Eduardo López-Collazo

Daily physical activity in patients with chronic obstructive pulmonary disease is mainly associated with dynamic hyperinflation.

RATIONALE Although the major limitation to exercise performance in patients with COPD is dynamic hyperinflation, little is known about its relation to daily physical activity. OBJECTIVES To analyze the contribution of dynamic hyperinflation, exercise tolerance, and airway oxidative stress to physical activity in patients with COPD. METHODS In a cross-sectional study, we included 110 patients with moderate to very severe COPD. Daily physical activity was measured using a triaxial accelerometer providing a mean of 1-minute movement epochs as vector magnitude units (VMU). Patients performed the 6-minute walk test, incremental exercise test with measurement of breathing pattern and operating lung volumes, and constant-work rate test at 75% of maximal work rate. MEASUREMENTS AND MAIN RESULTS Using the GOLD stage and BODE index, we determined arterial blood gases, lung volumes, diffusing capacity, and biomarkers in exhaled breath condensate. Daily physical activity was lower in the 89 patients who developed dynamic hyperinflation than in the 21 who did not (n =161 [SD 70] vs. n = 288 [SD 85] VMU; P = 0.001). Physical activity was mainly related to distance walked in 6 minutes (r = 0.72; P = 0.001), Vo(2) (r = 0.63; P = 0.001), change in end-expiratory lung volume during exercise (r = -0.73; P = 0.001), endurance time (r = 0.61; P = 0.001), and 8-isoprostane in exhaled breath condensate (r = -0.67; P = 0.001). In a multivariate linear regression analysis using VMU as a dependent variable, dynamic hyperinflation, change in end-expiratory lung volume, and distance walked in 6 minutes were retained in the prediction model (r(2) = 0.84; P = 0.001). CONCLUSIONS Daily physical activity of patients with COPD is mainly associated with dynamic hyperinflation, regardless of severity classification.

Tumor Cells Deactivate Human Monocytes by Up-Regulating IL-1 Receptor Associated Kinase-M Expression via CD44 and TLR4

Although blood monocytes possess significant cytotoxic activity against tumor cells, tumor-infiltrating monocytes are commonly deactivated in cancer patients. Monocytes pre-exposed to tumor cells show significantly decreased expression levels of TNF-α, IL-12p40, and IL-1R-associated kinase (IRAK)-1. Activation of the Ser/Thr kinase IRAK-1 is an important event in several inflammatory processes. By contrast, another IRAK family member, IRAK-M, negatively regulates this pathway, and is up-regulated in cultures of endotoxin-tolerant monocytes and in monocytes from septic patients within the timeframe of tolerance. In this study, we show that IRAK-M expression is enhanced at the mRNA and protein level in human monocytes cultured in the presence of tumor cells. IRAK-M was induced in monocytes upon coculturing with different tumor cells, as well as by fixed tumor cells and medium supplemented with the supernatant from tumor cell cultures. Moreover, blood monocytes from patients with chronic myeloid leukemia and patients with metastasis also overexpressed IRAK-M. Low concentrations of hyaluronan, a cell surface glycosaminoglycan released by tumor cells, also up-regulated IRAK-M. The induction of IRAK-M by hyaluronan and tumor cells was abolished by incubation with anti-CD44 or anti-TLR4 blocking Abs. Furthermore, down-regulation of IRAK-M expression by small interfering RNAs specific for IRAK-M reinstates both TNF-α mRNA expression and protein production in human monocytes re-exposed to a tumor cell line. Altogether, our findings indicate that deactivation of human monocytes in the presence of tumor cells involves IRAK-M up-regulation, and this effect appears to be mediated by hyaluronan through the engagement of CD44 and TLR4.

Metalloproteinases Shed TREM-1 Ectodomain from Lipopolysaccharide-Stimulated Human Monocytes

Triggering receptors expressed on myeloid cell (TREM) proteins are a family of cell surface receptors that participate in diverse cellular processes such as inflammation, coagulation, and bone homeostasis. TREM-1, in particular, is expressed on neutrophils and monocytes and is a potent amplifier of inflammatory responses. LPS and other microbial products induce up-regulation of cell surface-localized TREM-1 and the release of its soluble form, sTREM-1. Two hypotheses have been advanced to explain the origin of sTREM-1: alternative splicing of TREM-1 mRNA and proteolytic cleavage(s) of mature, membrane-anchored TREM-1. In this report, we present conclusive evidence in favor of the proteolytic mechanism of sTREM-1 generation. No alternative splicing forms of TREM-1 were detected in monocytes/macrophages. Besides, metalloproteinase inhibitors increased the stability of TREM-1 at the cell surface while significantly reducing sTREM-1 release in cultures of LPS-challenged human monocytes and neutrophils. We conclude that metalloproteinases are responsible for shedding of the TREM-1 ectodomain through proteolytic cleavage of its long juxtamembrane linker.

WIP1 phosphatase is a negative regulator of NF-κB signalling

Post-translational modifications of NF-κB through phosphorylations enhance its transactivation potential. Much is known about the kinases that phosphorylate NF-κB, but little is known about the phosphatases that dephosphorylate it. By using a genome-scale siRNA screen, we identified the WIP1 phosphatase as a negative regulator of NF-κB signalling. WIP1-mediated regulation of NF-κB occurs in both a p38-dependent and independent manner. Overexpression of WIP1 resulted in decreased NF-κB activation in a dose-dependent manner, whereas WIP1 knockdown resulted in increased NF-κB function. We show that WIP1 is a direct phosphatase of Ser 536 of the p65 subunit of NF-κB. Phosphorylation of Ser 536 is known to be essential for the transactivation function of p65, as it is required for recruitment of the transcriptional co-activator p300. WIP1-mediated regulation of p65 regulated binding of NF-κB to p300 and hence chromatin remodelling. Consistent with our results, mice lacking WIP1 showed enhanced inflammation. These results provide the first genetic proof that a phosphatase directly regulates NF-κB signalling in vivo.

Role for MyD88-Independent, TRIF Pathway in Lipid A/TLR4-Induced Endotoxin Tolerance

Repeated exposure to low doses of endotoxin results in progressive hyporesponsiveness to subsequent endotoxin challenge, a phenomenon known as endotoxin tolerance. In spite of its clinical significance in sepsis and characterization of the TLR4 signaling pathway as the principal endotoxin detection mechanism, the molecular determinants that induce tolerance remain obscure. We investigated the role of the TRIF/IFN-β pathway in TLR4-induced endotoxin tolerance. Lipid A-induced homotolerance was characterized by the down-regulation of MyD88-dependent proinflammatory cytokines TNF-α and CCL3, but up-regulation of TRIF-dependent cytokine IFN-β. This correlated with a molecular phenotype of defective NF-κB activation but a functional TRIF-dependent STAT1 signaling. Tolerance-induced suppression of TNF-α and CCL3 expression was significantly relieved by TRIF and IFN regulatory factor 3 deficiency, suggesting the involvement of the TRIF pathway in tolerance. Alternatively, selective activation of TRIF by poly(I:C)-induced tolerance to lipid A. Furthermore, pretreatment with rIFN-β also induced tolerance, whereas addition of IFN-β-neutralizing Ab during the tolerization partially alleviated tolerance to lipid A but not TLR2-induced endotoxin homo- or heterotolerance. Furthermore, IFNAR1−/− murine embryonal fibroblast and bone-marrow derived macrophages failed to induce tolerance. Together, these observations constitute evidence for a role of the TRIF/IFN-β pathway in the regulation of lipid A/TLR4-mediated endotoxin homotolerance.

Human Monocytes Undergo Functional Re-programming during Sepsis Mediated by Hypoxia-Inducible Factor-1α

Sepsis is characterized by a dysregulated inflammatory response to infection. Despite studies in mice, the cellular and molecular basis of human sepsis remains unclear and effective therapies are lacking. Blood monocytes serve as the first line of host defense and are equipped to recognize and respond to infection by triggering an immune-inflammatory response. However, the response of these cells in human sepsis and their contribution to sepsis pathogenesis is poorly understood. To investigate this, we performed a transcriptomic, functional, and mechanistic analysis of blood monocytes from patients during sepsis and after recovery. Our results revealed the functional plasticity of monocytes during human sepsis, wherein they transited from a pro-inflammatory to an immunosuppressive phenotype, while enhancing protective functions like phagocytosis, anti-microbial activity, and tissue remodeling. Mechanistically, hypoxia inducible factor-1α (HIF1α) mediated this functional re-programming of monocytes, revealing a potential mechanism for their therapeutic targeting to regulate human sepsis.

Pathophysiology of endotoxin tolerance: mechanisms and clinical consequences

Endotoxin tolerance was first described in a study that exposed animals to a sublethal dose of bacterial endotoxin. The animals subsequently survived a lethal injection of endotoxin. This refractory state is associated with the innate immune system and, in particular, with monocytes and macrophages, which act as the main participants. Several mechanisms are involved in the control of endotoxin tolerance; however, a full understanding of this phenomenon remains elusive. A number of recent reports indicate that clinical examples of endotoxin tolerance include not only sepsis but also diseases such as cystic fibrosis and acute coronary syndrome. In these pathologies, the risk of new infections correlates with a refractory state. This review integrates the molecular basis and clinical implications of endotoxin tolerance in various pathologies.

Protective effect of cyclosporin A and FK506 from nitric oxide-dependent apoptosis in activated macrophages.

Activation of macrophages with lipopolysaccharide (LPS) and low doses of interferon‐γ (IFN‐γ) induced apoptotic death through a nitric oxide‐dependent pathway. Treatment of cells with the immunosuppressors cyclosporin A (CsA) or FK506 inhibited the activation‐dependent apoptosis. These drugs decreased the up‐regulation of p53 and Bax characteristic of activated macrophages. Moreover, incubation of activated macrophages with CsA and FK506 contributed to maintain higher levels of Bcl‐2 than in LPS/IFN‐γ treated cells. The inhibition of apoptosis exerted by CsA and FK506 in macrophages was also observed when cell death was induced by treatment with chemical nitric oxide donors. Incubation of macrophages with LPS/IFN‐γ barely affected caspase‐1 but promoted an important activation of caspase‐3. Both CsA and FK506 inhibited pathways leading to caspase‐3 activation. Moreover, the cleavage of poly(ADP‐ribose) polymerase, a well established caspase substrate, was reduced by these immunosuppressive drugs. CsA and FK506 reduced the release of cytochrome c to the cytosol and the activation of caspase‐3 in cells treated with nitric oxide donors. These results indicate that CsA and FK506 protect macrophages from nitric oxide‐dependent apoptosis and suggest a contribution of the macrophage to innate immunity under conditions of immunosuppression of the host.

Monocytes from Cystic Fibrosis Patients Are Locked in an LPS Tolerance State: Down-Regulation of TREM-1 as Putative Underlying Mechanism

Cystic Fibrosis (CF) is an inherited pleiotropic disease that results from abnormalities in the gene that codes for the chloride channel, Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). CF patients are frequently colonized by several pathogens, but the mechanisms that allow colonization in spite of apparently functional immune systems are incompletely understood. In this paper we show that blood peripheral monocytes isolated from CF patients are found in an endotoxin tolerance state, yet this is not due to a deficient TLR activation. On the other hand, levels of the amplifier of inflammatory responses, TREM-1 (Triggering Receptor Expressed on Myeloid cells), are notably down-regulated in monocytes from patients, in comparison to those extracted from healthy volunteers. Furthermore, the soluble form of TREM-1 (sTREM-1) was not detected in the sera of patients. Additionally, and in strict contrast to patients who suffer from Chronic Obstructive Pulmonary Disease (COPD), CF monocytes challenged ex vivo with LPS neither up-regulated membrane-anchored TREM-1 nor sTREM-1. Finally, similar levels of PGE2 expression and p65 translocation into the nucleus were found in both patients and healthy volunteers, thus suggesting that TREM-1 regulation is neither controlled by PGE2 levels nor by p65 activation in this case. However, PU.1 translocation into the nucleus was significantly higher in CF monocytes than in controls, suggesting a role for this transcription factor in the control of TREM-1 expression. We conclude that down-regulation of TREM-1 expression in cystic fibrosis patients is at least partly responsible for the endotoxin tolerance state in which their monocytes are locked.

Inflammatory responses associated with acute coronary syndrome up-regulate IRAK-M and induce endotoxin tolerance in circulating monocytes.

Acute coronary syndrome (ACS) groups different cardiac diseases whose development is associated with inflammation. Here we have analyzed the levels of inflammatory cytokines and of members of the TLR/IRAK pathway including IRAK-M in monocytes from ACS patients classified as either UA (unstable angina), STEMI (ST-elevation myocardial infarction) or NSTEMI (non-ST-elevation myocardial infarction). Circulating monocytes from all patients, but not from healthy individuals, showed high levels of pro-inflammatory cytokines, TNF-α and IL-6, as well as of IRAK-M and IL-10. TLR4 was also up-regulated, but IRAK-1, IRAK-4 and MyD88 levels were similar in patients and controls. Further, we investigated the consequences of cytokines/IRAK-M expression on the innate immune response to endotoxin. Ex vivo responses to LPS were markedly attenuated in patient monocytes compared to controls. Control monocytes cultured for 6 h in supplemented medium (10% serum from ACS patients) expressed IRAK-M, and LPS stimulation failed to induce TNF-α and IL-6 in these cultures. Pre-incubation of the serum with a blocking anti-TNF-α antibody reduced this endotoxin tolerance effect, suggesting that TNF-α controls this phenomenon, at least partially. We show for the first time that inflammatory responses associated with ACS induce an unresponsiveness state to endotoxin challenge in circulating monocytes, which correlates with expression of IRAK-M, TLR4 and IL-10. The magnitude of this response varies according to the clinical condition (UA, STEMI or NSTEMI), and is regulated by TNF-α.