Kcnn4 is a regulator of macrophage multinucleation in bone homeostasis and inflammatory disease.

Macrophages can fuse to form osteoclasts in bone or multinucleate giant cells (MGCs) as part of the immune response. We use a systems genetics approach in rat macrophages to unravel their genetic determinants of multinucleation and investigate their role in both bone homeostasis and inflammatory disease. We identify a trans-regulated gene network associated with macrophage multinucleation and Kcnn4 as being the most significantly trans-regulated gene in the network and induced at the onset of fusion. Kcnn4 is required for osteoclast and MGC formation in rodents and humans. Genetic deletion of Kcnn4 reduces macrophage multinucleation through modulation of Ca(2+) signaling, increases bone mass, and improves clinical outcome in arthritis. Pharmacological blockade of Kcnn4 reduces experimental glomerulonephritis. Our data implicate Kcnn4 in macrophage multinucleation, identifying it as a potential therapeutic target for inhibition of bone resorption and chronic inflammation.

Distinct roles for MyD88 and Toll-like receptors 2, 5, and 9 in phagocytosis of Borrelia burgdorferi and cytokine induction.

The contribution of Toll-like receptors (TLRs) to phagocytosis of Borrelia burgdorferi has not been extensively studied. We show that bone marrow-derived macrophages (BMDM) from MyD88(-/-) mice or Raw cells transfected with a dominant-negative MyD88 were unable to efficiently internalize B. burgdorferi. Knockouts of TLR2 and TLR9 or knockdown of TLR5 by small interfering RNA produced no defects in phagocytosis of B. burgdorferi. Production of inflammatory cytokines was greatly diminished in MyD88(-/-) BMDM but only partially affected in TLR2(-/-) BMDM or knockdown of TLR5 and unaffected in TLR9(-/-) BMDM. Cytochalasin D reduced cytokine induction, but not to the level of the MyD88(-/-) BMDM. Addition of cytochalasin D to TLR2(-/-) BMDM inhibited inflammatory responses to B. burgdorferi to the level of MyD88(-/-) BMDM, consistent with a role for TLR2 in both recognition of extracellular products and lysosomal sampling by TLR2 after processing of the organism. Cytochalasin D had no impact on cytokine production in cells undergoing TLR5 knockdown. These results suggest that MyD88, but not TLR2, TLR5, and TLR9, is important for the uptake of B. burgdorferi and that MyD88 affects inflammatory responses through both its effects on phagocytosis and its role in transducing signals from TLR2 and TLR5.

Borrelia burgdorferi BBB07 interaction with integrin alpha3beta1 stimulates production of pro-inflammatory mediators in primary human chondrocytes.

Borrelia burgdorferi, the causative agent of Lyme disease, activates multiple signalling pathways leading to induction of pro-inflammatory mediators at sites of inflammation. Binding of B. burgdorferi to integrin alpha(3)beta(1) on human chondrocytes activates signalling leading to release of several pro-inflammatory mediators, but the B. burgdorferi protein that binds integrin alpha(3)beta(1) and elicits this response has remained unknown. A search of the B. burgdorferi genome for a canonical integrin binding motif, the RGD (Arg-Gly-Asp) tripeptide, revealed several candidate ligands for integrins. In this study we show that one of these candidates, BBB07, binds to integrin alpha(3)beta(1) and inhibits attachment of intact B. burgdorferi to the same integrin. BBB07 is expressed during murine infection as demonstrated by recognition by infected mouse sera. Recombinant purified BBB07 induces pro-inflammatory mediators in primary human chondrocyte cells by interaction with integrin alpha(3)beta(1). This interaction is specific, as P66, another integrin ligand of B. burgdorferi, does not activate signalling through alpha(3)beta(1). In summary, we have identified a B. burgdorferi protein, BBB07, that interacts with integrin alpha(3)beta(1) and stimulates production of pro-inflammatory mediators in primary human chondrocyte cells.

Role of novel protein kinase C isoforms in Lyme arthritis.

Inflammation caused by Borrelia burgdorferi infection occurs as a result of induction of pro-inflammatory cytokines from activation of multiple signalling pathways. It has previously been shown that mitogen-activated protein kinase (MAPK) and Janus kinase/signal transducer and activator of transcription signalling pathways are activated by B. burgdorferi in cultured human chondrocytes. Protein kinase C (PKC) signalling pathways are potential candidates that may control these downstream signalling pathways. Here we show that B. burgdorferi infection leads to phosphorylation and activation of novel PKC isoforms (PKC delta, epsilon, eta and theta) in a time-dependent manner. A specific inhibitor of novel PKC isoforms blocked the induction of pro-inflammatory molecules in response to B. burgdorferi infection as did transient transfection of novel PKC dominant-negative plasmids into chondrocytes. B. burgdorferi-induced p38 MAPK phosphorylation was also significantly inhibited by an inhibitor of novel PKC isoforms, suggesting that PKC activation occurs upstream of p38 activation. In vivo, administration of an inhibitor of classical and novel PKC isoforms to C3H/HeN mice infected with B. burgdorferi resulted in significantly reduced ankle inflammation and swelling. In conclusion, these data suggest that novel PKC isoforms are specifically activated by B. burgdorferi infection and this can contribute to the regulation of inflammation in vitro and in vivo.

Role of aggrecanase 1 in Lyme arthritis.

OBJECTIVE: Arthritis is one of the hallmarks of late-stage Lyme disease. Previous studies have shown that infection with Borrelia burgdorferi, the causative agent of Lyme disease, results in degradation of proteoglycans and collagen in cartilage. B burgdorferi do not appear to produce any exported proteases capable of digesting proteoglycans and collagen, but instead, induce and activate host proteases, such as matrix metalloproteinases (MMPs), which results in cartilage degradation. The role of aggrecanases in Lyme arthritis has not yet been determined. We therefore sought to delineate the contribution of aggrecanases to joint destruction in Lyme arthritis. METHODS: We examined the expression patterns of aggrecanases 1 and 2 (ADAMTS 4 and 5, respectively) in B burgdorferi-infected primary human chondrocyte cell cultures, in synovial fluid samples from patients with active Lyme arthritis, and in the joints of mice by real-time quantitative reverse transcription-polymerase chain reaction and immunoblotting techniques. Bovine cartilage explants were used to determine the role of aggrecanases in B burgdorferi-induced cartilage degradation. RESULTS: ADAMTS-4, but not ADAMTS-5, was induced in human chondrocytes infected with B burgdorferi. The active forms of ADAMTS-4 were increased in synovial fluid samples from patients with active Lyme arthritis and were elevated in the joints of mice infected with B burgdorferi. Using cartilage explant models of Lyme arthritis, it appeared that the cleavage of aggrecan was predominantly mediated by "aggrecanases" rather than MMPs. CONCLUSION: The induction of ADAMTS-4 by B burgdorferi results in the cleavage of aggrecan, which may be an important first step that leads to permanent degradation of cartilage.

Identification of a TLR-independent pathway for Borrelia burgdorferi-induced expression of matrix metalloproteinases and inflammatory mediators through binding to integrin alpha 3 beta 1.

Borrelia burgdorferi stimulates a robust inflammatory response at sites of localization. Binding of borrelial lipoproteins to TLR-2 is one pathway important in the host response to B. burgdorferi. However, while TLR-2 is clearly important in control of infection, inflammation is actually worsened in the absence of TLR-2 or the shared TLR adapter molecule, MyD88, suggesting that there are alternative pathways regulating inflammation. Integrins are cell surface receptors that play an important role in cell to cell communications and that can activate inflammatory signaling pathways. In this study, we report for the first time that B. burgdorferi binds to integrin alpha(3)beta(1) and that binding of B. burgdorferi to this integrin results in induction of proinflammatory cytokines, chemokines, and end-effector molecules such as matrix metalloproteinases in primary human chondrocyte cells. Expression of these same molecules is not affected by the absence of MyD88 in murine articular cartilage, suggesting that the two pathways act independently in activating host inflammatory responses to B. burgdorferi. B. burgdorferi-induced alpha(3) signaling is mediated by JNK, but not p38 MAPK. In summary, we have identified a new host receptor for B. burgdorferi, integrin alpha(3)beta(1); binding of B. burgdorferi to integrin alpha(3)beta(1) results in the release of inflammatory mediators and is proposed as a TLR-independent pathway for activation of the innate immune response by the organism.

MyD88 deficiency results in tissue-specific changes in cytokine induction and inflammation in interleukin-18-independent mice infected with Borrelia burgdorferi.

Toll-like receptors (TLRs) play an important role in the control of infection with Borrelia burgdorferi. Deficiencies in TLR-2 or the shared TLR adapter molecule MyD88 have been shown to result in greatly increased bacterial burdens in mice. However, although in vitro studies have shown that the activation of TLR pathways by B. burgdorferi results in the release of inflammatory cytokines, studies in deficient mice have shown either no change or increased rather than decreased inflammation in infected animals. In this study, we looked at mechanisms to explain the increase in inflammation in the absence of MyD88. We found that MyD88-deficient mice infected with B. burgdorferi did not show increased inflammation at sites typically associated with Lyme disease (joints and heart). However, there was markedly increased inflammation in the muscles, kidneys, pancreas, and lungs of deficient animals. Muscle inflammation was typically seen perivascularly and perineuronally similar to that seen in infected humans. Chemotactic chemokines and cytokines were greatly increased in the muscle and kidneys of MyD88-deficient animals but not in the joints or heart tissue, suggesting that MyD88-independent pathways for recognizing B. burgdorferi and inducing these chemokines are present in the muscle and kidneys. Interleukin-18 signaling through MyD88 does not appear to play a role in either control of infection or inflammation.

Induction of host matrix metalloproteinases by Borrelia burgdorferi differs in human and murine lyme arthritis.

Matrix metalloproteinases (MMPs) are induced from host tissues in response to Borrelia burgdorferi. Upregulation of MMPs may play a role in the dissemination of the organism through extracellular matrix tissues, but it can also result in destructive pathology. Although mice are a well-accepted model for Lyme arthritis, there are significant differences compared to human disease. We sought to determine whether MMP expression could account for some of these differences. MMP expression patterns following B. burgdorferi infection were analyzed in primary human chondrocytes, synovial fluid samples from patients with Lyme arthritis, and cartilage tissue from Lyme arthritis-susceptible and -resistant mice by using a gene array, real-time PCR, an enzyme-linked immunosorbent assay, and immunohistochemistry. B. burgdorferi infection significantly induced transcription of MMP-1, -3, -13, and -19 from primary human chondrocyte cells. Transcription of MMP-10 and tissue inhibitor of metalloprotease 1 was increased with B. burgdorferi infection, but protein expression was only minimally increased. The synovial fluid levels of MMPs from patients with high and low spirochete burdens were consistent with results seen in the in vitro studies. B. burgdorferi-susceptible C3H/HeN mice infected with B. burgdorferi showed induction of MMP-3 and MMP-19 but no other MMP or tissue inhibitor of metalloprotease. As determined by immunohistochemistry, MMP-3 expression was increased only in chondrocytes near the articular surface. The levels of MMPs were significantly lower in the more Lyme arthritis-resistant BALB/c and C57BL/6 mice. Differences between human and murine Lyme arthritis may be related to the lack of induction of collagenases, such MMP-1 and MMP-13, in mouse joints.

Borrelia burgdorferi-induced expression of matrix metalloproteinases from human chondrocytes requires mitogen-activated protein kinase and Janus kinase/signal transducer and activator of transcription signaling pathways.

Elevations in matrix metalloproteinase 1 (MMP-1) and MMP-3 have been found in patients with Lyme arthritis and in in vitro models of Lyme arthritis using cartilage explants and chondrocytes. The pathways by which B. burgdorferi, the causative agent of Lyme disease, induces the production of MMP-1 and MMP-3 have not been elucidated. We examined the role of the extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK) and the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways in MMP induction by B. burgdorferi. Infection with B. burgdorferi results in rapid phosphorylation of p38 and JNK within 15 to 30 min. Inhibition of JNK and p38 MAPK significantly reduced B. burgdorferi-induced MMP-1 and MMP-3 expression. Inhibition of ERK1/2 completely inhibited the expression of MMP-3 in human chondrocytes following B. burgdorferi infection but had little effect on the expression of MMP-1. B. burgdorferi infection also induced phosphorylation and nuclear translocation of STAT-3 and STAT-6 in primary human chondrocytes. Expression of MMP-1 and MMP-3 was significantly inhibited by inhibition of JAK3 activity. Induction of MMP-1 and -3 following MAPK and JAK/STAT activation was cycloheximide sensitive, suggesting synthesis of intermediary proteins is required. Inhibition of tumor necrosis factor alpha (TNF-alpha) significantly reduced MMP-1 but not MMP-3 expression from B. burgdorferi-infected cells; inhibition of interleukin-1beta (IL-1beta) had no effect. Treatment of B. burgdorferi-infected cells with JAK and MAPK inhibitors significantly inhibited TNF-alpha induction, consistent with at least a partial role for TNF-alpha in B. burgdorferi-induced MMP-1 expression in chondrocytes.

Chitosan IFN-gamma-pDNA Nanoparticle (CIN) Therapy for Allergic Asthma.

BACKGROUND: Allergic subjects produce relatively low amounts of IFN-gamma, a pleiotropic Th-1 cytokine that downregulates Th2-associated airway inflammation and hyperresponsiveness (AHR), the hallmarks of allergic asthma. Adenovirus-mediated IFN-gamma gene transfer reduces AHR, Th2 cytokine levels and lung inflammation in mice, but its use would be limited by the frequency of gene delivery required; therefore, we tested chitosan/IFN-gamma pDNA nanoparticles (CIN) for in situ production of IFN-gamma and its in vivo effects. METHODS: CIN were administered to OVA-sensitized mice to investigate the possibility of using gene transfer to modulate ovalbumin (OVA)-induced inflammation and AHR. RESULTS: Mice treated with CIN exhibit significantly lower AHR to methacholine challenge and less lung histopathology. Production of IFN-gamma is increased after CIN treatment while the Th2-cytokines, IL-4 and IL-5, and OVA-specific serum IgE are reduced compared to control mice. AHR and eosinophilia are also significantly reduced by CIN therapy administered therapeutically in mice with established asthma. CIN was found to inhibit epithelial inflammation within 6 hours of delivery by inducing apoptosis of goblet cells. Experiments performed on STAT4-defective mice do not show reduction in AHR with CIN treatment, thus implicating STAT4 signaling in the mechanism of CIN action. CONCLUSION: These results demonstrate that mucosal CIN therapy can effectively reduce established allergen-induced airway inflammation and AHR.

Respiratory syncytial virus infection activates STAT signaling in human epithelial cells.

Acute respiratory syncytial virus (RSV) infection causes airway inflammation and exacerbates asthma, but the mechanism of inflammation is poorly understood. The role of the STAT-signaling pathway in RSV infection in epithelial cells was examined in this study. DNA microarray analyses of RSV-infected human alveolar type II (A549) epithelial cells identified several genes whose expression was altered from -5.5 to +56.4-fold. Four of the highly expressed genes contained STAT-binding elements. In A549 and normal human bronchial epithelial cells (NHBE), RSV induced phosphorylation and nuclear translocation of STAT-1alpha that was abrogated when RSV attachment was blocked. Treatment with a JAK-2 inhibitor or transfection with dominant-negative STAT-1alpha blocked STAT-1alpha activation and RSV infection. RSV also activated STAT-3 and IL-6 specific antibodies blocked this activation. Thus, activation of the STAT-1alpha and STAT-3 pathways play a role in RSV infection.

Atrial natriuretic peptide gene transfer by means of intranasal administration attenuates airway reactivity in a mouse model of allergic sensitization.

Atrial natriuretic peptide (ANP) is a bronchodilator; however, the short half-life of ANP in vivo limited its therapeutic utility to treat asthma. The efficacy of intranasally administered plasmid DNA-expressing ANP (pANP; amino acid 99-126; Acc. No. XM131840) on the prevention of allergen-induced airway hyperresponsiveness (AHR) was examined in this study by using a mouse model of asthma. Ovalbumin-sensitized mice were treated with pANP versus control plasmids, and AHR was monitored after ovalbumin challenge for 5 weeks on 10-day intervals starting 4 days after gene transfer. Mice administered pANP demonstrated significantly less AHR for 20 days after treatment. The results demonstrate that pANP gene transfer protects against AHR and might be useful in the treatment of asthma.

Adenovirus-mediated interferon gamma gene therapy for allergic asthma: involvement of interleukin 12 and STAT4 signaling.

Allergic asthma is associated with airway inflammation and hyperresponsiveness caused by the dysregulated production of cytokines secreted by allergen-specific helper T type 2 (Th2) cells. Allergic subjects produce relatively low amounts of interferon gamma (IFN-gamma), a pleiotropic Th1 cytokine that downregulates Th2-associated responses. In this study, we examined the possibility of modulating ovalbumin (OVA)-induced inflammation and airway hyperreactivity (AHR) by recombinant adenovirus-mediated IFN-gamma (Ad-IFN-gamma) gene transfer. OVA-sensitized mice treated with Ad-IFN-gamma exhibit significantly lower levels of Th2 cytokines interleukin 4 (IL-4) and IL-5, OVA-specific serum IgE, lung eosinophilia, and AHR in response to methacholine challenge compared with control mice. The lung sections of the treated mice show less epithelial damage, mucous plugging, and eosinophil infiltration than controls. In contrast, Ad-IFN-gamma-treated mice express significantly higher levels of IFN-gamma and IL-12 when compared with controls. Moreover, administration of Ad-IFN-gamma to mice with established AHR significantly reduced AHR, Th2 cytokines, and lung inflammation. The IFN-gamma effects were dependent on IL-12 and STAT4 (signal transducer and activator of transcription 4), as mice treated with antibodies to IL-12 and STAT4 deficient mice show attenuated Ad-IFN-gamma responses. Thus, these results demonstrate that mucosal Ad-IFN-gamma gene transfer can effectively attenuate established allergen-induced airway inflammation and AHR, predominantly through an IL-12- and STAT4-dependent mechanism.

Intranasal gene transfer by chitosan-DNA nanospheres protects BALB/c mice against acute respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) infection is often associated in infancy with life-threatening bronchiolitis, which is also a major risk factor for the development of asthma. At present, no effective prophylaxis is available against RSV infection. Herein, we describe an effective prophylactic intranasal gene transfer strategy utilizing chitosan-DNA nanospheres (IGT), containing a cocktail of plasmid DNAs encoding all RSV antigens, except L. A single administration of IGT (25 microg/mouse) induces expression of the mRNA and proteins of all antigens in the lung and results in a significant reduction of viral titers and viral antigen load after acute RSV infection of these mice. IGT-administered mice show no significant change in airway reactivity to methacholine and no apparent pulmonary inflammation. Furthermore, IGT results in significant induction of RSV-specific IgG antibodies, nasal IgA antibodies, cytotoxic T lymphocytes, and interferon-gamma production in the lung and splenocytes compared with controls. Together, these results demonstrate the potential of IGT against acute RSV infection.

2'-5' Oligoadenylate synthetase plays a critical role in interferon-gamma inhibition of respiratory syncytial virus infection of human epithelial cells.

Respiratory syncytial virus (RSV), associated with bronchiolitis and asthma, is resistant to the antiviral effects of type-I interferons (IFN), but not IFN-gamma. However, the antiviral mechanism of IFN-gamma action against RSV infection is unknown. The molecular mechanism of IFN-gamma-induced antiviral activity was examined in this study using human epithelial cell lines HEp-2 and A549. Exposure of these cells to 100-1000 units/ml of IFN-gamma, either before or after RSV infection, results in a significant decrease in RSV infection. After 1 h of exposure, IFN-gamma induces protein expression of IFN regulatory factor-1 (IRF-1) but not IRF-2, double-stranded RNA-activated protein kinase, and inducible nitric-oxide synthase in these cells. The mRNA for IRF-1, p40, and p69 isoforms of 2'-5' oligoadenylate synthetase (2-5 AS) are detectable, respectively, at 1 and 4 h of IFN-gamma exposure. Studies using cycloheximide and antisense oligonucleotides to IRF-1 indicate a direct role of IRF-1 in activating 2-5 AS. Cells transfected with 2-5 AS antisense oligonucleotides inhibit the antiviral effect of IFN-gamma. A stable cell line of HEp-2 overexpressing RNase L inhibitor, RLI-14, which exhibits an IFN-gamma-induced gene expression pattern similar to that of the parent cell line, shows a significant reduction in RNase L activity and IFN-gamma-mediated antiviral effect, compared with HEp-2 cells. These results provide direct evidence of the involvement of 2-5 AS in IFN-gamma-mediated antiviral activity in these cells.