Lipopolysaccharide (LPS) stimulation of Pancreatic Ductal Adenocarcinoma (PDAC) and macrophages activates the NLRP3 inflammasome that influences the levels of pro-inflammatory cytokines in a co-culture model.

Modified macrophages, tumor-associated macrophages (TAMs), are key contributors to the survival, growth, and metastatic behavior of pancreatic ductal adenocarcinoma (PDAC) cells. Central to the role of inflammation and TAMs lies the NLRP3 inflammasome. This study investigated the effects of LPS-stimulated inflammation on cell proliferation, levels of pro-inflammatory cytokines, and the NLRP3 inflammasome pathway in a co-culture model using PDAC cells and macrophages in the presence or absence of MCC950, a NLRP3-specific inhibitor. The effects of LPS-stimulated inflammation were tested on two PDAC cell lines (Panc 10.05 and SW 1990) co-cultured with RAW 264.7 macrophages. Cell proliferation was determined using the MTT assay. Levels of pro-inflammatory cytokines, IL-1ß, and TNF-α were determined by ELISA. Western blot analyses were used to examine the expression of NLRP3 in both PDAC cells and macrophages. The co-culture and interaction between PDAC cell lines and macrophages led to pro-inflammatory microenvironment under LPS stimulation as evidenced by high levels of secreted IL-1ß and TNF-α. Inhibition of the NLRP3 inflammasome by MCC950 counteracted the effects of LPS stimulation on the regulation of the NLRP3 inflammasome and pro-inflammatory cytokines in PDAC and macrophages. However, MCC950 differentially modified the viability of the metastatic vs primary PDAC cell lines. LPS stimulation increased PDAC cell viability by regulating the NLRP3 inflammasome and pro-inflammatory cytokines in the tumor microenvironment of PDAC cells/macrophages co-cultures. The specific inhibition of the NLRP inflammasome by MCC950 effectively counteracted the LPS-stimulated inflammation.

Lactoferrin modulates the biofilm formation and bap gene expression of methicillin-resistant Staphylococcus epidermidis.

Lactoferrin is an innate glycoprotein with broad antibacterial and antibiofilm properties. The autonomous antibiofilm activity of lactoferrin against Gram-positive bacteria is postulated to involve the cell wall and biofilm components. Thus, the prevention of biomass formation and eradication of preformed biofilms by lactoferrin was investigated using a methicillin-resistant Staphylococcus epidermidis (MRSE) strain. Additionally, the ability of lactoferrin to modulate the expression of the biofilm-associated protein gene (bap) was studied. The bap gene regulates the production of biofilm-associated proteins responsible for bacterial adhesion and aggregation. In the in vitro biofilm assays, lactoferrin prevented biofilm formation and eradicated established biofilms for up to 24 and 72 h, respectively. Extensive eradication of MRSE biofilm biomass was accompanied by the significant upregulation of bap gene expression. These data suggest the interaction of lactoferrin with the biofilm components and cell wall of MRSE, including the biofilm-associated protein.

Pentacyclic Triterpenoids as Antibiofilm Agents against Methicillinresistant and Biofilm-forming Staphylococcus aureus (MRSA).

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) has been constantly evolv-ing and developing resistance against conventional antibiotics. One of the key features of MRSA that enables it to develop resistance to antibiotics and host immune system is its ability to form biofilm in indwelling medical devices. In previous studies, the antimicrobial activity and mechanisms of action of three known pentacyclic triterpenoids α-amyrin, betulinic acid and betulinaldehyde against planktonic cells of MRSA were determined and elucidated. OBJECTIVE: This study was carried out to evaluate the ability of the three compounds to significantly reduce the biomass of pre-formed biofilms of MRSA and metabolic activity of the bacterial cells in the biofilm. METHODS: The anti-biofilm activity of α-amyrin, betulinic acid and betulinaldehyde, individually and in combination with oxacillin or vancomycin, against reference strain of MRSA in pre-formed biofilm were evaluated using the crystal violet and resazurin assays. RESULTS: α-amyrin and betulinic acid significantly reduced the biomass of pre-formed biofilms of MRSA as individual compounds and in combination with oxacillin or vancomycin. Although betulinaldehyde individually increased the biomass, selected combinations with oxacillin and vancomycin were able to reduce the biomass. All three compounds did not show cytotoxic properties on normal mammalian cells. CONCLUSION: The three pentacyclic triterpenoids could significantly reduce pre-formed biofilm of MRSA with no cytotoxic effects on normal mammalian cells. These findings demonstrated that pentacyclic triterpenoids have the potential to be developed further as antibiofilm agents against MRSA cells in bio-films, to combat infections caused by multidrug-resistant and biofilm-forming S. aureus.

Macrophages sense and kill bacteria through carbon monoxide-dependent inflammasome activation.

Microbial clearance by eukaryotes relies on complex and coordinated processes that remain poorly understood. The gasotransmitter carbon monoxide (CO) is generated by the stress-responsive enzyme heme oxygenase-1 (HO-1, encoded by Hmox1), which is highly induced in macrophages in response to bacterial infection. HO-1 deficiency results in inadequate pathogen clearance, exaggerated tissue damage, and increased mortality. Here, we determined that macrophage-generated CO promotes ATP production and release by bacteria, which then activates the Nacht, LRR, and PYD domains-containing protein 3 (NALP3) inflammasome, intensifying bacterial killing. Bacterial killing defects in HO-1-deficient murine macrophages were restored by administration of CO. Moreover, increased CO levels enhanced the bacterial clearance capacity of human macrophages and WT murine macrophages. CO-dependent bacterial clearance required the NALP3 inflammasome, as CO did not increase bacterial killing in macrophages isolated from NALP3-deficient or caspase-1-deficient mice. IL-1ß cleavage and secretion were impaired in HO-1-deficient macrophages, and CO-dependent processing of IL-1ß required the presence of bacteria-derived ATP. We found that bacteria remained viable to generate and release ATP in response to CO. The ATP then bound to macrophage nucleotide P2 receptors, resulting in activation of the NALP3/IL-1ß inflammasome to amplify bacterial phagocytosis by macrophages. Taken together, our results indicate that macrophage-derived CO permits efficient and coordinated regulation of the host innate response to invading microbes.

Carbon monoxide induced PPARγ SUMOylation and UCP2 block inflammatory gene expression in macrophages.

Carbon monoxide (CO) dampens pro-inflammatory responses in a peroxisome proliferator-activated receptor-γ (PPARγ) and p38 mitogen-activated protein kinase (MAPK) dependent manner. Previously, we demonstrated that CO inhibits lipopolysaccharide (LPS)-induced expression of the proinflammatory early growth response-1 (Egr-1) transcription factor in macrophages via activation of PPARγ. Here, we further characterize the molecular mechanisms by which CO modulates the activity of PPARγ and Egr-1 repression. We demonstrate that CO enhances SUMOylation of PPARγ which we find was attributed to mitochondrial ROS generation. Ectopic expression of a SUMOylation-defective PPARγ-K365R mutant partially abolished CO-mediated suppression of LPS-induced Egr-1 promoter activity. Expression of a PPARγ-K77R mutant did not impair the effect of CO. In addition to PPARγ SUMOylation, CO-activated p38 MAPK was responsible for Egr-1 repression. Blocking both CO-induced PPARγ SUMOylation and p38 activation, completely reversed the effects of CO on inflammatory gene expression. In primary macrophages isolated form C57/BL6 male mice, we identify mitochondrial ROS formation by CO as the upstream trigger for the observed effects on Egr-1 in part through uncoupling protein 2 (UCP2). Macrophages derived from bone marrow isolated from Ucp2 gene Knock-Out C57/BL6 mice (Ucp2(-/-)), produced significantly less ROS with CO exposure versus wild-type macrophages. Moreover, absence of UCP2 resulted in a complete loss of CO mediated Egr-1 repression. Collectively, these results indentify p38 activation, PPARγ-SUMOylation and ROS formation via UCP2 as a cooperative system by which CO impacts the inflammatory response.

Carbon monoxide is a poison... to microbes! CO as a bactericidal molecule.

Inflammation and immunity result in a wide range of disease processes, including chronic obstructive pulmonary disease, ischemia-reperfusion injury, atherosclerosis, vascular thrombosis and sepsis. Heme oxygenase-1 (HO-1) is a key enzyme that is indispensable for the temporal and spatial regulation of host response and, together with its essential metabolite carbon monoxide (CO), is crucial for maintaining homeostasis, inhibition of inflammation and the preservation of function and life. The biology of HO-1 is being discussed in this review series by Soares and colleagues and thus will not be reviewed here. Rather we will complement the HO-1 overview with a comprehensive discussion of CO as perhaps the one product of HO-1 that has been most studied. Of the numerous physiologic effects observed with CO, in the past five years it has become apparent that CO has been ascribed an additional novel role as a 'bactericidal agent'. Its role in the maintenance of homeostasis remains intact; however, the designation necessitates the paradoxical induction of the inflammatory response and binding to hemoproteins in order to restore homeostasis and sustain life. In this article, we review and discuss reports that have propelled and challenged the paradoxical use of CO, once viewed as a toxic molecule, now as a host defense molecule agent against microbes.

Carbon monoxide signals via inhibition of cytochrome c oxidase and generation of mitochondrial reactive oxygen species.

Carbon monoxide (CO), which is produced endogenously in the breakdown of heme, has been recognized as an important physiological second messenger similar to NO. Additionally, pharmacological delivery of CO is protective in numerous models of injury, including ischemia/reperfusion, transplantation, hemorrhagic shock, and endotoxemia. However, the mechanism of action of CO is only partially elucidated focused primarily on how it modulates the cellular response to stress. The purpose of these investigations is to test the hypothesis that CO acts via inhibition of cytochrome c oxidase leading to the generation of low levels of reactive oxygen species (ROS) that in turn mediate subsequent adaptive signaling. We show here that CO increases ROS generation in RAW 264.7 cells, which is inhibited by antimycin A and is absent in respiration-deficient rho0 cells. CO inhibits cytochrome c oxidase, while maintaining cellular ATP levels and increasing mitochondrial membrane potential. The addition of antioxidants or inhibition of complex III of the electron transport chain by antimycin A attenuates the inhibitory effects of CO on lipopolysaccharide (LPS)-induced TNF-alpha and blocked CO-induced p38 MAPK phosphorylation, which we previously have shown to be important in the anti-inflammatory effects of CO.

Carbon monoxide reverses established pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is an incurable disease characterized by a progressive increase in pulmonary vascular resistance leading to right heart failure. Carbon monoxide (CO) has emerged as a potently protective, homeostatic molecule that prevents the development of vascular disorders when administered prophylactically. The data presented in this paper demonstrate that CO can also act as a therapeutic (i.e., where exposure to CO is initiated after pathology is established). In three rodent models of PAH, a 1 hour/day exposure to CO reverses established PAH and right ventricular hypertrophy, restoring right ventricular and pulmonary arterial pressures, as well as the pulmonary vascular architecture, to near normal. The ability of CO to reverse PAH requires functional endothelial nitric oxide synthase (eNOS/NOS3) and NO generation, as indicated by the inability of CO to reverse chronic hypoxia-induced PAH in eNOS-deficient (nos3-/-) mice versus wild-type mice. The restorative function of CO was associated with a simultaneous increase in apoptosis and decrease in cellular proliferation of vascular smooth muscle cells, which was regulated in part by the endothelial cells in the hypertrophied vessels. In conclusion, these data demonstrate that CO reverses established PAH dependent on NO generation supporting the use of CO clinically to treat pulmonary hypertension.

Carbon monoxide orchestrates a protective response through PPARgamma.

Carbon monoxide (CO) suppresses proinflammatory responses in macrophages reacting to LPS. We hypothesize that CO acts by inducing a molecule(s) that suppresses the inflammatory response to subsequent stress. Exposure of macrophages to CO alone in vitro produced a brief burst of mitochondrial-derived ROS, which led to expression of PPARgamma. PPARgamma expression proved essential for mediating the anti-inflammatory effects of CO. Blocking the CO-mediated increase in ROS generation prevented PPARgamma induction, and blocking PPARgamma prevented CO's anti-inflammatory effects. In a model of acute lung injury in mice, CO blocked expression of Egr-1, a central mediator of inflammation, and decreased tissue damage; inhibition of PPARgamma abrogated both effects. These data identify the mitochondrial oxidases as an (perhaps the) initial cellular target of CO and demonstrate that CO upregulates expression of PPARgamma via the mitochondria, which assures that a subsequent stress stimulus will lead to a cytoprotective as opposed to a proinflammatory phenotype.

Bilirubin can induce tolerance to islet allografts.

Induction of heme oxygenase-1 (HO-1) expression in recipients of allogeneic islets can lead to long-term survival (>100 d) of those islets. We tested whether administration of bilirubin would substitute for the beneficial effects of HO-1 expression in islet transplantation. Administering bilirubin to the recipient (B6AF1) or incubating islets in a bilirubin-containing solution ex vivo led to long-term survival of allogeneic islets in a significant percentage of cases. In addition, administering bilirubin to only the donor frequently led to long-term survival of DBA/2 islets in B6AF1 recipients and significantly prolonged graft survival of BALB/c islets in C57BL/6 recipients. Donor treatment with bilirubin up-regulated mRNA expression of protective genes such as HO-1 and bcl-2 and suppressed proinflammatory and proapoptotic genes including monocyte chemoattractant protein-1 and caspase-3 and -8 in the islet grafts before transplantation. Furthermore, treatment of only the donor suppressed the expression of proinflammatory cytokines including TNF-alpha, inducible nitric oxide synthase, monocyte chemoattractant protein-1, and other proapoptotic and proinflammatory genes normally seen in the islets after transplantation. Donor treatment also reduced the number of macrophages that infiltrated the islet grafts in the recipients. Preincubation of betaTC3 cells with bilirubin also protected the cells from lipid peroxidation. Our data suggests that the potent antioxidant and antiinflammatory actions of bilirubin may contribute to islet survival.

Transcriptional regulation of the HO-1 gene in cultured macrophages exposed to model airborne particulate matter.

Respirable particulate matter generated during incomplete combustion of fossil fuels may principally target the cells found in the distal region of the lung. This study characterizes some of the effects that a model particulate matter has on the induction of heme oxygenase (HO)-1 in macrophages. HO-1 is a highly inducible stress response gene that has been demonstrated to modulate chemical, physical, and environmental stimuli. Cultured macrophages (RAW 264.7 cells) exposed continuously to a well-defined model of particulate matter (benzo[a]pyrene adsorbed onto carbon black) induced HO-1 gene expression in a time-dependent manner. Likewise, the addition of benzo[a]pyrene-1,6-quinone, a redox cycling metabolite of benzo[a]pyrene, to RAW cells also induced HO-1. This particle-induced gene expression of HO-1 was found to correlate with a corresponding increase in protein levels. Gene regulation studies were performed to delineate the transcriptional regulation of HO-1 after exposure to model particulate matter. Deletional analysis of the HO-1 gene and mutational analysis of activator protein (AP)-1 regulatory element on both distal enhancers demonstrated the importance of this transcriptional factor in mediating HO-1 gene transcription in response to model particulate matter. These results were supported by gel shift analysis demonstrating increased AP-1 binding activity after exposure to particulate matter. In summary, this study demonstrates that model particulate matter enhanced the expression of HO-1. This inductive process may be mediated by AP-1 activation of the regulatory elements on both the 5'-distal enhancers.