stimulation. Accordingly, we demonstrated that H3K27 on the IRAK-M promoter is trimethylated in unstimulated cells and that this silencing epigenetic mark is removed upon LPS stimulation. Our data propose a mechanism for IRAK-M transcriptional regulation according to which, in the naive state, polycomb recessive complex 2 repressed the IRAK-M promoter, allowing low levels of expression; following LPS stimulation, the IRAK-M promoter is derepressed, and transcription is induced to allow its expression.

Chemotherapy-Induced IL34 Enhances Immunosuppression by Tumor-Associated Macrophages and Mediates Survival of Chemoresistant Lung Cancer Cells.

The ability of tumor cells to escape immune destruction and their acquired resistance to chemotherapy are major obstacles to effective cancer therapy. Although immune checkpoint therapies such as anti-PD-1 address these issues in part, clinical responses remain limited to a subpopulation of patients. In this report, we identified IL34 produced by cancer cells as a driver of chemoresistance. In particular, we found that IL34 modulated the functions of tumor-associated macrophages to enhance local immunosuppression and to promote the survival of chemoresistant cancer cells by activating AKT signaling. Targeting IL34 in chemoresistant tumors resulted in a remarkable inhibition of tumor growth when accompanied with chemotherapy. Our results define a pathogenic role for IL34 in mediating immunosuppression and chemoresistance and identify it as a tractable target for anticancer therapy. Cancer Res; 76(20); 6030-42. ©2016 AACR.

L-4F Inhibits Oxidized Low-density Lipoprotein-induced Inflammatory Adipokine Secretion via Cyclic AMP/Protein Kinase A-CCAAT/Enhancer Binding Protein ß Signaling Pathway in 3T3-L1 Adipocytes.

BACKGROUND: Adipocytes behave like a rich source of pro-inflammatory cytokines including monocyte chemoattractant protein-1 (MCP-1). Oxidized low-density lipoprotein (oxLDL) participates in the local chronic inflammatory response, and high-density lipoprotein could counterbalance the proinflammatory function of oxLDL, but the underlying mechanism is not completely understood. This study aimed to evaluate the effect of apolipoprotein A-I mimetic peptide L-4F on the secretion and expression of MCP-1 in fully differentiated 3T3-L1 adipocytes induced by oxLDL and to elucidate the possible mechanisms. METHODS: Fully differentiated 3T3-L1 adipocytes were incubated in the medium containing various concentration of L-4F (0-50 µg/ml) with oxLDL (50 µg/ml) stimulated, with/without protein kinase A (PKA) inhibitor H-89 (10 µmol/L) preincubated. The concentrations of MCP-1 in the supernatant, the mRNA expression of MCP-1, the levels of CCAAT/enhancer binding protein α (C/EBPα), and CCAAT/enhancer binding protein ß (C/EBPß) were evaluated. The monocyte chemotaxis assay was performed by micropore filter method using a modified Boyden chamber. RESULTS: OxLDL stimulation induced a significant increase of MCP-1 expression and secretion in 3T3-L1 adipocytes, which were inhibited by L-4F preincubation in a dose-dependent manner. PKA inhibitor H-89 markedly reduced the oxLDL-induced MCP-1 expression, but no further decrease was observed when H-89 was used in combination with L-4F (50 µg/ml) (P > 0.05). OxLDL stimulation showed no significant effect on C/EBPα protein level but increased C/EBPß protein level in a time-dependent manner. H-89 and L-4F both attenuated C/EBPß protein level in oxLDL-induced 3T3-L1 adipocytes. CONCLUSIONS: OxLDL induces C/EBPß protein synthesis in a time-dependent manner and enhances MCP-1 secretion and expression in 3T3-L1 adipocytes. L-4F dose-dependently counterbalances the pro-inflammatory effect of oxLDL, and cyclic AMP/PKA-C/EBPß signaling pathway may participate in it.

C/EBPß regulates sensitivity to bortezomib in prostate cancer cells by inducing REDD1 and autophagosome-lysosome fusion.

The purpose of this study was to ascertain the mechanisms by which advanced prostate cancer cells resist bortezomib therapy. Several independent studies have shown that cells are protected from proteasome inhibition by increased autophagic activity. We investigated whether C/EBPß, a transcription factor involved in the control of autophagic gene expression, regulates resistance to proteasome inhibition. In PC3 cells over-expressing C/EBPß, turnover of autophagic substrates and expression of core autophagy genes were increased. Conversely, C/EBPß knockdown suppressed autophagosome-lysosome fusion. We also found that C/EBPß knockdown suppressed REDD1 expression to delay early autophagy, an effect rescued by exogenous REDD1. Cells with suppressed C/EBPß levels showed delayed autophagy activation upon bortezomib treatment. Knockdown of C/EBPß sensitized PC3 cells to bortezomib, and blockade of autophagy by chloroquine did not further increase cell death in cells expressing shRNA targeting C/EBPß. Lastly, we observed a decreased growth of PC3 cells and xenografts with C/EBPß knockdown and such xenografts were sensitized to bortezomib treatment. Our results demonstrate that C/EBPß is a critical effector of autophagy via regulation of autolysosome formation and promotes resistance to proteasome inhibitor treatment by increasing autophagy.

[C/EBPß promotes NF-κB-mediated invasion and migration of human renal carcinoma 786-O cells].

OBJECTIVE: To explore the regulatory role of CCAAT/enhancer-binding protein beta (C/EBPß) in invasion and migration of 786-O human renal cancer cells. METHODS: C/EBPß adenoviral overexpression vector (pAd-C/EBPß) and C/EBPß siRNA were constructed and respectively transfered into 786-O cells. Wound healing and Transwell(TM) invasion methods were used to detect the invasion and migration of the cells. Laser scanning confocal microscopy was applied to determine the translocation of nuclear factor κB (NF-κB), the central regulator of epithelial-mesenchymal transition (EMT); Western blotting was performed to examine the levels of EMT key markers E-cadherin, vimentin and tenascin. RESULTS: When C/EBPß was overexpressed, invasion and migration of 786-O cells were enhanced, translocation of NF-κB increased, E-cadherin level was reduced, and vimentin and tenascin levels were elevated. When C/EBPß was knocked down, the results were completely opposite to those of C/EBPß overexpression: invasion and migration of 786-O cells were inhibited, translocation of NF-κB decreased, E-cadherin level was elevated, and vimentin and tenascin expressions were suppressed. Moreover, the above changes were not found in the cells that co-treated with pAd-C/EBPß and C/EBPß siRNA. CONCLUSION: C/EBPß could promote the invasion and migration of human renal carcinoma 786-O cells, and had an inducing effect on nuclear translocation of NF-κB in 786-O cells.

Accelerated apoptosis of peripheral blood monocytes in Cebpb-deficient mice.

The CCAAT/enhancer-binding protein ß (C/EBPß) transcription factor is required for granulopoiesis under stress conditions. However, little is known about its roles in steady state hematopoiesis. Here, we analyzed the peripheral blood and bone marrow of Cebpb(-/-) mice at steady state by flow cytometry and unexpectedly found that the number of peripheral blood monocytes was severely reduced, while the number of bone marrow monocytes was maintained. The ability of Cebpb(-/-) bone marrow cells to give rise to macrophages/monocytes in vitro was comparable to that of wild-type bone marrow cells. Apoptosis of monocytes was enhanced in the peripheral blood, but not in the bone marrow of Cebpb(-/-) mice. These results indicate that C/EBPß is required for the survival of monocytes in peripheral blood.

C/EBPß and Nuclear Factor of Activated T Cells Differentially Regulate Adamts-1 Induction by Stimuli Associated with Vascular Remodeling.

Emerging evidence indicates that the metalloproteinase Adamts-1 plays a significant role in the pathophysiology of vessel remodeling, but little is known about the signaling pathways that control Adamts-1 expression. We show that vascular endothelial growth factor (VEGF), angiotensin-II, interleukin-1ß, and tumor necrosis factor α, stimuli implicated in pathological vascular remodeling, increase Adamts-1 expression in endothelial and vascular smooth muscle cells. Analysis of the intracellular signaling pathways implicated in this process revealed that VEGF and angiotensin-II upregulate Adamts-1 expression via activation of differential signaling pathways that ultimately promote functional binding of the NFAT or C/EBPß transcription factors, respectively, to the Adamts-1 promoter. Infusion of mice with angiotensin-II triggered phosphorylation and nuclear translocation of C/EBPß proteins in aortic cells concomitantly with an increase in the expression of Adamts-1, further underscoring the importance of C/EBPß signaling in angiotensin-II-induced upregulation of Adamts-1. Similarly, VEGF promoted NFAT activation and subsequent Adamts-1 induction in aortic wall in a calcineurin-dependent manner. Our results demonstrate that Adamts-1 upregulation by inducers of pathological vascular remodeling is mediated by specific signal transduction pathways involving NFAT or C/EBPß transcription factors. Targeting of these pathways may prove useful in the treatment of vascular disease.

C/EBPß is a transcriptional key regulator of IL-36α in murine macrophages.

Interleukin (IL)-36α - one of the novel members of the IL-1 family of cytokines - is a potent regulator of dendritic and T cells and plays an important role in inflammatory processes like experimental skin inflammation in mice and in mouse models for human psoriasis. Here, we demonstrate that C/EBPß, a transcription factor required for the selective expression of inflammatory genes, is a key activator of the Il36A gene in murine macrophages. RNAi-mediated suppression of C/EBPß expression in macrophages (C/EBPß(low) cells) significantly impaired Il36A gene induction following challenge with LPS. Despite the presence of five predicted C/EBP binding sites, luciferase reporter assays demonstrated that C/EBPß confers responsiveness to LPS primarily through a half-CRE•C/EBP element in the proximal Il36A promoter. Electrophoretic mobility shift assays showed that C/EBPß but not CREB proteins interact with this critical half-CRE•C/EBP element. In addition, overexpression of C/EBPß in C/EBPß(low) cells enhanced the expression of Il36A whereas CREB-1 had no effect. Finally, chromatin immunoprecipitation confirmed that C/EBPß but neither CREB-1, ATF-2 nor ATF4 is directly recruited to the proximal promoter region of the Il36A gene. Together, these findings demonstrate an essential role of C/EBPß in the regulation of the Il36A gene via the proximal half-CRE•C/EBP element in response to inflammatory stimuli.

Non-steady-state hematopoiesis regulated by the C/EBPß transcription factor.

Steady-state hematopoiesis responds to extracellular stimuli to meet changing demands and also to pathologically altered intracellular signaling. Granulocyte production increases following infection or in response to cytokine stimulation, and activation of the CCAAT/enhancer-binding protein ß (C/EBPß) transcription factor is required for such stress-induced granulopoiesis, whereas C/EBPα plays a critical role in maintaining steady-state granulopoiesis. Different roles of these C/EBP transcription factors in different modes of hematopoiesis are evolutionally conserved from zebrafish to humans. In addition to reactions against infections, C/EBPß is responsible for cancer-driven myelopoiesis, which promotes cancer progression, at least in part, by abrogating the immune response in the cancer microenvironment. The BCR-ABL fusion protein activates emergency-specific pathway of granulopoiesis by upregulating C/EBPß. This in turn causes chronic phase chronic myeloid leukemia, which is characterized by myeloid expansion. The C/EBPß transcription factor also plays a role in other hematological malignancies of both myeloid and lymphoid lineage origin. Thus, elucidation of the upstream and downstream networks surrounding C/EBPß will lead to the development of novel therapeutic strategies for diseases mediated by non-steady-state hematopoiesis.

Microgravity activates p38 MAPK-C/EBPß pathway to regulate the expression of arginase and inflammatory cytokines in macrophages.

OBJECTIVE AND DESIGN: Molecular mechanisms of microgravity-caused immunosuppression are not fully elucidated. In the present study, we investigated the effects of simulated microgravity on macrophage functions and tried to identify the related intracellular signal pathways. MATERIAL OR SUBJECTS: Primary mouse macrophages were used in the present study. The gene expression and function of IL-4-treated mouse macrophages were detected after simulated microgravity or 1 g control. METHODS: Freshly isolated primary mouse macrophages were cultured in a standard simulated microgravity situation using a rotary cell culture system (RCCS-1) and 1 g control conditions. Real-time PCR, western blots and flow cytometry were used to investigate the related intracellular signals and molecule expression. RESULTS: The arginase mRNA and protein levels in freshly isolated primary mouse macrophages under simulated microgravity using RCCS-1 were significantly higher than those under normal gravity. Meanwhile, simulated microgravity induced over-expression of C/EBPß, a transcription factor of arginase promoter, and activation of p38 MAPK, which could increase C/EBPß expression. Furthermore, up-regulation of Interleukin-6 (IL-6) and down-regulation of IL-12 p40 (IL-12B) in LPS-stimulated macrophages were also detected after simulated microgravity, which is regulated by C/EBPß. CONCLUSIONS: Simulated microgravity activates a p38 MAPK-C/EBPß pathway in macrophages to up-regulate arginase and IL-6 expression and down-regulate IL-12B expression. Both increased arginase expression and decreased IL-12B expression in macrophages during inflammation could result in immunosuppression under microgravity.