Inositol hexakisphosphate kinase 1 regulates neutrophil function in innate immunity by inhibiting phosphatidylinositol-(3,4,5)-trisphosphate signaling.

Inositol phosphates are widely produced throughout animal and plant tissues. Diphosphoinositol pentakisphosphate (InsP7) contains an energetic pyrophosphate bond. Here we demonstrate that disruption of inositol hexakisphosphate kinase 1 (InsP6K1), one of the three mammalian inositol hexakisphosphate kinases (InsP6Ks) that convert inositol hexakisphosphate (InsP6) to InsP7, conferred enhanced phosphatidylinositol-(3,4,5)-trisphosphate (PtdIns(3,4,5)P3)-mediated membrane translocation of the pleckstrin homology domain of the kinase Akt and thus augmented downstream PtdIns(3,4,5)P3 signaling in mouse neutrophils. Consequently, these neutrophils had greater phagocytic and bactericidal ability and amplified NADPH oxidase-mediated production of superoxide. These phenotypes were replicated in human primary neutrophils with pharmacologically inhibited InsP6Ks. In contrast, an increase in intracellular InsP7 blocked chemoattractant-elicited translocation of the pleckstrin homology domain to the membrane and substantially suppressed PtdIns(3,4,5)P3-mediated cellular events in neutrophils. Our findings establish a role for InsP7 in signal transduction and provide a mechanism for modulating PtdIns(3,4,5)P3 signaling in neutrophils.

Leukotriene B4-driven neutrophil recruitment to the skin is essential for allergic skin inflammation.

Scratching triggers skin flares in atopic dermatitis. We demonstrate that scratching of human skin and tape stripping of mouse skin cause neutrophil influx. In mice, this influx was largely dependent on the generation of leukotriene B4 (LTB4) by neutrophils and their expression of the LTB4 receptor BLT1. Allergic skin inflammation in response to epicutaneous (EC) application of ovalbumin to tape-stripped skin was severely impaired in Ltb4r1(-/-) mice and required expression of BLT1 on both T cells and non-T cells. Cotransfer of wild-type (WT) neutrophils, but not neutrophils deficient in BLT1 or the LTB4-synthesizing enzyme LTA4H, restored the ability of WT CD4(+) effector T cells to transfer allergic skin inflammation to Ltb4r1(-/-) recipients. Pharmacologic blockade of LTB4 synthesis inhibited allergic skin inflammation elicited by cutaneous antigen challenge in previously EC-sensitized mice. Our results demonstrate that a neutrophil-T cell axis reliant on LTB4-BLT1 interaction is required for allergic skin inflammation.

Inhibition of PAX3 by TGF-beta modulates melanocyte viability.

The protein encoded by paired-box homeotic gene 3 (PAX3) is a key regulator of the microphthalmia-associated transcription factor (Mitf) in the melanocyte lineage. Here, we show that PAX3 expression in skin is directly inhibited by TGF-beta/Smads. UV irradiation represses TGF-beta in keratinocytes, and the repression of TGF-beta/Smads upregulates PAX3 in melanocytes, which is associated with a UV-induced melanogenic response and consequent pigmentation. Furthermore, the TGF-beta-PAX3 signaling pathway interacts with the p53-POMC/MSH-MC1R signaling pathway, and both are crucial in melanogenesis. The activation of p53-POMC/MSH-MC1R signaling is required for the UV-induced melanogenic response because PAX3 functions in synergy with SOX10 in a cAMP-response element (CRE)-dependent manner to regulate the transcription of Mitf. This study will provide a rich foundation for further research on skin cancer prevention by enabling us to identify targeted small molecules in the signaling pathways of the UV-induced melanogenic response that are highly likely to induce naturally protective pigmentation.

Deactivation of Akt by a small molecule inhibitor targeting pleckstrin homology domain and facilitating Akt ubiquitination.

The phosphatidylinositol-3,4,5-triphosphate (PIP3) binding function of pleckstrin homology (PH) domain is essential for the activation of oncogenic Akt/PKB kinase. Following the PIP3-mediated activation at the membrane, the activated Akt is subjected to other regulatory events, including ubiquitination-mediated deactivation. Here, by identifying and characterizing an allosteric inhibitor, SC66, we show that the facilitated ubiquitination effectively terminates Akt signaling. Mechanistically, SC66 manifests a dual inhibitory activity that directly interferes with the PH domain binding to PIP3 and facilitates Akt ubiquitination. A known PH domain-dependent allosteric inhibitor, which stabilizes Akt, prevents the SC66-induced Akt ubiquitination. A cancer-relevant Akt1 (e17k) mutant is unstable, making it intrinsically sensitive to functional inhibition by SC66 in cellular contexts in which the PI3K inhibition has little inhibitory effect. As a result of its dual inhibitory activity, SC66 manifests a more effective growth suppression of transformed cells that contain a high level of Akt signaling, compared with other inhibitors of PIP3/Akt pathway. Finally, we show the anticancer activity of SC66 by using a soft agar assay as well as a mouse xenograft tumor model. In conclusion, in this study, we not only identify a dual-function Akt inhibitor, but also demonstrate that Akt ubiquitination could be chemically exploited to effectively facilitate its deactivation, thus identifying an avenue for pharmacological intervention in Akt signaling.

Pretreatment with phosphatase and tensin homolog deleted on chromosome 10 (PTEN) inhibitor SF1670 augments the efficacy of granulocyte transfusion in a clinically relevant mouse model.

The clinical outcome of granulocyte transfusion therapy is often hampered by short ex vivo shelf life, inefficiency of recruitment to sites of inflammation, and poor pathogen-killing capability of transplanted neutrophils. Here, using a recently developed mouse granulocyte transfusion model, we revealed that the efficacy of granulocyte transfusion can be significantly increased by elevating intracellular phosphatidylinositol (3,4,5)-trisphosphate signaling with a specific phosphatase and tensin homolog deleted on chromosome 10 (PTEN) inhibitor SF1670. Neutrophils treated with SF1670 were much sensitive to chemoattractant stimulation. Neutrophil functions, such as phagocytosis, oxidative burst, polarization, and chemotaxis, were augmented after SF1670 treatment. The recruitment of SF1670-pretreated transfused neutrophils to the inflamed peritoneal cavity and lungs was significantly elevated. In addition, transfusion with SF1670-treated neutrophils led to augmented bacteria-killing capability (decreased bacterial burden) in neutropenic recipient mice in both peritonitis and bacterial pneumonia. Consequently, this alleviated the severity of and decreased the mortality of neutropenia-related pneumonia. Together, these observations demonstrate that the innate immune responses can be enhanced and the severity of neutropenia-related infection can be alleviated by augmenting phosphatidylinositol (3,4,5)-trisphosphate in transfused neutrophils with PTEN inhibitor SF1670, providing a therapeutic strategy for improving the efficacy of granulocyte transfusion.

PTEN negatively regulates engulfment of apoptotic cells by modulating activation of Rac GTPase.

Efficient clearance of apoptotic cells by phagocytes (efferocytosis) is critical for normal tissue homeostasis and regulation of the immune system. Apoptotic cells are recognized by a vast repertoire of receptors on macrophage that lead to transient formation of phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] and subsequent cytoskeletal reorganization necessary for engulfment. Certain PI3K isoforms are required for engulfment of apoptotic cells, but relatively little is known about the role of lipid phosphatases in this process. In this study, we report that the activity of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a phosphatidylinositol 3-phosphatase, is elevated upon efferocytosis. Depletion of PTEN in macrophage results in elevated PtdIns(3,4,5)P(3) production and enhanced phagocytic ability both in vivo and in vitro, whereas overexpression of wild-type PTEN abrogates this process. Loss of PTEN in macrophage leads to activation of the pleckstrin homology domain-containing guanine-nucleotide exchange factor Vav1 and subsequent activation of Rac1 GTPase, resulting in increased amounts of F-actin upon engulfment of apoptotic cells. PTEN disruption also leads to increased production of anti-inflammatory cytokine IL-10 and decreased production of proinflammatory IL-6 and TNF-α upon engulfment of apoptotic cells. These data suggest that PTEN exerts control over efferocytosis potentially by regulating PtdIns(3,4,5)P(3) levels that modulate Rac GTPase and F-actin reorganization through Vav1 exchange factor and enhancing apoptotic cell-induced anti-inflammatory response.

Small-molecule screen identifies reactive oxygen species as key regulators of neutrophil chemotaxis.

Neutrophil chemotaxis plays an essential role in innate immunity, but the underlying cellular mechanism is still not fully characterized. Here, using a small-molecule functional screening, we identified NADPH oxidase-dependent reactive oxygen species as key regulators of neutrophil chemotactic migration. Neutrophils with pharmacologically inhibited oxidase, or isolated from chronic granulomatous disease (CGD) patients and mice, formed more frequent multiple pseudopodia and lost their directionality as they migrated up a chemoattractant concentration gradient. Knocking down NADPH oxidase in differentiated neutrophil-like HL60 cells also led to defective chemotaxis. Consistent with the in vitro results, adoptively transferred CGD murine neutrophils showed impaired in vivo recruitment to sites of inflammation. Together, these results present a physiological role for reactive oxygen species in regulating neutrophil functions and shed light on the pathogenesis of CGD.

Targeted deletion of tumor suppressor PTEN augments neutrophil function and enhances host defense in neutropenia-associated pneumonia.

Neutropenia and related infections are the most important dose-limiting toxicities in anticancer chemotherapy and radiotherapy. In this study, we explored a new strategy for augmenting host defense in neutropenia-related pneumonia. Phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) signaling in neutrophils was elevated by depleting PTEN, a phosphatidylinositol 3'-phosphatase that hydrolyzes PtdIns(3,4,5)P(3). In myeloid-specific PTEN knockout mice, significantly more neutrophils were recruited to the inflamed lungs during neutropenia-associated pneumonia. Using an adoptive transfer technique, we demonstrated that this enhancement could be caused directly by PTEN depletion in neutrophils. In addition, disruption of PTEN increased the recruitment of macrophages and elevated proinflammatory cytokines/chemokine levels in the inflamed lungs, which could also be responsible for the enhanced neutrophil recruitment. Depleting PTEN also significantly delayed apoptosis and enhanced the bacteria-killing capability of the recruited neutrophils. Finally, we provide direct evidence that enhancement of neutrophil function by elevating PtdIns(3,4,5)P(3) signaling can alleviate pneumonia-associated lung damage and decrease pneumonia-elicited mortality. Collectively, these results not only provide insight into the mechanism of action of PTEN and PtdIns(3,4,5)P(3) signaling pathway in modulating neutrophil function during lung infection and inflammation, but they also establish PTEN and related pathways as potential therapeutic targets for treating neutropenia-associated pneumonia.

Myeloid-specific deletion of tumor suppressor PTEN augments neutrophil transendothelial migration during inflammation.

Phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) is a second messenger that is involved in a number of cell activities including cell growth, proliferation, and motility. PIP(3) is produced by PI3K and regulated by PTEN (phosphatase and tensin homolog deleted on chromosome 10) and SHIP lipid phosphatases. Evidence from our experiments shows that enhanced PIP(3) production results in elevated neutrophil recruitment under inflammatory conditions. However, the mechanism of this elevation is not well understood. We used intravital video microscopy to investigate neutrophil recruitment in the cremaster venules of wild-type and PTEN knockout (KO) mice. Neutrophil transmigration was augmented in PTEN KO mice 4 h after TNF-alpha intrascrotal injection. PTEN KO neutrophils also showed significantly enhanced transmigration 2 h after MIP-2 intrascrotal injection, an effect that dramatically decreased when PI3K or Src kinase inhibitor treatments preceded MIP-2 stimulation. Similarly, fMLP superfusion of the cremaster muscle lead to enhanced emigration in PTEN KO mice. The observed elevation in neutrophil emigration was likely caused by increased speed of crawling, crossing the venular wall, and migrating through the muscular tissue in PTEN KO mice because the effect of PTEN depletion on neutrophil rolling or adhesion was minimal. Interestingly, chemoattractant-induced release of gelatinase and elastase was also elevated in PTEN null neutrophils, providing a potential mechanism for the enhanced neutrophil migration in the PTEN KO mice. Collectively, these results demonstrate that PTEN deletion in neutrophils enhances their invasivity and recruitment to inflamed sites more likely by raising the cell physical capability to cross the vascular and tissue barriers.

Focal adhesion kinase regulates pathogen-killing capability and life span of neutrophils via mediating both adhesion-dependent and -independent cellular signals.

Various neutrophil functions such as phagocytosis, superoxide production, and survival are regulated by integrin signaling. Despite the essential role of focal adhesion kinase (FAK) in mediating this signaling pathway, its exact function in neutrophils is ill defined. In this study, we investigated the role of FAK in neutrophils using a myeloid-specific conditional FAK knockout mouse. As reported in many other cell types, FAK is required for regulation of focal adhesion dynamics when neutrophils adhere to fibronectin or ICAM-1. Adhesion on VCAM-1-coated surfaces and chemotaxis after adhesion were not altered in FAK null neutrophils. In addition, we observed significant reduction in NADPH oxidase-mediated superoxide production and complement-mediated phagocytosis in FAK null neutrophils. As a result, these neutrophils displayed decreased pathogen killing capability both in vitro and in vivo in a mouse peritonitis model. In adherent cells, the defects associated with FAK deficiency are likely due to suppression of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) signaling and chemoattractant-elicited calcium signaling. Disruption of FAK also reduced chemoattractant-elicited superoxide production in suspended neutrophils in the absence of cell adhesion. This may be solely caused by suppression of PtdIns(3,4,5)P3 signaling in these cells, because the fMLP-elicited calcium signal was not altered. Consistent with decreased PtdIns(3,4,5)P3/Akt signaling in FAK null neutrophils, we also observed accelerated spontaneous death in these cells. Taken together, our results revealed previously unrecognized roles of FAK in neutrophil function and provided a potential therapeutic target for treatment of a variety of infectious and inflammatory diseases.

Inositol trisphosphate 3-kinase B (InsP3KB) as a physiological modulator of myelopoiesis.

Inositol trisphosphate 3-kinase B (InsP3KB) belongs to a family of kinases that convert inositol 1,4,5-trisphosphate (Ins(1,4,5)P3 or IP3) to inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4). Previous studies have shown that disruption of InsP3KB leads to impaired T cell and B cell development as well as hyperactivation of neutrophils. Here, we demonstrate that InsP3KB is also a physiological modulator of myelopoiesis. The InsP3KB gene is expressed in all hematopoietic stem/progenitor cell populations. In InsP3KB null mice, the bone marrow granulocyte monocyte progenitor (GMP) population was expanded, and GMP cells proliferated significantly faster. Consequently, neutrophil production in the bone marrow was enhanced, and the peripheral blood neutrophil count was also substantially elevated in these mice. These effects might be due to enhancement of PtdIns(3,4,5)P3/Akt signaling in the InsP3KB null cells. Phosphorylation of cell cycle-inhibitory protein p21(cip1), one of the downstream targets of Akt, was augmented, which can lead to the suppression of the cell cycle-inhibitory effect of p21.

[Inhibition of dexamethasone, indomethacin and resveratrol on matrix metalloproteinase-9 and the mechanism of inhibition].

AIM: To investigate the expression of matrix metalloproteinase-9 (MMP-9) in mouse ears induced with croton oil and the inhibitory effect of dexamethasone, indomethacin and resveratrol on MMP-9 expression, and further explore the relationship between anti-inflammation and MMP-9 inhibition of these three medicines. METHODS: Immuno-histochemistry was used to detect the expression of MMP-9 in mouse ears. Expression of MMP-9 in U937 cells was analyzed by gelatin zymography. RESULTS: Mouse ear edema induced with croton oil was inhibited significantly by dexamethasone and indomethacin at the dose of 10 mg.kg-1 and resveratrol at 50 mg.kg-1 administered subcutaneously. The inhibitory rate was 76.2% (P < 0.001), 56.7% (P < 0.001) and 36.9% (P < 0.001) respectively. The MMP-9 expression increased in mouse ears induced with croton oil and inhibited by dexamethasone, indomethacin and resveratrol at above doses. Gelatin zymography results showed that MMP-9 expression in U937 cells increased significantly after exposed to PMA at 1 x 10(-8) mol.L-1 (P < 0.001); MMP-9 expression induced with phorbol myristate acetate(PMA) was inhibited by dexamethasone at 1 x 10(-9), 1 x 10(-7) and 1 x 10(-5) mol.L-1, indomethacin at 1 x 10(-6) and 1 x 10(-5) mol.L-1 and resveratrol at 1 x 10(-6) and 1 x 10(-5) mol.L-1. CONCLUSION: The inhibition of MMP-9 expression may be one of the anti-inflammatory mechanisms of dexamethasone, indomethacin and resveratrol.

[Effect of lipopolysaccharide on expression of matrix metalloproteinase-9 in human synoviocyte from patients with rheumatoid arthritis].

AIM: To study the effects of lipopolysaccharide (LPS), the supernatant of U937 cells stimulated with LPS and dexamethasone on matrix metalloproteinase-9 (MMP-9) expression in the synoviocyte from patients with rheumatoid arthritis(RA). METHODS: Fibroblast-like cells (FLS) from the joint tissue of patients with rheumatoid arthritis were cultured and incubated for 24 h with LPS (1 mg.L-1) or the supernatant of U937 cells stimulated with LPS (1 mg.L-1) for 24 h. Dexamethasone was added to the supernatant of U937 cells and FLS was incubated for 24 h. The activity of MMP-9 was analyzed by gelatin zymography. Protein expression of MMP-9 was detected by Western blot using special polyclonal antibodies. The mRNA expression of MMP-9 was detected by RT-PCR. RESULTS: The expression of MMP-9 was not markedly changed in FLS treated with LPS. The MMP-9 activity, MMP-9 secretion and MMP-9 mRNA expression were significantly increased in FLS cultured with the supernatant from U937 cell treated with LPS. Dexamethasone markedly inhibited the activity, protein secretion and mRNA expression of MMP-9 in FLS cultured with the supernatant from U937 cell stimulated with LPS, and the inhibitory effects were increased as the concentration of dexamethasone increased. CONCLUSION: LPS did not directly affect the expression of MMP-9 in FLS, but it was found to indirectly cause the increase of MMP-9 expression in FLS by stimulating U937 cell. Dexamethasone was found to inhibit this increase of MMP-9 expression.

[Effect of lipopolysaccharide on expression of interleukin-6 in human synoviocyte from patients with rheumatoid arthritis].

AIM: To study the effects of lipopolysaccharide (LPS), the supernatant of U937 cells stimulated with LPS and dexamethasone on interleukin-6 (IL-6) expression in the synoviocyte from patients with rheumatoid arthritis (RA). METHODS: Fibroblast-like synoviocytes (FLS) from the joint tissue of patients with rheumatoid arthritis were cultured and incubated for 24 h with LPS (1 mg.L-1) or the supernatant of U937 cells stimulated with LPS (1 mg.L-1) for 24 h. Dexamethasone was added to the supernatant of U937 cells and FLS was incubated for 24 h. The expression of IL-6 protein was detected by radioimmunoassay. The mRNA expression of IL-6 was accessed by RT-PCR. RESULTS: The growth of FLS was not markedly affected by LPS, and the protein secretion and mRNA expression of IL-6 were not markedly changed in FLS treated with LPS. The IL-6 secretion and IL-6 mRNA expression were significantly increased in FLS cultured with the supernatant from U937 cell treated with LPS. Dexamethasone markedly inhibited the protein secretion and mRNA expression of IL-6 in FLS cultured with the supernatant from U937 cell stimulated with LPS. The inhibitory effects were increased as the concentration of dexamethasone increased. CONCLUSION: LPS was not shown to directly affect the expression of IL-6 in FLS, but it indirectly causes the increase of the IL-6 expression in FLS by stimulating U937 cell. Dexamethasone can inhibit this increase of the IL-6 expression.

Anti-inflammatory effect of amurensin H on asthma-like reaction induced by allergen in sensitized mice.

AIM: To explore the anti-inflammatory effects of amurensin H on asthma-like reaction induced by allergen in sensitized mice. METHODS: BALB/c mice were sensitized by ovalbumin (OVA, ip) on d 0 and d 14 and challenged with 1% OVA on d 18 to 22. Mice developed airway eosinophilia, mucus hypersecretion, and elevation in cytokine levels. Mice were administered amurensin H orally at the doses of 49, 70, or 100 mg/kg once every day from d 15 to the last day. Bronchoalveolar lavage fluid (BALF) were collected at 24 h and 48 h after the last OVA challenge. Levels of tumor necrosis factor-alpha (TNF-alpha), interleukin 4 (IL-4), interleukin 5 (IL-5), and interleukin 13 (IL-13) in BALF were measured using ELISA method. Differential cell counts of macrophages, lymphocytes, neutrophils and eosinophils were performed in 200 cells per slide (one slide per animal). Lung tissue sections of 6-mum thickness were stained with Mayer's hematoxylin and eosin for assessment of cell infiltration, mucus production, and tissue damage. RESULTS: Oral administration of amurensin H significantly inhibited OVA-induced increases in total cell counts, eosinophil counts, and TNF- alpha, IL-4, IL-5 and IL-13 levels in BALF. In addition, amuresin H dramatically decreased OVA-induced lung tissue damage and mucus production. CONCLUSION: Amurensin H may have therapeutic potential for the treatment of allergic airway inflammation.

Resveratrol inhibits matrix metalloproteinase-9 transcription in U937 cells.

AIM: To examine the inhibitory effect of resveratrol on matrix metalloroteinase-9 (MMP-9) and explore its mechanism. METHODS: MMP-9 activity was analyzed by gelatin zymography; MMP-9 protein was detected by Western blot; MMP-9 mRNA expression was investigated by RT-PCR. Activation of activator protein -1 (AP-1) was measured by electrophoretic mobility shift assay (EMSA). RESULTS: MMP-9 activity in U937 cells increased significantly after exposed to PMA at 10 nmol/L for 24 h without FCS (P<0.01). Resveratrol at 1 and 10 micromol/L showed significant inhibition on MMP-9 activity (P<0.05 and P<0.01, respectively). Western blot and RT-PCR experiments displayed that MMP-9 protein (P<0.01) and mRNA expression (P<0.01) increased significantly in PMA-treated U937 cells. Resveratrol at 1 and 10 micromol/L showed inhibitory effects on MMP-9 protein production and MMP-9 mRNA expression (P<0.05). The activation of AP-1 induced by PMA was also extensively inhibited by resveratrol at 0.1, 1, and 10 micromol/L. CONCLUSION: The inhibitory effect of resveratrol on MMP-9 activity may be partly through suppression of activation of nuclear transcription factor AP-1, and inhibition of MMP-9 mRNA expression and MMP-9 protein production.

Suppression of IL-8 gene transcription by resveratrol in phorbol ester treated human monocytic cells.

Resveratrol (3,4',5-trihydroxy-trans-silbene), a natural phytoalexin found in grapes and other food products, has promising anti-inflammatory and anticancer effects. To observe the modulation of interleukin-8 (IL-8) production in human monocytic cells by resveratrol and explore its mechanism at the gene transcription level, U937 cells were stimulated with phorbol 12-myristate 13-acetate (PMA) for 24h. IL-8 protein in supernatants was measured by radioimmunoassay. The cytotoxicity of PMA, dexamethasone and resveratrol was accessed by MTT cell proliferation assay. The RNA level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and IL-8 were detected by RT-PCR using specific primers. DNA binding activities of NF-kappaB and AP-1 were examined by electrophoretic mobility shift assay (EMSA). 0.01-100 nM PMA could significantly induce IL-8 production in U937 cells; 10 microM Dexamethasone and 10, 1, 0.1 microM resveratrol could inhibit PMA-induced IL-8 protein production and mRNA accumulation. The cytotoxicity did not contribute to their inhibitory effect. The DNA binding activity of AP-1 was inhibited by dexamethasone and resveratrol, but resveratrol has little effect on PMA-induced NF-kappaB activation. Resveratrol could inhibit PMA-induced IL-8 production in U937 cells at protein and mRNA levels. The suppression of IL-8 gene transcription by resveratrol was, at least partly, due to inhibition of AP-1 activation.