Characterization of pulmonary immune responses to hyperoxia by high-dimensional mass cytometry analyses.

Prolonged exposure to hyperoxia has deleterious effects on the lung, provoking both inflammation and alveolar injury. The elements of hyperoxic injury, which result in high rates of lethality in experimental models, are thought to include multicellular immune responses. To characterize these alterations in immune cell populations, we performed time-of-flight mass cytometry (CyTOF) analysis of CD45-expressing immune cells in whole lung parenchyma and the bronchoalveolar space of mice, exposed to 48 hours of hyperoxia together with normoxic controls. At the tested time point, hyperoxia exposure resulted in decreased abundance of immunoregulatory populations (regulatory B cells, myeloid regulatory cells) in lung parenchyma and markedly decreased proliferation rates of myeloid regulatory cells, monocytes and alveolar macrophages. Additionally, hyperoxia caused a shift in the phenotype of alveolar macrophages, increasing proportion of cells with elevated CD68, CD44, CD11c, PD-L1, and CD205 expression levels. These changes occurred in the absence of histologically evident alveolar damage and abundance of neutrophils in the parenchyma or alveolar space did not change at these time points. Collectively, these findings demonstrate that pulmonary response to hyperoxia involves marked changes in specific subsets of myeloid and lymphoid populations. These findings have important implications for therapeutic targeting in acute lung injury.

Danger signals from mitochondrial DAMPS in trauma and post-injury sepsis.

In all multicellular organisms, immediate host responses to both sterile and infective threat are initiated by very primitive systems now grouped together under the general term 'danger responses'. Danger signals are generated when primitive 'pattern recognition receptors' (PRR) encounter activating 'alarmins'. These molecular species may be of pathogenic infective origin (pathogen-associated molecular patterns) or of sterile endogenous origin (danger-associated molecular patterns). There are many sterile and infective alarmins and there is considerable overlap in their ability to activate PRR, but in all cases the end result is inflammation. It is the overlap between sterile and infective signals acting via a relatively limited number of PRR that generally underlies the great clinical similarity we see between sterile and infective systemic inflammatory responses. Mitochondria (MT) are evolutionarily derived from bacteria, and thus they sit at the crossroads between sterile and infective danger signal pathways. Many of the molecular species in mitochondria are alarmins, and so the release of MT from injured cells results in a wide variety of inflammatory events. This paper discusses the known participation of MT in inflammation and reviews what is known about how the major.

Heme oxygenase-1 derived carbon monoxide permits maturation of myeloid cells.

Critical functions of the immune system are maintained by the ability of myeloid progenitors to differentiate and mature into macrophages. We hypothesized that the cytoprotective gas molecule carbon monoxide (CO), generated endogenously by heme oxygenases (HO), promotes differentiation of progenitors into functional macrophages. Deletion of HO-1, specifically in the myeloid lineage (Lyz-Cre:Hmox1(flfl)), attenuated the ability of myeloid progenitors to differentiate toward macrophages and decreased the expression of macrophage markers, CD14 and macrophage colony-stimulating factor receptor (MCSFR). We showed that HO-1 and CO induced CD14 expression and efficiently increased expansion and differentiation of myeloid cells into macrophages. Further, CO sensitized myeloid cells to treatment with MCSF at low doses by increasing MCSFR expression, mediated partially through a PI3K-Akt-dependent mechanism. Exposure of mice to CO in a model of marginal bone marrow transplantation significantly improved donor myeloid cell engraftment efficiency, expansion and differentiation, which corresponded to increased serum levels of GM-CSF, IL-1α and MCP-1. Collectively, we conclude that HO-1 and CO in part are critical for myeloid cell differentiation. CO may prove to be a novel therapeutic agent to improve functional recovery of bone marrow cells in patients undergoing irradiation, chemotherapy and/or bone marrow transplantation.

Intraoperative administration of inhaled carbon monoxide reduces delayed graft function in kidney allografts in Swine.

Ischemia/reperfusion injury and delayed graft function (DGF) following organ transplantation adversely affect graft function and survival. A large animal model has not been characterized. We developed a pig kidney allograft model of DGF and evaluated the cytoprotective effects of inhaled carbon monoxide (CO). We demonstrate that donor warm ischemia time is a critical determinant of DGF as evidenced by a transient (4-6 days) increase in serum creatinine and blood urea nitrogen following transplantation before returning to baseline. CO administered to recipients intraoperatively for 1 h restored kidney function more rapidly versus air-treated controls. CO reduced acute tubular necrosis, apoptosis, tissue factor expression and P-selectin expression and enhanced proliferative repair as measured by phosphorylation of retinol binding protein and histone H3. Gene microarray analyses with confirmatory PCR of biopsy specimens showed that CO blocked proinflammatory gene expression of MCP-1 and heat shock proteins. In vitro in pig renal epithelial cells, CO blocks anoxia-reoxygenation-induced cell death while promoting proliferation. This large animal model of DGF can be utilized for testing therapeutic strategies to reduce or prevent DGF in humans. The efficacy of CO on improving graft function posttransplant validates the model and offers a potentially important therapeutic strategy to improve transplant outcomes.

Molecular pathways in the progression of hormone-independent and metastatic prostate cancer.

Once prostate cancer becomes castration resistant, cancer cells may rapidly gain the ability to invade and to metastasize to lymph nodes and distant organs. The progression through hormone-dependent to hormone-independent/castration-resistant and metastatic PCa is poorly understood. In this review paper, we provide an overview on the cellular and molecular mechanisms underlying the process of tumor cell invasion and metastasis in prostate cancer. We specifically present the most recent findings on the role of multiple cellular signaling pathways including androgen receptor (AR), mitogen-activated protein kinases (MAPK), Akt, transforming growth factor b (TGFb interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) in the development of hormone-independent/castration-resistant prostate cancer. In addition, we also discuss the recent findings on signatures of gene expression during prostate cancer progression. Our overviews on the novel findings will help to gain better understanding of the complexity of molecular mechanisms that may play an essential role in the development of castration-resistant and metastatic prostate cancer. It will also shed light on the identification of specific targets and the design of effective therapeutic drug candidates.

Carbon monoxide increases macrophage bacterial clearance through Toll-like receptor (TLR)4 expression.

Carbon monoxide (CO), a catabolic product of heme degradation, is an efficacious cytoprotectant and potent anti-inflammatory molecule. One of the important cellular targets of carbon monoxide is the macrophage, a key modulator of inflammation. In this study we investigated the effects of CO on the ability of cultured macrophages to phagocytose E. coli. Exposure to CO augmented E. coli phagocytosis but had no effect on inert particulate matter internalization. The ability of CO to increase uptake of the bacteria was in part mediated by the redistribution and increased expression of Toll-like receptor 4 (TLR4) on the cell surface. Furthermore, inhibition of p38 MAPK attenuated CO/E. coli-induced surface expression of TLR4 and abrogated the CO effects on E. coli phagocytosis. Collectively these data show that CO enhances the rate of E. coli phagocytosis via p38-mediated surface expression of TLR4 and suggest that CO may be a potential therapeutic modality by which to increase bacterial clearance.

Carbon monoxide pretreatment prevents respiratory derangement and ameliorates hyperacute endotoxic shock in pigs.

Endotoxic shock, one of the most prominent causes of mortality in intensive care units, is characterized by pulmonary hypertension, systemic hypotension, heart failure, widespread endothelial activation/injury, and clotting culminating in disseminated intravascular coagulation and multi-organ system failure. In the last few years, studies in rodents have shown that administration of low concentrations of carbon monoxide (CO) exerts potent therapeutic effects in a variety of diseases/disorders. In this study, we have administered CO (one our pretreatment at 250 ppm) in a clinically relevant, well-characterized model of LPS-induced acute lung injury in pigs. Pretreatment only with inhaled CO significantly ameliorated several of the acute pathological changes induced by endotoxic shock. In terms of lung physiology, CO pretreatment corrected the LPS-induced changes in resistance and compliance and improved the derangement in pulmonary gas exchange. In terms of coagulation and inflammation, CO reduced the development of disseminated intravascular coagulation and completely suppressed serum levels of the proinflammatory IL-1beta in response to LPS, while augmenting the anti-inflammatory cytokine IL-10. Moreover, the effects of CO blunted the deterioration of kidney and liver function, suggesting a beneficial effect in terms of end organ damage associated with endotoxic shock. Lastly, CO pretreatment prevents LPS-induced ICAM expression on lung endothelium and inhibits leukocyte marginalization on lung parenchyma.

Immunomodulatory effects of inhaled carbon monoxide on rat syngeneic small bowel graft motility.

BACKGROUND: Intestinal transplantation provokes an intense inflammatory response within the graft muscularis that causes intestinal ileus. We hypothesised that endogenously produced anti-inflammatory substances could be utilised as novel therapeutics. Therefore, we tested the protective effects of inhaled carbon monoxide (CO) and an endogenous haeme oxygenase 1 (HO-1) anti-inflammatory mediator on transplant induced inflammatory responses and intestinal ileus in the rat. METHODS: Gastrointestinal transit of non-absorbable FITC labelled dextran and in vitro jejunal circular muscle contractions were measured in controls and syngeneic orthotopic transplanted animals with and without CO inhalation (250 ppm for 25 hours). Inflammatory mRNAs for interleukin (IL)-6, IL-1beta, tumour necrosis factor alpha (TNF-alpha), intercellular adhesion molecule 1 (ICAM-1), inducible nitric oxide (iNOS), cyclooxygenase 2 (COX-2), and IL-10 were quantified by real time reverse transcriptase-polymerase chain reaction and HO-1 by northern blot. Histochemical stains characterised neutrophil infiltration and enterocyte apoptosis. RESULTS: Transplantation delayed transit and suppressed jejunal circular muscle contractility. Transplantation induced dysmotility was significantly improved by CO inhalation. Transplantation initiated a significant upregulation in IL-6, IL-1beta, TNF-alpha, ICAM-1, iNOS, COX-2, and HO-1 mRNAs with the graft muscularis. CO inhalation significantly decreased expression of IL-6, IL-1beta, iNOS, and COX-2 mRNAs. CO also significantly decreased serum nitrite levels (iNOS activity). CONCLUSIONS: CO inhalation significantly improved post-transplant motility and attenuated the inflammatory cytokine milieu in the syngeneic rat transplant model. Thus clinically providing CO, the end product of the anti-inflammatory HO-1 pathway, may prove to be an effective therapeutic adjunct for clinical small bowel transplantation.

Carbon monoxide attenuates aeroallergen-induced inflammation in mice.

Carbon monoxide (CO) generated by catalysis of heme by heme oxygenase is increased in the exhaled air of asthmatic patients. Based on recent studies demonstrating that asthma is an inflammatory disease associated with increased oxidants and that CO confers cytoprotection in oxidant-induced lung injury and inflammation, we sought to better understand the functional role of CO in asthma by using an aeroallergen model. Mice were sensitized to ovalbumin, challenged with aerosolized ovalbumin, and maintained in either CO (250 parts/million) or room air for 48 h. The differential effects of CO on bronchoalveolar lavage (BAL) fluid cell types were observed, with a marked attenuation of BAL fluid eosinophils in the CO-treated animals at 24 and 48 h. A marked reduction of the proinflammatory cytokine interleukin-5 was observed in the CO-treated mice, with no significant changes for other proinflammatory cytokines. These differential effects of CO were also observed with leukotrienes (LTs) and prostaglandins in that CO significantly decreased BAL fluid PGE2, and LTB4 but exerted negligible effect on thromboxane B2 or LTC4/D4/E4. Our data suggest a putative immunoregulatory role for CO in aeroallergen-induced inflammation in mice.

Activated Akt protects the lung from oxidant-induced injury and delays death of mice.

Oxidant-induced injury to the lung causes extensive damage to lung epithelial cells. Impaired protection and repair of the lung epithelium can result in death. The serine-threonine kinase Akt has been implicated in inhibiting cell death induced by different stimuli including growth factor withdrawal, cell cycle discordance, DNA damage, and loss of cell adhesion in different cell types. However, the in vivo relevance of this prosurvival pathway has not been explored. Here we show that a constitutively active form of Akt introduced intratracheally into the lungs of mice by adenovirus gene transfer techniques protects mice from hyperoxic pulmonary damage and delays death of mice. This is the first demonstration of the in vivo protective function of Akt in the context of oxidant-induced lung injury.

Heme oxygenase: colors of defense against cellular stress.

The discovery of the gaseous molecule nitric oxide in 1987 unraveled investigations on its functional role in the pathogenesis of a wide spectrum of biological and pathological processes. At that time, the novel concept that an endogenous production of a gaseous substance such as nitric oxide can impart such diverse and potent cellular effects proved to be very fruitful in enhancing our understanding of many disease processes including lung disorders. Interestingly, we have known for a longer period of time that there exists another gaseous molecule that is also generated endogenously; the heme oxygenase (HO) enzyme system generates the majority if not all of the endogenously produced carbon monoxide. This enzyme system also liberates two other by-products, bilirubin and ferritin, each possessing important biological functions and helping to define the uniqueness of the HO enzyme system. In recent years, interest in HO has emerged in numerous disciplines including the central nervous system, cardiovascular physiology, renal and hepatic systems, and transplantation. We review the functional role of HO in lung biology and its real potential application to lung diseases.

Carbon monoxide generated by heme oxygenase 1 suppresses endothelial cell apoptosis.

Heme oxygenase 1 (HO-1) inhibits apoptosis by regulating cellular prooxidant iron. We now show that there is an additional mechanism by which HO-1 inhibits apoptosis, namely by generating the gaseous molecule carbon monoxide (CO). Overexpression of HO-1, or induction of HO-1 expression by heme, protects endothelial cells (ECs) from apoptosis. When HO-1 enzymatic activity is blocked by tin protoporphyrin (SnPPIX) or the action of CO is inhibited by hemoglobin (Hb), HO-1 no longer prevents EC apoptosis while these reagents do not affect the antiapoptotic action of bcl-2. Exposure of ECs to exogenous CO, under inhibition of HO-1 activity by SnPPIX, substitutes HO-1 in preventing EC apoptosis. The mechanism of action of HO-1/CO is dependent on the activation of the p38 mitogen-activated protein kinase (MAPK) signaling transduction pathway. Expression of HO-1 or exposure of ECs to exogenous CO enhanced p38 MAPK activation by TNF-alpha. Specific inhibition of p38 MAPK activation by the pyridinyl imidazol SB203580 or through overexpression of a p38 MAPK dominant negative mutant abrogated the antiapoptotic effect of HO-1. Taken together, these data demonstrate that the antiapoptotic effect of HO-1 in ECs is mediated by CO and more specifically via the activation of p38 MAPK by CO.

Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway.

The stress-inducible protein heme oxygenase-1 provides protection against oxidative stress. The anti-inflammatory properties of heme oxygenase-1 may serve as a basis for this cytoprotection. We demonstrate here that carbon monoxide, a by-product of heme catabolism by heme oxygenase, mediates potent anti-inflammatory effects. Both in vivo and in vitro, carbon monoxide at low concentrations differentially and selectively inhibited the expression of lipopolysaccharide-induced pro-inflammatory cytokines tumor necrosis factor-alpha, interleukin-1beta, and macrophage inflammatory protein-1beta and increased the lipopolysaccharide-induced expression of the anti-inflammatory cytokine interleukin-10. Carbon monoxide mediated these anti-inflammatory effects not through a guanylyl cyclase-cGMP or nitric oxide pathway, but instead through a pathway involving the mitogen-activated protein kinases. These data indicate the possibility that carbon monoxide may have an important protective function in inflammatory disease states and thus has potential therapeutic uses.

Heme oxygenase-1 inhibits TNF-alpha-induced apoptosis in cultured fibroblasts.

Heme oxygenase (HO)-1 catalyzes the oxidative cleavage of heme to yield equimolar amounts of biliverdin, iron, and carbon monoxide. HO-1 is a stress response protein, the induction of which is associated with protection against oxidative stress. The mechanism(s) of protection is not completely elucidated, although it is suggested that one or more of the catalytic by-products provide antioxidant functions either directly or indirectly. The involvement of reactive oxygen species in apoptosis raised the question of a possible role for HO-1 in programmed cell death. Using the tetracycline-regulated expression system, we show here that conditional overexpression of HO-1 prevents tumor necrosis factor-alpha-induced apoptosis in murine L929 fibroblasts. Inhibition of apoptosis was not observed in the presence of tin protoporphyrin, a specific inhibitor of HO activity, and in cells overexpressing antisense HO-1. Interestingly, exogenous administration of a low concentration of carbon monoxide also prevented tumor necrosis factor-alpha-induced apoptosis in L929 fibroblasts. Inhibition of tumor necrosis factor-alpha-induced apoptosis by HO-1 overexpression was reversed by 1H-(1,2, 4)oxadiazolo(4,3-a)quinoxalin-1-one, an inhibitor of guanylate cyclase, which is a target enzyme for carbon monoxide. Taken together, our data suggest that the antiapoptotic effect of HO-1 may be mediated via carbon monoxide.

Mitogen-activated protein kinase pathway mediates hyperoxia-induced apoptosis in cultured macrophage cells.

We have previously demonstrated that the lungs of mice can exhibit increased programmed cell death or apoptosis after hyperoxic exposure in vivo. In this report, we show that hyperoxic exposure in vitro can also induce apoptosis in cultured murine macrophage cells (RAW 264.7) as assessed by DNA-laddering, terminal deoxynucleotidyltransferase dUTP nick end-labeling, and nucleosomal assays. To further delineate the signaling pathway of hyperoxia-induced apoptosis in RAW 264.7 macrophages, we first show that hyperoxia can activate the mitogen-activated protein kinase (MAPK) pathway, the extracellular signal-regulated kinases (ERKs) p42/p44, in a time-dependent manner as assessed by increased phosphorylation of ERK1/ERK2 by Western blot analyses. Neither the c-Jun NH(2)-terminal kinase/stress-activated protein kinase nor the p38 MAPK was activated by hyperoxia in these cells. Chemical or genetic inhibition of the ERK p42/p44 MAPK pathway by PD-98059, a selective inhibitor of MAPK kinase, and dominant negative mutants of ERK, respectively, attenuated hyperoxia-induced apoptosis as assessed by DNA laddering and nucleosomal ELISAs. Taken together, our data suggest that hyperoxia can induce apoptosis in cultured murine macrophages and that the MAPK pathway mediates hyperoxia-induced apoptosis.

Carbon monoxide provides protection against hyperoxic lung injury.

Findings in recent years strongly suggest that the stress-inducible gene heme oxygenase (HO)-1 plays an important role in protection against oxidative stress. Although the mechanism(s) by which this protection occurs is poorly understood, we hypothesized that the gaseous molecule carbon monoxide (CO), a major by-product of heme catalysis by HO-1, may provide protection against oxidative stress. We demonstrate here that animals exposed to a low concentration of CO exhibit a marked tolerance to lethal concentrations of hyperoxia in vivo. This increased survival was associated with highly significant attenuation of hyperoxia-induced lung injury as assessed by the volume of pleural effusion, protein accumulation in the airways, and histological analysis. The lungs were completely devoid of lung airway and parenchymal inflammation, fibrin deposition, and pulmonary edema in rats exposed to hyperoxia in the presence of a low concentration of CO. Furthermore, exogenous CO completely protected against hyperoxia-induced lung injury in rats in which endogenous HO enzyme activity was inhibited with tin protoporphyrin, a selective inhibitor of HO. Rats exposed to CO also exhibited a marked attenuation of hyperoxia-induced neutrophil infiltration into the airways and total lung apoptotic index. Taken together, our data demonstrate, for the first time, that CO can be therapeutic against oxidative stress such as hyperoxia and highlight possible mechanism(s) by which CO may mediate these protective effects.

Exogenous administration of heme oxygenase-1 by gene transfer provides protection against hyperoxia-induced lung injury.

Heme oxygenase-1 (HO-1) confers protection against a variety of oxidant-induced cell and tissue injury. In this study, we examined whether exogenous administration of HO-1 by gene transfer could also confer protection. We first demonstrated the feasibility of overexpressing HO-1 in the lung by gene transfer. A fragment of the rat HO-1 cDNA clone containing the entire coding region was cloned into plasmid pAC-CMVpLpA, and recombinant adenoviruses containing the rat HO-1 cDNA fragment Ad5-HO-1 were generated by homologous recombination. Intratracheal administration of Ad5-HO-1 resulted in a time-dependent increase in expression of HO-1 mRNA and protein in the rat lungs. Increased HO-1 protein expression was detected diffusely in the bronchiolar epithelium of rats receiving Ad5-HO-1, as assessed by immunohistochemical studies. We then examined whether ectopic expression of HO-1 could confer protection against hyperoxia-induced lung injury. Rats receiving Ad5-HO-1, but not AdV-betaGal, a recombinant adenovirus expressing Escherichia coli beta-galactosidase, before exposure to hyperoxia (>99% O2) exhibited marked reduction in lung injury, as assessed by volume of pleural effusion and histological analyses (significant reduction of edema, hemorrhage, and inflammation). In addition, rats receiving Ad5-HO-1 also exhibited increased survivability against hyperoxic stress when compared with rats receiving AdV-betaGal. Expression of the antioxidant enzymes manganese superoxide dismutase (Mn-SOD) and copper-zinc superoxide dismutase (CuZn-SOD) and of L-ferritin and H-ferritin was not affected by Ad5-HO-1 administration. Furthermore, rats treated with Ad5-HO-1 exhibited attenuation of hyperoxia-induced neutrophil inflammation and apoptosis. Taken together, these data suggest the feasibility of high-level HO-1 expression in the rat lung by gene delivery. To our knowledge, we have demonstrated for the first time that HO-1 can provide protection against hyperoxia-induced lung injury in vivo by modulation of neutrophil inflammation and lung apoptosis.

Pulmonary apoptosis in aged and oxygen-tolerant rats exposed to hyperoxia.

Accumulating evidence demonstrates that genotoxic and oxidant stress can induce programmed cell death or apoptosis in cultured cells. However, little is known about whether oxidative stress resulting from the deleterious effects of hyperoxia can induce apoptosis in vivo and even less is known regarding the functional significance of apoptosis in vivo in response to hyperoxia. Using hyperoxia as a model of oxidant-induced lung injury in the rat, we show that hyperoxic stress results in marked apoptotic signals in the lung. Lung tissue sections obtained from rats exposed to hyperoxia exhibit increased apoptosis in a time-dependent manner by terminal transferase dUTP nick end labeling assays. To examine whether hyperoxia-induced apoptosis in the lung correlated with the extent of lung injury or tolerance (adaptation) to hyperoxia, we investigated the pattern of apoptosis with a rat model of age-dependent tolerance to hyperoxia. We show that apoptosis is associated with increased survival of aged rats to hyperoxia and with decreased levels of lung injury as measured by the volume of pleural effusion, wet-to-dry lung weight, and myeloperoxidase content in aged rats compared with young rats after hyperoxia. We also examined this relationship in an alternate model of tolerance to hyperoxia. Lipopolysaccharide (LPS)-treated young rats not only demonstrated tolerance to hyperoxia but also exhibited a significantly lower apoptotic index compared with saline-treated rats after hyperoxia. To further separate the effects of aging and tolerance, we show that aged rats pretreated with LPS did not exhibit a significant level of tolerance against hyperoxia. Furthermore, similar to the hyperoxia-tolerant LPS-pretreated young rats, the nontolerant LPS-pretreated aged rats also exhibited a significantly reduced apoptotic index compared with aged rats exposed to hyperoxia alone. Taken together, our data suggest that hyperoxia-induced apoptosis in vivo can be modulated by both aging and tolerance effects. We conclude that there is no overall relationship between apoptosis and tolerance.