Early p38 Activation Regulated by MKP-1 Is Determinant for High Levels of IL-10 Expression Through TLR2 Activation.

Toll-like receptors (TLRs) play a crucial role in the recognition of pathogen-derived components as a first line of defense against infections. It has been suggested that depending on the nature of the pathogens, TLRs activation induce a distinct cytokine profile that may contribute to the polarization of the acquired immune response. Here, we investigated the early MAPK signaling activation via TLR4 and TLR2 receptors and its impact in differential cytokine profile by macrophages. We found that TLR2 ligands activated MAPKs p38 and ERK earlier compared to the TLR4 ligand LPS in macrophages. Higher IL-10/IL-12 and IL-10/TNF-α ratios were also observed at later time points in response to TLR2 ligands compared to LPS. The results also indicate an earlier activation of the phosphatase MKP-1 and that MKP-1 KO macrophages show a prolongation in p38 phosphorylation in response to TLR2 stimulation. Furthermore, p38 is critical for IL-10 expression in response to TLR2 ligands, which triggers the macrophage change to a M2 and regulatory phenotype in contrast to the M1 phenotype induced by TLR4 activation. Therefore, the early TLR2-mediated p38 induction contributes for the high IL-10 production, likely as a virulence strategy to suppress host Th1 response against certain types of pathogens.

Inhibition of PTP1B Promotes M2 Polarization via MicroRNA-26a/MKP1 Signaling Pathway in Murine Macrophages.

Sepsis is a life-threatening condition that often occurs in the intensive care unit. The excessive activation of the host's immune system at early stages contributes to multiple organ damage. Mitogen-activated protein kinase phosphatase-1 (MKP1) exerts an important effect on the inflammatory process. In our recent bioinformatic analysis, we confirmed that the inhibition of protein tyrosine phosphatase-1B (PTP1B) significantly promoted the expression of MKP1 in murine macrophages. However, the underlying mechanism and its effect on macrophage polarization remain unclear. In this study, we show that the suppression of PTP1B induced upregulation of MKP1 in M1 macrophages. A RayBiotech mouse inflammation antibody assay further revealed that MKP1-knockdown promoted pro-inflammatory cytokine (IL-1ß, IL12p70, IL-17, IL-21, IL-23, and TNF-α) secretion but suppressed anti-proinflammatory cytokine (IL-10) production in M2 macrophages. Phospho-proteomics analysis further identified ERK1/2 and p38 as downstream molecules of MKP1. Moreover, we found that the inhibition of PTP1B lowered the expression of miR-26a, showing a negative correlation with MKP1 protein expression. Thus, we concluded that the inhibition of PTP1B contributes to M2 macrophage polarization via reducing mir-26a and afterwards enhancing MKP1 expression in murine macrophages.

Effects of silencing the DUSP1 gene using lentiviral vector-mediated siRNA on the release of proinflammatory cytokines through regulation of the MAPK signaling pathway in mice with acute pancreatitis.

The present study investigated the effects of dual specificity phosphatase 1 (DUSP1) gene silencing using lentiviral vector-mediated small interfering (si)RNA on the release of proinflammatory cytokines through the regulation of the mitogen­activated protein kinase (MAPK) signaling pathway in mice with acute pancreatitis (AP). Two siRNA­DUSP1 sequences and one scramble siRNA sequence were designed, and the expression of DUSP1 was detected using western blot analysis to screen for the one with a higher interference rate. An AP mouse model was established, and KM mice were assigned to either a control, siRNA, AP, AP+PD98059, AP+scramble, AP+siRNA or AP+PD98059+siRNA group. The expression of proinflammatory cytokines, including tumor necrosis factor (TNF)­α, interleukin (IL)­1ß and IL­6, high mobility group box 1 (HMGB1), and S100A12 in serum samples were detected using an enzyme­linked immunosorbent assay at 12, 24 and 48 h post­modeling. The serum amylase levels were also detected. The expression levels of DUSP1, TNF­α, IL­1ß, IL­6, HMGB1, S100A12, phosphorylated (p­) extracellular signal­regulated kinase (ERK), p­c­Jun N­terminal kinase (JNK), p­p38, ERK, JNK and p38 in pancreatic, liver, kidney and lung tissues were detected using reverse transcription­quantitative polymerase chain reaction and western blot analysis. Compared with the control group, the siRNA group demonstrated marginally upregulated serum amylase, lipase, urinary trypsinogen­2, and proinflammatory cytokines, HMGB1 and S100A12 in serum and tissues, with no statistically significant difference, elevated expression levels of p­ERK, p­JNK and p­p38, and decreased expression of DUSP1. The other five groups demonstrated increased expression levels of TNF­α, IL­1ß, IL­6, HMGB1, S100A12, amylase, lipase and urinary trypsinogen­2 in serum, and increased expression levels of DUSP1, TNF­α, IL­1ß, IL­6, HMGB1, S100A12, p­ERK, p­JNK and p­p38 in tissues. Compared with the AP group, the AP+PD98059+siRNA group had decreased expression of DUSP1 in tissues, whereas the AP+PD98059 group had decreased serum expression levels of TNF­α, IL­1ß, IL­6, HMGB1, S100A12 and amylase, lipase and urinary trypsinogen­2. The expression levels of TNF­α, IL­1ß, IL­6, HMGB1, S100A12, p­ERK, p­JNK, p­p38 in tissues, and edema of pancreatic tissue were alleviated, whereas the opposite results were observed in the AP+siRNA group with the decreased expression of DUSP1. The results suggested that DUSP1 gene silencing promoted the release of proinflammatory cytokines through activation of the MAPK signaling pathway in mice with AP.

Anti- trachea inflammatory effects of diosgenin from Dioscorea nipponica through interactions with glucocorticoid receptor α.

Asthma is a heterogeneous disease characterized by symptoms of chronic inflammation and airway structural and functional changes. It affects about 300 million people worldwide and causes 250 000 deaths annually, but its symptoms can be greatly relieved by regular use of inhaled glucocorticoids (GCs). GCs exert their function through interacting with glucocorticoid receptors (GRs). Diosgenin is a naturally occurring steroidal saponin abundantly present in many medicinal plants, including Dioscorea nipponica, which shares a similar steroidal structure with GC. In this study, ovalbumin (OVA)-induced asthmatic mice and primary tracheal epithelial cells (TECs) were used as research models. ELISAs were applied to measure the secretion of TNF-α, IL-1ß, and IL-6, while quantitative PCR and western blotting were applied to evaluate expression of GRs SLPI, TTP, GILZ, MKP-1, and NF-κB. Our data demonstrated that diosgenin suppressed the secretion of TNF-α, IL-1ß, and IL-6 by enhancing the expression of GRs, SLPI, GILZ, and MKP-1, and inhibiting the expression of HSP70. These data provide some evidence on the molecular mechanism of diosgenin, which might facilitate its clinical applications.

Dual-Specificity Phosphatase 1 and Tristetraprolin Cooperate To Regulate Macrophage Responses to Lipopolysaccharide.

Dual-specificity phosphatase (DUSP) 1 dephosphorylates and inactivates members of the MAPK superfamily, in particular, JNKs, p38α, and p38ß MAPKs. It functions as an essential negative regulator of innate immune responses, hence disruption of the Dusp1 gene renders mice extremely sensitive to a wide variety of experimental inflammatory challenges. The principal mechanisms behind the overexpression of inflammatory mediators by Dusp1(-/-) cells are not known. In this study, we use a genetic approach to identify an important mechanism of action of DUSP1, involving the modulation of the activity of the mRNA-destabilizing protein tristetraprolin. This mechanism is key to the control of essential early mediators of inflammation, TNF, CXCL1, and CXCL2, as well as the anti-inflammatory cytokine IL-10. The same mechanism also contributes to the regulation of a large number of transcripts induced by treatment of macrophages with LPS. These findings demonstrate that modulation of the phosphorylation status of tristetraprolin is an important physiological mechanism by which innate immune responses can be controlled.

Effect of the plant derivative Compound A on the production of corticosteroid-resistant chemokines in airway smooth muscle cells.

Preclinical models of human conditions including asthma showed the therapeutic potential of Compound A (CpdA), a dissociated glucocorticoid (GC) receptor (GRα) ligand. Whether CpdA inhibits GC resistance, a central feature of severe asthma, has not been addressed. We investigated whether CpdA modulates cytokine-induced GC resistance in human airway smooth muscle (ASM) cells. Healthy and asthmatic ASM cells were treated with TNF-α/IFN-γ for 24 hours in the presence or absence of CpdA. ELISA and quantitative PCR assays were used to assess the effect of CpdA on chemokine expression. Activation of GRα by CpdA was assessed by quantitative PCR, immunostaining, and receptor antagonism using RU486. An effect of CpdA on the transcription factor interferon regulatory factor 1 (IRF-1) was investigated using immunoblot, immunostaining, and small interfering RNA (siRNA) knockdown. CpdA inhibited production of fluticasone-resistant chemokines CCL5, CX3CL1, and CXCL10 at protein and mRNA levels in both asthmatic and healthy cells. CpdA failed to induce expression of GC-induced Leucine Zipper while transiently inducing mitogen-activated protein kinase phosphatase 1 (MKP-1) at both mRNA and protein levels. CpdA inhibitory action was not associated with GRα nuclear translocation, nor was it prevented by RU486 antagonism. Activation of IRF-1 by TNF-α/IFN-γ was inhibited by CpdA. IRF-1 siRNA knockdown reduced cytokine-induced CCL5 and CX3CL1 production. siRNA MKP-1 prevented the inhibitory effect of CpdA on cytokine-induced CXCL10 production. For the first time, we show that CpdA inhibits the production of GC-resistant chemokines via GRα-independent mechanisms involving the inhibition of IRF-1 and up-regulation of MKP-1. Thus, targeting CpdA-sensitive pathways in ASM cells represents an alternative therapeutic approach to treat GC resistance in asthma.

Prolonged proinflammatory cytokine production in monocytes modulated by interleukin 10 after influenza vaccination in older adults.

We evaluated in vivo innate immune responses in monocyte populations from 67 young (aged 21-30 years) and older (aged ≥65 years) adults before and after influenza vaccination. CD14(+)CD16(+) inflammatory monocytes were induced after vaccination in both young and older adults. In classical CD14(+)CD16(-) and inflammatory monocytes, production of tumor necrosis factor α and interleukin 6, as measured by intracellular staining, was strongly induced after vaccination. Cytokine production was strongly associated with influenza vaccine antibody response; the highest levels were found as late as day 28 after vaccination in young subjects and were substantially diminished in older subjects. Notably, levels of the anti-inflammatory cytokine interleukin 10 (IL-10) were markedly elevated in monocytes from older subjects before and after vaccination. In purified monocytes, we found age-associated elevation in phosphorylated signal transducer and activator of transcription-3, and decreased serine 359 phosphorylation of the negative IL-10 regulator dual-specificity phosphatase 1. These findings for the first time implicate dysregulated IL-10 production in impaired vaccine responses in older adults.

Obesity impairs apoptotic cell clearance in asthma.

BACKGROUND: Asthma in obese adults is typically more severe and less responsive to glucocorticoids than asthma in nonobese adults. OBJECTIVE: We sought to determine whether the clearance of apoptotic inflammatory cells (efferocytosis) by airway macrophages was associated with altered inflammation and reduced glucocorticoid sensitivity in obese asthmatic patients. METHODS: We investigated the relationship of efferocytosis by airway (induced sputum) macrophages and blood monocytes to markers of monocyte programming, in vitro glucocorticoid response, and systemic oxidative stress in a cohort of adults with persistent asthma. RESULTS: Efferocytosis by airway macrophages was assessed in obese (n=14) and nonobese (n=19) asthmatic patients. Efferocytosis by macrophages was 40% lower in obese than nonobese subjects, with a mean efferocytic index of 1.77 (SD, 1.07) versus 3.00 (SD, 1.25; P<.01). A similar reduction of efferocytic function was observed in blood monocytes of obese participants. In these monocytes there was also a relative decrease in expression of markers of alternative (M2) programming associated with efferocytosis, including peroxisome proliferator-activated receptor δ and CX3 chemokine receptor 1. Macrophage efferocytic index was significantly correlated with dexamethasone-induced mitogen-activated protein kinase phosphatase 1 expression (ρ=0.46, P<.02) and baseline glucocorticoid receptor α expression (ρ=0.44, P<.02) in PBMCs. Plasma 4-hydroxynonenal levels were increased in obese asthmatic patients at 0.33 ng/mL (SD, 0.15 ng/mL) versus 0.16 ng/mL (SD, 0.08 ng/mL) in nonobese patients (P=.006) and was inversely correlated with macrophage efferocytic index (ρ=-0.67, P=.02). CONCLUSIONS: Asthma in obese adults is associated with impaired macrophage/monocyte efferocytosis. Impairment of this anti-inflammatory process is associated with altered monocyte/macrophage programming, reduced glucocorticoid responsiveness, and systemic oxidative stress.

MicroRNA-101, mitogen-activated protein kinases and mitogen-activated protein kinases phosphatase-1 in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is the prototype of human autoimmune disease in which various inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1, IL-6 and interferon (IFN) play crucial pathogenic roles. The production of these cytokines is responsible for the mitogen-activated protein kinases (MAPKs), which can also generate mitogen-activated protein kinases phosphatases (MKPs). MKP-1, a prototypical member of the MKP family that can influence outcomes of autoimmune diseases and reduce the inflammatory cytokines by dephosphorylation of p38 and JNK MAPK, plays a critical role in the expression of inflammatory mediators at transcriptional and post-transcriptional levels. MicroRNA-101 (miR) is a small non-coding RNA that regulates the MAPK response by targeting MKP-1 mRNA 3'-UTR, and affects the secretion of the downstream inflammatory cytokines. However, the interaction among the above three in the pathogenesis of SLE has not previously been reported. This review discusses the current understanding of the role of the MAPK/MKP/miR-101 axis in regulating immune responses and the pathogenesis of SLE to provide new ideas for clinical treatment of SLE.

Mechanisms of IFN-γ-induced apoptosis of human skin keratinocytes in patients with atopic dermatitis.

BACKGROUND: Enhanced apoptosis of keratinocytes is the main cause of eczema and spongiosis in patients with the common inflammatory skin disease atopic dermatitis (AD). OBJECTIVE: The aim of the study was to investigate molecular mechanisms of AD-related apoptosis of keratinocytes. METHODS: Primary keratinocytes isolated from patients with AD and healthy donors were used to study apoptosis by using annexin V/7-aminoactinomycin D staining. Illumina mRNA Expression BeadChips, quantitative RT-PCR, and immunofluorescence were used to study gene expression. In silico analysis of candidate genes was performed on genome-wide single nucleotide polymorphism data. RESULTS: We demonstrate that keratinocytes of patients with AD exhibit increased IFN-γ-induced apoptosis compared with keratinocytes from healthy subjects. Further mRNA expression analyses revealed differential expression of apoptosis-related genes in AD keratinocytes and skin and the upregulation of immune system-related genes in skin biopsy specimens of chronic AD lesions. Three apoptosis-related genes (NOD2, DUSP1, and ADM) and 8 genes overexpressed in AD skin lesions (CCDC109B, CCL5, CCL8, IFI35, LYN, RAB31, IFITM1, and IFITM2) were induced by IFN-γ in primary keratinocytes. The protein expression of IFITM1, CCL5, and CCL8 was verified in AD skin. In line with the functional studies and AD-related mRNA expression changes, in silico analysis of genome-wide single nucleotide polymorphism data revealed evidence of an association between AD and genetic markers close to or within the IFITM cluster or RAB31, DUSP1, and ADM genes. CONCLUSION: Our results demonstrate increased IFN-γ responses in skin of patients with AD and suggest involvement of multiple new apoptosis- and inflammation-related factors in the development of AD.

Mitogen-activated protein kinase phosphatase (MKP)-1 in immunology, physiology, and disease.

Mitogen-activated protein kinases (MAPKs) are key regulators of cellular physiology and immune responses, and abnormalities in MAPKs are implicated in many diseases. MAPKs are activated by MAPK kinases through phosphorylation of the threonine and tyrosine residues in the conserved Thr-Xaa-Tyr domain, where Xaa represents amino acid residues characteristic of distinct MAPK subfamilies. Since MAPKs play a crucial role in a variety of cellular processes, a delicate regulatory network has evolved to control their activities. Over the past two decades, a group of dual specificity MAPK phosphatases (MKPs) has been identified that deactivates MAPKs. Since MAPKs can enhance MKP activities, MKPs are considered as an important feedback control mechanism that limits the MAPK cascades. This review outlines the role of MKP-1, a prototypical MKP family member, in physiology and disease. We will first discuss the basic biochemistry and regulation of MKP-1. Next, we will present the current consensus on the immunological and physiological functions of MKP-1 in infectious, inflammatory, metabolic, and nervous system diseases as revealed by studies using animal models. We will also discuss the emerging evidence implicating MKP-1 in human disorders. Finally, we will conclude with a discussion of the potential for pharmacomodulation of MKP-1 expression.

Attenuation of the acute inflammatory response by dual specificity phosphatase 1 by inhibition of p38 MAP kinase.

Dual specificity phosphatase 1 (DUSP1) dephosphorylates and, hence, regulates the activity of MAP kinases. The present study investigated the effect of DUSP1 on inflammatory gene expression and on the development of carrageenan-induced inflammation. It was found that DUSP1 expression was increased by LPS, and the down-regulation of DUSP1 by siRNA enhanced the phosphorylation of p38 MAPK, while JNK phosphorylation was not affected in murine macrophages. LPS-induced interleukin (IL)-6, tumor-necrosis factor (TNF) and cyclooxygenase-2 (COX2) expression were enhanced in bone marrow-derived macrophages (BMMs) from DUSP1(-/-) mice as compared to those from wild-type mice. In addition, down-regulation of DUSP1 by siRNA enhanced IL-6, TNF and COX2 expression in J774 macrophages, while p38 MAPK inhibitors SB202190 and BIRB 796 inhibited the expression of those inflammatory factors. In vivo, the intensity of the carrageenan-induced paw edema reaction was increased in DUSP1(-/-) mice as compared to the wild-type animals. In conclusion, DUSP1 is an important negative regulator of the acute inflammatory response by limiting p38 MAPK, and compounds which enhance DUSP1 expression or activity may hold a promise as anti-inflammatory drugs.

Dysregulation of p38 and MKP-1 in response to NOD1/TLR4 stimulation in sarcoid bronchoalveolar cells.

RATIONALE: Sarcoidosis is a systemic inflammatory disorder characterized by distinct up-regulation of Th1 cytokines, such as tumor necrosis factor (TNF)-α and IL-12. The mechanism underlying this up-regulation remains unclear. Recognition of microbial moieties through Toll-like or Nod-like receptors evokes sequential activation of mitogen-activated protein kinases (MAPKs), which plays a role in Th1-immune response. OBJECTIVES: To test the hypothesis that dysregulation in MAPK signaling in response to microbial stimulation is important in mediating Th1 response in sarcoidosis. METHODS: Ex vivo cultured bronchoalveolar lavage (BAL) cells isolated from patients with sarcoidosis and control subjects were stimulated with low-dose Toll-like receptor 4 (TLR4) and nucleotide-binding oligomerization domain 1 (NOD1) ligands as a model of microbial stimulation, and MAPK signaling and inflammatory response were analyzed. MEASUREMENTS AND MAIN RESULTS: BAL cells from patients with sarcoidosis exhibited higher basal p38 activity, greater p38 phosphorylation, and more robust production of TNF-α and IL-12/IL-23p40 on stimulation with NOD1 and TLR4 agonists than cells isolated from control subjects. In contrast, control BAL cells had greater basal extracellular signal-regulated kinase (ERK) activity and NOD1 and TLR4 agonists preferentially activated the ERK pathway. Inhibition of p38, but not ERK, attenuated production of both IL12/IL23p40 and TNF-α. Interestingly, stimulation of cells from patients with sarcoidosis with either NOD1 or TLR4 ligand failed to induce MAPK phosphatase 1 (MKP-1). Adenovirus-mediated overexpression of MKP-1 attenuated p38 activation and decreased the production of IL12/IL23p40 and TNF-α in sarcoid BAL cells. CONCLUSIONS: Our results suggest that enhanced p38 signaling in response to microbial products is caused by abnormal regulation of MKP-1 and contributes to heightened inflammation in sarcoidosis.

MicroRNA-101 targets MAPK phosphatase-1 to regulate the activation of MAPKs in macrophages.

MAPK phosphatase-1 (MKP-1) is an archetypical member of the dual-specificity phosphatase family that deactivates MAPKs. Induction of MKP-1 has been implicated in attenuating the LPS- or peptidoglycan-induced biosynthesis of proinflammatory cytokines, but the role of noncoding RNA in the expression of the MKP-1 is still poorly understood. In this study, we show that MKP-1 is a direct target of microRNA-101 (miR-101). Transfection of miR-101 attenuates induction of MKP-1 by LPS as well as prolonged activation of p38 and JNK/stress-activated protein kinase, whereas inhibition of miR-101 enhances the expression of MKP-1 and shortens p38 and JNK activation. We also found that expression of miR-101 is induced by multiple TLR ligands, including LPS, peptidoglycan, or polyinosinic-polycytidylic acid, and that inhibition of PI3K/Akt by LY294002 or Akt RNA interference blocks the induction of miR-101 by LPS in RAW264.7 macrophage cells. Moreover, treatment of cells with dexamethasone, a widely used anti-inflammatory agent, markedly inhibits miR-101 expression and enhances the expression of MKP-1 in LPS-stimulated macrophages. Together, these results indicate that miR-101 regulates the innate immune responses of macrophages to LPS through targeting MKP-1.

Phosphatidylinositol 3-kinase activation attenuates the TLR2-mediated macrophage proinflammatory cytokine response to Francisella tularensis live vaccine strain.

An inadequate innate immune response appears to contribute to the virulence of Francisella tularensis following pulmonary infection. Studies in mice suggest that this poor response results from suppression of proinflammatory cytokine production early during infection, but the mechanisms involved are not understood. PI3K is known to regulate proinflammatory cytokine expression, but its exact role (positive versus negative) is controversial. We sought to clarify the role of PI3K in regulating proinflammatory signaling and cytokine production during infection with F. tularensis live vaccine strain (LVS). In this study, we demonstrate that the induction of TNF and IL-6 expression by LVS in mouse bone marrow-derived macrophages was markedly enhanced when PI3K activity was inhibited by either of the well-known chemical inhibitors, wortmannin or LY294002. The enhanced cytokine expression was accompanied by enhanced activation of p38 MAPK and ERK1/2, both of which were critical for LVS-induced expression of TNF and IL-6. LVS-induced MAPK activation and cytokine production were TLR2- and MyD88- dependent. PI3K/Akt activation was MyD88-dependent, but was surprisingly TLR2-independent. LVS infection also rapidly induced MAPK phosphatase-1 (MKP-1) expression; PI3K and TLR2 signaling were required. Peak levels of MKP-1 correlated closely with the decline in p38 MAPK and ERK1/2 phosphorylation. These data suggest that infection by LVS restrains the TLR2-triggered proinflammatory response via parallel activation of PI3K, leading to enhanced MKP-1 expression, accelerated deactivation of MAPKs, and suppression of proinflammatory cytokine production. This TLR2-independent inhibitory pathway may be an important mechanism by which Francisella suppresses the host's innate immune response.

NF-kappaB-mediated expression of MAPK phosphatase-1 is an early step in desensitization to TLR ligands in enterocytes.

Toll-like receptor (TLR) signaling in naive enterocytes is rapidly inhibited, leading to the establishment of tolerance. To gain insight into tolerance at the level of the proinflammatory mitogen-activated protein kinase (MAPK) p38, we characterized TLR-mediated induction of the p38-specific phosphatase MKP-1. In cultured enterocytes, ligands of TLR3, TLR4, TLR5, and TLR9, but not TLR2, induce MKP-1 at 30-60 min, coincident with dephosphorylation of p38 following the peak of TLR ligand-induced phosphorylation. Induction of MKP-1 is blocked by inhibitors of nuclear factor (NF)-kappaB, but not of MAPK. Small interfering RNA knockdown of IkBalpha prolongs the expression of MKP-1. Rat MKP-1 promoter contains two NF-kappaB-binding sites, mutations in which additively impair lipopolysaccharide-induced transcription from the MKP-1 promoter. In the intestine, MKP-1 is expressed in the crypts, the epithelial compartment that also displays bacteria-dependent activating phosphorylation of p38. Thus, NF-kappaB-dependent expression of MKP-1 may contribute, by desensitization of p38, to the rapid establishment of unresponsiveness to several TLR ligands in enterocytes.

The RNA binding protein tristetraprolin influences the activation state of murine dendritic cells.

Dendritic cells (DCs) serve to maintain peripheral tolerance under steady state conditions. Upon triggering by activation signals they initiate strong immune responses. The activation of DCs is accompanied by a rapid upregulation of proinflammatory cytokines, which were shown in other cell types to be regulated by mechanisms at the transcriptional and posttranscriptional level. Tristetraprolin (TTP), an important RNA binding protein, is involved in the regulation of mRNA stability of such cytokines. In this study we analyzed the significance of TTP for mouse DCs, which were derived from TTP(-/-) and WT bone marrow progenitor cells (BM-DCs). Unstimulated BM-DCs of TTP(-/-) mice expressed lower levels of mRNAs encoding the costimulatory molecules CD40 and CD86 and surprisingly also the canonical TTP targets TNF-alpha and IL-10 as compared with WT DCs. On the protein level, both DC populations expressed comparable amounts of CD80 and CD86 and of either cytokine, but TTP(-/-) DCs expressed less MHCII than WT DCs. On the other hand, TTP(-/-) DCs displayed elevated expression of other TTP target mRNAs like IL-1beta, c-fos and Mkp-1. Stimulation of BM-DCs of either genotype with lipopolysaccharide resulted in a rapid upregulation to a comparable extent of all molecules monitored so far, except for c-fos mRNA. Subsequent mRNA decay analysis revealed gene-specific differences in mRNA stability, which was influenced by the presence of TTP and the activation state of the DCs. Unstimulated TTP(-/-) DCs exerted a markedly lower allogeneic T cell stimulatory potential than WT DCs. Moreover, TTP(-/-) DCs induced an altered cytokine pattern in cocultures of DCs and T cells. However, allogeneic T cells primed by unstimulated DCs of either genotype were equally refractory to restimulation and suppressed the proliferation of naive T cells to the same extent. Thus, the findings of this study lend support to the interpretation that without external stimulation antigen presenting activity in DCs in the presence of TTP is more pronounced than in its absence and that posttranscriptional regulation contributes to the control of gene expression in DCs.

Increased inflammation, impaired bacterial clearance, and metabolic disruption after gram-negative sepsis in Mkp-1-deficient mice.

MAPKs are crucial for TNF-alpha and IL-6 production by innate immune cells in response to TLR ligands. MAPK phosphatase 1 (Mkp-1) deactivates p38 and JNK, abrogating the inflammatory response. We have previously demonstrated that Mkp-1(-/-) mice exhibit exacerbated inflammatory cytokine production and increased mortality in response to challenge with LPS and heat-killed Staphylococcus aureus. However, the function of Mkp-1 in host defense during live Gram-negative bacterial infection remains unclear. We challenged Mkp-1(+/+) and Mkp-1(-/-) mice with live Escherichia coli i.v. to examine the effects of Mkp-1 deficiency on animal survival, bacterial clearance, metabolic activity, and cytokine production. We found that Mkp-1 deficiency predisposed animals to accelerated mortality and was associated with more robust production of TNF-alpha, IL-6 and IL-10, greater bacterial burden, altered cyclooxygenase-2 and iNOS expression, and substantial changes in the mobilization of energy stores. Likewise, knockout of Mkp-1 also sensitized mice to sepsis caused by cecal ligation and puncture. IL-10 inhibition by neutralizing Ab or genetic deletion alleviated increased bacterial burden. Treatment with the bactericidal antibiotic gentamicin, given 3 h after Escherichia coli infection, protected Mkp-1(+/+) mice from septic shock but had no effect on Mkp-1(-/-) mice. Thus, during Gram-negative bacterial sepsis Mkp-1 not only plays a critical role in the regulation of cytokine production but also orchestrates the bactericidal activities of the innate immune system and controls the metabolic response to stress.

MAP kinase phosphatase-1 protects against inflammatory bone loss.

The mitogen-activated protein (MAP) kinase phosphatase (MKP) family plays an important function in regulating the pro-inflammatory cytokines by deactivating MAP kinases. MKP-1 is essential for the dephosphorylation of p38 MAP kinase that regulates expression of IL-6, TNF-alpha, and IL-1 beta. We hypothesized that MKP-1 regulates inflammatory bone loss in experimental periodontitis. Wild-type and Mkp-1(-/-) mice received A. actinomycetemcomitans LPS injection in the palatal region or PBS control 3 times/wk for 30 days. Mice were killed, and maxillae were assessed by microcomputed tomography, histological analysis, and TRAP staining for measurement of bone loss, extent of inflammation, and degree of osteoclastogenesis. Results indicated that, in LPS-injected Mkp-1(-/-) mice, significantly greater bone loss occurred with more inflammatory infiltrate and a significant increase in osteoclastogenesis compared with Mkp-1(-/-) control sites or either wild-type group. Analysis of these data indicates that MKP-1 plays a key role in the regulation of inflammatory bone loss.

Induction of IRAK-M is associated with lipopolysaccharide tolerance in a human endotoxemia model.

Recent in vitro and murine in vivo studies have identified several potential LPS tolerance factors. In this study, we describe the expression kinetics of these LPS tolerance factors in standardized human endotoxemia models using i.v. LPS bolus administration. Responsiveness to LPS as well as the expression of potential regulators of LPS signaling were determined in peripheral whole blood. Intravenous LPS administration (4 ng/kg) resulted in peak plasma levels of TNF-alpha at 1.5 h followed by subsequent peaks of the classic negative feedback inhibitors A20 and IL-10 at 2 and 3 h, respectively. Circulating blood monocyte counts decimated during the initial inflammatory response, but normalized in the period between 4 and 8 h post-LPS. The LPS response as determined by ex vivo TNF release per monocyte in whole blood was profoundly decreased at 6-8 h post-LPS injection despite cessation of A20 and IL-10 expression after 4 h. Analysis of MyD88short, IL-1R-associated kinase (IRAK)-1, IRAK-M, ST2, suppressor of cytokine signaling-1 and -3, SHIP-1, and MAP kinase phosphatase-1 expression indicated that the observed LPS tolerance was associated with decreased IRAK-1 and elevated IRAK-M expression in this human model. Interestingly, a lower dose of LPS (1 ng/kg) induced LPS tolerance accompanied with IRAK-M up-regulation but without depletion of IRAK-1. In vitro studies in whole blood showed that IRAK-M up-regulation by LPS is largely dependent on TNF-alpha. The observed rise of IRAK-M transcription in the human endotoxemia model appeared much greater compared with in vitro-stimulated whole blood. In conclusion, LPS tolerance in human endotoxemia models is associated with IRAK-M up-regulation.