Knockdown Macrophages Research Articles

Kruppel-Like Factor 2-Mediated Suppression of MicroRNA-155 Reduces the Proinflammatory Activation of Macrophages.

ObjectiveRecent evidence indicates that significant interactions exist between Kruppel-like factor 2 (KLF2) and microRNAs (miRNAs) in endothelial cells. Because KLF2 is known to exert anti-inflammatory effects and inhibit the pro-inflammatory activation of monocytes, we sought to identify how inflammation-associated miR-155 is regulated by KLF2 in macrophages.Approach and ResultsPeritoneal macrophages from wild-type (WT) C57Bl/6 mice were transfected with either recombinant adenovirus vector expressing KLF2 (Ad-KLF2) or siRNA targeting KLF2 (KLF2-siRNA) for 24 h–48 h, then stimulated with oxidized low-density lipoproteins (ox-LDL, 50 μg/mL) for 24 h. Quantitative real-time polymerase chain reaction showed that KLF2 markedly reduced the expression of miR-155 in quiescent/ox-LDL-stimulated macrophages. We also found that the increased expression of miR-155, monocyte chemoattractant protein (MCP-1) and interleukin (IL)-6 and the decreased expression of the suppressor of cytokine signaling (SOCS)-1 and IL-10 in ox-LDL-treated macrophages were significantly suppressed by KLF2. Most importantly, over-expression of miR-155 could partly reverse the suppressive effects of KLF2 on the inflammatory response of macrophages. Conversely, the suppression of miR-155 in KLF2 knockdown macrophages significantly overcame the pro-inflammatory properties associated with KLF2 knockdown. Finally, Ad-KLF2 significantly attenuated the diet-induced formation of atherosclerotic lesions in apolipoprotein E-deficient (apoE-/-) mice, which was associated with a significantly reduced expression of miR-155 and its relative inflammatory cytokine genes in the aortic arch and in macrophages.ConclusionKLF2-mediated suppression of miR-155 reduced the inflammatory response of macrophages.

Biliverdin Reductase A (BVRA) Mediates Macrophage Expression of Interleukin-10 in Injured Kidney.

Biliverdin reductase A is an enzyme, with serine/threonine/tyrosine kinase activation, converting biliverdin (BV) to bilirubin (BR) in heme degradation pathway. It has been reported to have anti-inflammatory and antioxidant effect in monocytes and human glioblastoma. However, the function of BVRA in polarized macrophage was unknown. This study aimed to investigate the effect of BVRA on macrophage activation and polarization in injured renal microenvironment. Classically activated macrophages (M1macrophages) and alternative activation of macrophages (M2 macrophages) polarization of murine bone marrow derived macrophage was induced by GM-CSF and M-CSF. M1 polarization was associated with a significant down-regulation of BVRA and Interleukin-10 (IL-10), and increased secretion of TNF-α. We also found IL-10 expression was increased in BVRA over-expressed macrophages, while it decreased in BVRA knockdown macrophages. In contrast, BVRA over-expressed or knockdown macrophages had no effect on TNF-α expression level, indicating BVRA mediated IL-10 expression in macrophages. Furthermore, we observed in macrophages infected with recombinant adenoviruses BVRA gene, which BVRA over-expressed enhanced both INOS and ARG-1 mRNA expression, resulting in a specific macrophage phenotype. Through in vivo study, we found BVRA positive macrophages largely existed in mice renal ischemia perfusion injury. With the treatment of the regular cytokines GM-CSF, M-CSF or LPS, excreted in the injured renal microenvironment, IL-10 secretion was significantly increased in BVRA over-expressed macrophages. In conclusion, the BVRA positive macrophage is a source of anti-inflammatory cytokine IL-10 in injured kidney, which may provide a potential target for treatment of kidney disease.

The Lipid-Modifying Enzyme SMPDL3B Negatively Regulates Innate Immunity.

SummaryLipid metabolism and receptor-mediated signaling are highly intertwined processes that cooperate to fulfill cellular functions and safeguard cellular homeostasis. Activation of Toll-like receptors (TLRs) leads to a complex cellular response, orchestrating a diverse range of inflammatory events that need to be tightly controlled. Here, we identified the GPI-anchored Sphingomyelin Phosphodiesterase, Acid-Like 3B (SMPDL3B) in a mass spectrometry screening campaign for membrane proteins co-purifying with TLRs. Deficiency of Smpdl3b in macrophages enhanced responsiveness to TLR stimulation and profoundly changed the cellular lipid composition and membrane fluidity. Increased cellular responses could be reverted by re-introducing affected ceramides, functionally linking membrane lipid composition and innate immune signaling. Finally, Smpdl3b-deficient mice displayed an intensified inflammatory response in TLR-dependent peritonitis models, establishing its negative regulatory role in vivo. Taken together, our results identify the membrane-modulating enzyme SMPDL3B as a negative regulator of TLR signaling that functions at the interface of membrane biology and innate immunity.

Mycobacterium tuberculosis 19-kDa lipoprotein induces Toll-like receptor 2-dependent peroxisome proliferator-activated receptor γ expression and promotes inflammatory responses in human macrophages.

Mycobacterium tuberculosis (M.tb) enhances its survival in macrophages by suppressing immune responses, in part through its complex cell wall structures. M.tb 19‑kDa lipoprotein (P19), a component of the complex cell wall structures of M.tb, is a Toll‑like receptor (TLR) agonist, and may induce immune responses through TLR2. Furthermore, the activation of peroxisome proliferator‑activated receptor γ (PPARγ) is also involved in M.tb‑induced immune responses in macrophages. In the present study, specific agonists/antagonists and siRNA were used to investigate the role of PPARγ in P19‑induced immune responses in human macrophages, including TLR2 activation, p38 phosphorylation and cytokine production. In the present study, PPARγ expression, p38 phosphorylation and cytokine production were upregulated following M.tb H37Rv infection or P19 treatment. By pretreating macrophages with a specific PPARγ agonist or antagonist, it was demonstrated that phosphorylation and IL‑6 production are modulated in macrophages by PPARγ activity. Following TLR2 knockdown in macrophages, the expression of PPARγ was significantly decreased in the presence or absence of P19 treatment. Furthermore, p38 phosphorylation and cytokine production were significantly reduced in TLR2 knockdown macrophages following P19 treatment. It was demonstrated in the current study that PPARγ was induced and activated by M.tb infection and that P19‑induced PPARγ expression, p38 phosphorylation and cytokine production in macrophages are dependent on TLR2. These findings suggest a role for PPARγ and TLR2 in P19‑induced p38 phosphorylation and cytokine production, thereby potentially influencing M.tb pathogenesis.

Blockade of the T cell immunoglobulin and mucin domain protein 3 pathway exacerbates sepsis-induced immune deviation and immunosuppression.

Sepsis is a life-threatening condition, but the pathophysiological basis and biomarkers for the monitoring of sepsis and as targets for therapy remain to be determined. We have shown previously that T cell immunoglobulin and mucin domain protein 3 (Tim-3), a negative immune regulator, is involved in the physiopathology of sepsis, but the underlying mechanisms remain unclear. In the present study, we showed that Tim-3 signalling modulated the response patterns of both macrophages and T helper cells in sepsis. Blockade of the Tim-3 pathway exacerbated sepsis-induced proinflammatory macrophage responses and lymphocyte apoptosis during the early phase of sepsis, and enhanced the shift to anti-inflammatory responses for both macrophages and T helper cells during the late phase of sepsis. Tim-3 signalling was found to regulate CD80 and CD86 expression on macrophages both in vivo and in vitro. Co-culture of T cells with Tim-3 knock-down macrophages led to a biased T helper type 2 (Th2) response, partially explaining how Tim-3 signalling shapes inflammation patterns in vivo. Further studies on this pathway might shed new light on the pathogenesis of sepsis and suggest new approaches for intervention.

Abstract 230: Stimulation of Cholesterol Efflux from Human Macrophage by Liver X Receptor Agonists is a 2-Step Mechanism Requiring ARL7 and ABCA1

Nuclear Liver X Receptors activation by synthetic agonists was proven to be atheroprotective in mice; an effect likely based on stimulation of cellular cholesterol efflux from arterial macrophages. However, mechanisms involved in free cholesterol efflux from mouse macrophages appear distinct from those operating in human macrophages. The objective of this study was to decipher precise cellular mechanisms controlling free cholesterol efflux from human macrophages upon LXR stimulation. In THP-1 and human monocyte-derived macrophages (HMDM), treatment with the LXR agonist GW3965 efficiently induced ARL7 expression (6-fold, p<0.05), an effect associated with an increased amount of plasma membrane free cholesterol available for efflux (+25%, p<0.05) and a higher lipid rafts formation (+10%, p<0.05). Both effects were abolished in ARL7 Knockdown (KD) macrophages, leading to a lack of stimulation of cholesterol efflux by GW3965. Specific targeting of each LXR isoforms, LXRα and LXRβ, by RNAi revealed that LXRα silencing in THP-1 and HMDM reduced significantly expression of cholesterol transporters ABCA1, ABCG1 and receptor SR-BI/Cla-1 mRNA levels, as well as free cholesterol efflux to apoA1 (-30%, p<0.05) and to HDL (-20%, p<0.05) upon stimulation with LXR, whereas LXRβ silencing has no impact. Interestingly, stimulation of cholesterol efflux to HDL by GW3965 was significantly reduced (-50%, p<0.05) in ABCA1 KD THP-1 macrophages; those cells being incapable to promote cholesterol efflux to apoA1. However, silencing of ABCG1 or SR-B1/Cla-1 had no impact on cholesterol efflux to HDL from either control or ABCA1 KD THP-1 macrophages treated or not with LXR agonist. By contrast stimulation of cholesterol efflux to HDL by GW3965 was completely abolished in LXRα/ABCA1 double KD macrophages, highlighting the major contribution of ABCA1 in cholesterol efflux from human macrophage. We conclude that LXR-mediated stimulation of cholesterol efflux from human macrophages is a two-steps mechanism. First, LXR activation promotes ARL7-dependent free cholesterol transport to plasma membrane, mostly in lipid raft domains. Then, membrane free cholesterol is exported to apoA1 and HDL acceptors through ABCA1; this latter step being controlled selectively by LXRα.

Abstract 18971: Changes in HDL Quality Rather Than Macrophage Function Mediate the Atheroprotective Phenotype in Usf1 Knockout Mice

We previously demonstrated that allelic variants of Usf1 are associated with myriad metabolic phenotypes in human populations and the knockout of Usf1 in mice leads to a favorable metabolic phenotype. Here, we have studied the role of macrophages and HDL particles in the development of atherosclerosis in the Usf1 -/- mice. A mouse strain of double knockout of Usf1 and Ldlr (Usf1 -/- Ldlr -/-) was generated, and mice along with their Usf1 +/+ Ldlr -/- littermates were fed with a high fat diet diet for 20 weeks. The double knockouts displayed a three-fold reduction in their atherosclerotic lesion area as demonstrated by the aortic longitudinal section based en face technique. To study the potential mechanisms behind the attenuated lesion development in the Usf1 -/- mice, we knocked down USF1 by constitutive expression of shRNA from human monocyte cell line (THP1). We differentiated monocytes into macrophages and loaded them with acetylated LDL for 48 hours. We observed no change in cholesterol efflux from USF1 knockdown macrophages to wild type mouse serum or HDL when compared to efflux from control cells expressing non-targeting shRNA. Furthermore, we performed bone marrow transplantation of Usf1 -/- and wild type bone marrow to Ldlr -/- mouse background. After 12 weeks of western diet we saw no change in atherosclerotic lesion development in the aortic root area in the mice with Usf1 -/- bone marrow compared to the mice with wild type bone marrow, indicating a macrophage independent mechanism for the atheroprotection in Usf1 -/- mice. The Usf1 -/- mice displayed both elevated cholesterol (p<0.001) and phospholipids (p<0.001) in their serum both on western and regular chow diet. Interestingly, cholesterol efflux from wild type THP1 macrophages to Usf1 -/- serum and HDL was significantly increased (p<0.05 and p<0.001, respectively) compared to serum and HDL from the wild type mice indicating that not only increased serum HDL concentration but also changes in HDL particle quality in the Usf1 -/- mice have properties increasing cholesterol efflux. Our study establishes the atheroprotective role for Usf1 deficiency that could be in part due to the favorable changes HDL particle quality rather than macrophage specific effects in the Usf1 -/- mice.

IRF3 and ERK MAP-kinases control nitric oxide production from macrophages in response to poly-I:C

Understanding nitric oxide (NO) in innate anti-viral immunity and immune-mediated pathology is hampered by incomplete details of its transcriptional and signaling factors. We found in macrophages that IRF3, ERK MAP-kinases, and PKR are essential to NO production in response to RNA-virus mimic, poly I:C, a TLR3 agonist. ERK’s role in NO induction may be through phosphorylation of serine-171 of IRF3 and expression of NO-inducing cytokines, IL-6 and IFN-β. However, these cytokines induced less NO in IRF3 knockout or knockdown macrophages. These findings show that ERK and IRF3 coordinate induction of NO by macrophages in response to stimulation of TLR3.

Autophagy restricts Chlamydia trachomatis growth in human macrophages via IFNG-inducible guanylate binding proteins

Interferon γ (IFNG) is a key host response regulator of intracellular pathogen replication, including that of Chlamydia spp The antichlamydial functions of IFNG manifest in a strictly host, cell-type and chlamydial strain dependent manner. It has been recently shown that the IFNG-inducible family of immunity-related GTPases (IRG) proteins plays a key role in the defense against nonhost adapted chlamydia strains in murine epithelial cells. In humans, IFN-inducible guanylate binding proteins (hGBPs) have been shown to potentiate the antichlamydial effect of IFNG; however, how hGBPs regulate this property of IFNG is unknown. In this study, we identified hGBP1/2 as important resistance factors against C. trachomatis infection in IFNG-stimulated human macrophages. Exogenous IFNG reduced chlamydial infectivity by 50 percent in wild-type cells, whereas shRNA hGBP1/2 knockdown macrophages fully supported chlamydial growth in the presence of exogenous IFNG. hGBP1/2 were recruited to bacterial inclusions in human macrophages upon stimulation with IFNG, which triggered rerouting of the typically nonfusogenic bacterial inclusions for lysosomal degradation. Inhibition of lysosomal activity and autophagy impaired the IFNG-mediated elimination of inclusions. Thus, hGBP1/2 are critical effectors of antichlamydial IFNG responses in human macrophages. Through their capacity to remodel classically nonfusogenic chlamydial inclusions and stimulate fusion with autophagosomes, hGBP1/2 disable a major chlamydial virulence mechanism and contribute to IFNG-mediated pathogen clearance.

High mobility group box 1 protein synergizes with lipopolysaccharide and peptidoglycan for nitric oxide production in mouse peritoneal macrophages in vitro

Extracellular high mobility group box 1 (HMGB1) protein and nitric oxide (NO) has been credited with multiple inflammatory functions using in vivo and in vitro systems. Therefore, delineating their regulation may be an important therapeutic strategy for the treatment of sepsis. In the present study, it is demonstrated that recombinant HMGB1 (rHMGB1) synergizes with sub threshold concentration of TLR2 agonist (PGN; 1μg/ml) as well as with TLR4 agonist (LPS; 1ng/ml) to induce NO release in mouse peritoneal macrophages. The enhanced iNOS expression was also observed at the transcription and translational level. Co-incubation of macrophages with rHMGB1 with either PGN or LPS showed enhanced expression of TLR2, TLR4 and RAGE. TLR2, TLR4 or RAGE knockdown macrophages effectively inhibited the rHMGB1+PGN or LPS induced NO synergy. It was further observed that the JNK MAPK inhibitor SP600125 attenuated the PGN+rHMGB1 induced iNOS/NO synergy whereas p38 MAPK inhibitor SB908912 inhibited iNOS/NO synergy induced by LPS+rHMGB1. It was also observed that the activation of NF-κB is essential for the synergy as the pharmacological inhibition or siRNA knockdown of NF-κB (cRel) significantly reduced the rHMGB1+PGN or rHMGB1+LPS induced enhanced iNOS/NO expression. Altogether, the data suggests that the co-incubation of macrophages with rHMGB1 with either LPS or PGN induces the synergistic effect on iNOS expression and NO release by the upregulation of surface receptors (TLR2, TLR4 and RAGE) which in turn amplifies the MAPKs (p38 and JNK) and NF-κB activation and results in enhanced iNOS expression and NO production.

Peptidoglycan induced expression of peroxisome proliferator-activated receptor γ in mouse peritoneal macrophages: Role of ERK and JNK MAP kinases

The peroxisome proliferator-activated receptor (PPAR) γ plays an important role in macrophage inflammatory homeostasis. Here we investigate the cross talk between PPARγ and TLR2 signaling pathway in mouse peritoneal macrophages. Real time RT-PCR and immunoblot analysis revealed that peptidoglycan (PGN) treatment of macrophages leads to biphasic effect on PPARγ expression i.e. an early upregulation and a late suppression. Inhibition of ERK MAP kinase by PD98059 abolished the early and rapid induction of PPARγ, while the inhibition of JNK MAP kinase by SP600125 nullifies the late inhibitory effect on the PPARγ expression in a dose-dependent manner. Furthermore, PPARγ knockdown macrophages showed enhanced NF-κB activity after PGN treatment. PGN treatment also enhances PPARγ interaction with p65 as observed by immunoprecipitation. This interaction may inhibit NF-κB (p65) activity as increased nuclear localization of p65 was observed in PPARγ knockdown macrophages after PGN treatment. PPARγ knockdown also increased the PGN-induced inflammatory cytokines (TNF-α, IL-1β, IL-12p40) production. Thus, our observations suggest that PGN induces PPARγ expression which is regulated by MAPKs activation and this enhanced PPARγ in turn attenuate NF-κB activity probably via enhancing p65 nuclear export. These results provide insight into how these pathways could be modulated in inflammatory diseases.

Outer Membrane Protein A (OmpA) of Shigella flexneri 2a Links Innate and Adaptive Immunity in a TLR2-dependent Manner and Involvement of IL-12 and Nitric Oxide

We determine that OmpA of Shigella flexneri 2a is recognized by TLR2 and consequently mediates the release of proinflammatory cytokines and activates NF-κB in HEK 293 cells transfected with TLR2. We also observe that in RAW macrophages TLR2 is essential to instigate the early immune response to OmpA via NF-κB activation and secretion of cytokines and NO. Consistent with these results, TLR2 knockdown using siRNA abolishes the initiation of immune responses. Processing and presentation of OmpA depend on TLR2; MHCII presentation of the processed antigen and expression of CD80 significantly attenuated in TLR2 knockdown macrophages. The optimum production of IFN-γ by the macrophages:CD4(+) T cells co-culture depends on both TLR2 activation and antigen presentation. So, TLR2 is clearly recognized as a decisive factor in initiating host innate immune response to OmpA for the development of CD4(+) T cell adaptive response. Furthermore, we demonstrate in vivo that intranasal immunization of mice with OmpA selectively enhances the release of IFN-γ and IL-2 by CD4(+) T cells. Importantly, OmpA increases the level of IFN-γ production in Ag-primed splenocytes. The addition of neutralizing anti-IL-12p70 mAb to cell cultures results in the decreased release of OmpA-enhanced IFN-γ by Ag-primed splenocytes. Moreover, coincubation with OmpA-pretreated macrophages enhances the production of IFN-γ by OmpA-primed CD4(+) T cells, representing that OmpA may enhance IFN-γ expression in CD4(+) T cells through the induction of IL-12 production in macrophages. These results demonstrate that S. flexneri 2a OmpA may play a critical role in the development of Th1 skewed adaptive immune response.

Coronin-1a inhibits autophagosome formation around Mycobacterium tuberculosis-containing phagosomes and assists mycobacterial survival in macrophages

Mycobacterium tuberculosis is an intracellular bacterium that can survive within macrophages. Such survival is potentially associated with Coronin-1a (Coro1a). We investigated the mechanism by which Coro1a promotes the survival of M. tuberculosis in macrophages and found that autophagy was involved in the inhibition of mycobacterial survival in Coro1a knock-down (KD) macrophages. Fluorescence microscopy and immunoblot analyses revealed that LC3, a representative autophagic protein, was recruited to M. tuberculosis-containing phagosomes in Coro1a KD macrophages. Thin-section electron microscopy demonstrated that bacilli were surrounded by the multiple membrane structures in Coro1a KD macrophages. The proportion of LC3-positive mycobacterial phagosomes colocalized with p62/SQSTM1, ubiquitin or LAMP1 increased in Coro1a KD macrophages during infection. These results demonstrate the formation of autophagosomes around M. tuberculosis in Coro1a KD macrophages. Phosphorylation of p38 mitogen-activated protein kinase (MAPK) was induced in response to M. tuberculosis infection in Coro1a KD macrophages, suggesting that Coro1a blocks the activation of the p38 MAPK pathway involved in autophagosome formation. LC3 recruitment to M. tuberculosis-containing phagosomes was also observed in Coro1a KD alveolar or bone marrow-derived macrophages. These results suggest that Coro1a inhibits autophagosome formation in alveolar macrophages, thereby facilitating M. tuberculosis survival within the lung.

Cathelicidin is involved in the intracellular killing of mycobacteria in macrophages

Macrophages have been shown to kill Mycobacterium tuberculosis through the action of the antimicrobial peptide cathelicidin (CAMP), whose expression was shown to be induced by 1,25-dihydroxyvitamin D3 (1,25D3). Here, we investigated in detail the antimycobacterial effect of murine and human cathelicidin against Mycobacterium smegmatis and M. bovis BCG infections. We have synthesized novel LL-37 peptide variants that exhibited potent in vitro bactericidal activity against M. smegmatis, M. bovis BCG and M. tuberculosis H37Rv, as compared with parental peptide. We show that the exogenous addition of LL-37 or endogenous overexpression of cathelicidin in macrophages significantly reduced the intracellular survival of mycobacteria relative to control cells. An upregulation of cathelicidin mRNA expression was observed that correlated with known M. smegmatis killing phases in J774 macrophages. Moreover, RNAi-based Camp knock-down macrophages and Camp(-/-) bone marrow derived mouse macrophages were significantly impaired in their ability to kill mycobacteria. M. smegmatis killing in Camp(-/-) macrophages was less extensive than in Camp(+/+) cells following activation with FSL-1, an inducer of cathelicidin expression. Finally we show that LL-37 and 1,25D3 treatment results in increase in colocalization of BCG-containing phagosomes with lysosomes. Altogether, these data demonstrate that cathelicidin plays an important role in controlling intracellular survival of mycobacteria.

3BP2 Adapter Protein Is Required for Receptor Activator of NFκB Ligand (RANKL)-induced Osteoclast Differentiation of RAW264.7 Cells

The adapter protein 3BP2 (also known as SH3BP2 and Abl SH3-binding protein 2) has been involved in leukocyte signaling and activation downstream immunoreceptors. Genetic studies have further associated 3BP2 mutations to the human disease cherubism and to inflammation and bone dysfunction in mouse. However, how wild type 3BP2 functions in macrophage differentiation remains poorly understood. In this study, using small interfering RNA-mediated silencing of 3BP2 in the RAW264.7 monocytic cell line, we show that 3BP2 was required for receptor activator of NFkappaB ligand (RANKL)-induced differentiation of RAW264.7 cells into multinucleated mature osteoclasts but not for granulocyte macrophage-colony stimulating factor/interleukin-4-induced differentiation into dendritic cells. 3BP2 silencing was associated with impaired activation of multiple signaling events downstream of RANK, including actin reorganization; Src, ERK, and JNK phosphorylation; and up-regulation of osteoclastogenic factors. In addition, 3BP2 knockdown cells induced to osteoclast by RANKL displayed a reduced increase of Src and nuclear factor of activated T cells (NFATc1) mRNA and protein expression. Importantly, 3BP2 interacted with Src, Syk, Vav, and Cbl in monocytic cells, and the introduction of constitutively active mutants of Src and NFATc1 in 3BP2-deficient cells restored osteoclast differentiation. Finally, the expression of a 3BP2 cherubism mutant was found to promote increased Src activity and NFAT-dependent osteoclast formation. Together, this study demonstrates that wild type 3BP2 is a key regulator of RANK-mediated macrophage differentiation into osteoclast through Src and NFATc1 activation.

Characterization of the Molecular Clock in Mouse Peritoneal Macrophages

Macrophages play essential roles in the innate immune system. In this study, we show that macrophage functions such as phagocytosis and cytokine/chemokine expressions display a circadian rhythm that is regulated by a molecular clock. Phagocytosis, a crucial early reaction by which macrophages protect their host against foreign particles, exhibited a circadian variation that peaks during the light period and bottoms during the dark period. These diurnal changes of phagocytosis activity in macrophages were induced without exogenous stimulants such as bacterial infection. The expression of the clock genes including brain and muscle Arnt-like protein-1 (BMAL1) exhibited robust circadian rhythms in macrophages. The expression patterns of the clock genes in macrophages were similar to those in the suprachiasmatic nucleus and other peripheral tissues. Among inflammation factors examined, the level of monocyte chemoattractant protein-1 (MCP-1/JE) mRNA exhibited most robust circadian oscillation. Expression of other cytokines such as IL-1beta, IL-6 and TNFalpha showed mild diurnal changes. Knockdown of the BMAL1 expression resulted in a decrease of the MCP-1/JE mRNA level in macrophages. BMAL1 increased significantly but weakly MCP-1/JE promoter activity. MCP-1/JE promoter activity is known to be regulated by nuclear factor-kappa B (NF-kappaB). NF-kappaB activity in BMAL1 knockdown macrophages was lower than that in control cells. Consequently, the circadian expression of MCP-1/JE in macrophages is regulated by BMAL1 through the activation of NF-kappaB. The results obtained in this study indicate that the innate immunoreactions involving macrophages are at least partly regulated by the autonomous clock machinery.