Akt-Mediated Proinflammatory Response of Mononuclear Phagocytes Infected with Burkholderia cenocepacia Occurs by a Novel GSK3 beta-Dependent, I kappa B Kinase-Independent Mechanism

The environmental bacterium Burkholderia cenocepacia causes opportunistic lung infections in immunocompromised individuals, particularly in patients with cystic fibrosis. Infections in these patients are associated with exacerbated inflammation leading to rapid decay of lung function, and in some cases resulting in cepacia syndrome, which is characterized by a fatal acute necrotizing pneumonia and sepsis. B. cenocepacia can survive intracellularly in macrophages by altering the maturation of the phagosome, but very little is known on macrophage responses to the intracellular infection. In this study, we have examined the role of the PI3K/Akt signaling pathway in B. cenocepacia-infected monocytes and macrophages. We show that PI3K/Akt activity was required for NF-kappa B activity and the secretion of proinflammatory cytokines during infection with B. cenocepacia. In contrast to previous observations in epithelial cells infected with other Gram-negative bacteria, Akt did not enhance I kappa B kinase or NF-kappa B p65 phosphorylation, but rather inhibited GSK3 beta, a negative regulator of NF-kappa B transcriptional activity. This novel mechanism of modulation of NF-kappa B activity may provide a unique therapeutic target for controlling excessive inflammation upon B. cenocepacia infection. The Journal of Immunology, 2011, 187: 635-643.

Determining the role of mononuclear phagocytes in prion neuroinvasion from the skin

Many prion diseases are acquired by peripheral exposure, and skin lesions are an effective route of transmission. Following exposure, early prion replication, upon FDCs in the draining LN is obligatory for the spread of disease to the brain. However, the mechanism by which prions are conveyed to the draining LN is uncertain. Here, transgenic mice were used, in which langerin(+) cells, including epidermal LCs and langerin(+) classical DCs, were specifically depleted. These were used in parallel with transgenic mice, in which nonepidermal CD11c(+) cells were specifically depleted. Our data show that prion pathogenesis, following exposure via skin scarification, occurred independently of LC and other langerin(+) cells. However, the depletion of nonepidermal CD11c(+) cells impaired the early accumulation of prions in the draining LN, implying a role for these cells in the propagation of prions from the skin. Therefore, together, these data suggest that the propagation of prions from the skin to the draining LN occurs via dermal classical DCs, independently of langerin(+) cells.

Age-related changes in non-receptor dependent generation of reactive oxygen species from phagocytes of healthy adults

Several authors have shown that neutrophil generation of reactive oxygen species (ROS) declines with advancing age. Similar changes have also been suggested in monocytes. In both cases alterations in second messenger activity have been implicated as the most likely explanation for these observations. The aim of this study was to investigate the effect of age on phagocyte ROS generation, stimulated by the direct activation of protein kinase C (PKC). Venous blood was drawn from normal healthy subjects, cells were separated on a double density gradient into mononuclear and polymorphonuclear (pmn) cells. Phorbol myristate acetate (PMA) was employed as a cell stimulus. Superoxide generation was measured by cytochrome c reduction and myeloperoxidase (MPO) products by measurement of peak luminol chemiluminescence (CL). Fifty-eight subjects, 25 males and 33 females, were studied, median age 49 years (range 26-88 years). Polymorphonuclear cell superoxide generation was significantly higher in males and there was a trend towards higher pmn MPO product generation in males. Using Spearman's ranked correlation coefficient, monocyte superoxide generation was negatively correlated with age (r = -0.473, P <0.001). No changes in the generation of MPO products was found. There were also trends towards a negative correlation of pmn cytochrome c reduction and peak luminol CL with age in males but not females. Since PMA directly activates protein kinase C, reduced monocyte superoxide generation with increasing age appears to be related to alterations in the ROS generating system downstream of the cell receptor. Impaired monocyte superoxide generation may have implications for non-specific defence against certain infections and early tumour growth in the elderly. Factors underlying these changes in monocyte function therefore require further study.

Paraquat-Induced Retinal Degeneration Is Exaggerated in CX3CR1-Deficient Mice and Is Associated with Increased Retinal Inflammation

PURPOSE:<br/>To investigate the role of the Fractalkine receptor CX3CR1 pathway in oxidative insults-mediated retinal degeneration and immune activation.<br/>METHODS:<br/>A prooxidant, paraquat (0.75 µM) was injected into the vitreous of C57BL/6J, CX3CR1(gpf/+), and CX3CR1(gfp/gfp) mice. Retinal lesions were investigated clinically by topic endoscopic fundus imaging and fluorescence angiography, and pathologically by light- and electron microscopy. Retinal immune gene expression was determined by real-time RT-PCR. Microglial activation and immune cell infiltration were examined by confocal microscopy of retinal flatmounts.<br/>RESULTS:<br/>Intravitreal injection of paraquat (0.75 µM) resulted in acute retinal capillary nonperfusion within 2 days, which improved from 4 days to 4 weeks postinjection (p.i.). Panretinal degeneration was observed at 4 days p.i. and progressed further at 4 weeks p.i. In the absence of CX3CR1, retinal degeneration was exaggerated and was accompanied by increased TNF-a, iNOS, IL-1ß, Ccl2, and Casp-1 gene expression. Confocal microscopy of retinal flatmounts revealed microglial activation and CD44(+)MHC-II(+) monocyte and GR1(+) neutrophil infiltration in paraquat-injected eyes. The number of activated microglia and infiltrating leukocytes was significantly higher in CX3CR1(gfp/gfp) mice than in CX3CR1(gfp/+) mice.<br/>CONCLUSIONS:<br/>Our results suggest that the CX3CR1 signaling pathway may play an important role in controlling retinal inflammation under oxidative and ischemia/reperfusion conditions. In the absence of CX3CR1, uncontrolled retinal inflammation results in exaggerated retinal degeneration.

Role of Bacterial Surface Structures on the Interaction of <i>Klebsiella pneumoniae</i> with Phagocytes

Phagocytosis is a key process of the immune system. The human pathogen Klebsiella pneumoniae is a well known example of a pathogen highly resistant to phagocytosis. A wealth of evidence demonstrates that the capsule polysaccharide (CPS) plays a crucial role in resistance to phagocytosis. The amoeba Dictyostelium discoideum shares with mammalian macrophages the ability to phagocytose and kill bacteria. The fact that K. pneumoniae is ubiquitous in nature and, therefore, should avoid predation by amoebae, poses the question whether K. pneumoniae employs similar means to counteract amoebae and mammalian phagocytes. Here we developed an assay to evaluate K. pneumoniae-D. discoideum interaction. The richness of the growth medium affected the threshold at which the cps mutant was permissive for Dictyostelium and only at lower nutrient concentrations the cps mutant was susceptible to predation by amoebae. Given the critical role of bacterial surface elements on host-pathogen interactions, we explored the possible contribution of the lipopolysaccharide (LPS) and outer membrane proteins (OMPs) to combat phagoyctosis by D. discoideum. We uncover that, in addition to the CPS, the LPS O-polysaccharide and the first core sugar participate in Klebsiella resistance to predation by D. discoideum. K. pneumoniae LPS lipid A decorations are also necessary to avoid predation by amoebae although PagP-dependent palmitoylation plays a more important role than the lipid A modification with aminoarabinose. Mutants lacking OMPs OmpA or OmpK36 were also permissive for D. discoideium growth. Except the LPS O-polysaccharide mutants, all mutants were more susceptible to phagocytosis by mouse alveolar macrophages. Finally, we found a correlation between virulence, using the pneumonia mouse model, and resistance to phagocytosis. Altogether, this work reveals novel K. pneumoniae determinants involved in resistance to phagocytosis and supports the notion that Dictyostelium amoebae might be useful as host model to measure K. pneumoniae virulence and not only phagocytosis. © 2013 March et al.

Age- and Light-Dependent Development of Localised Retinal Atrophy in CCL2(-/-)CX3CR1(GFP/GFP) Mice

Previous studies have shown that CCL2/CX3CR1 deficient mice on C57BL/6N background (with rd8 mutation) have an early onset (6 weeks) of spontaneous retinal degeneration. In this study, we generated CCL2(-/-)CX3CR1(GFP/GFP) mice on the C57BL/6J background. Retinal degeneration was not detected in CCL2(-/-)CX3CR1(GFP/GFP) mice younger than 6 months. Patches of whitish/yellowish fundus lesions were observed in 17~60% of 12-month, and 30~100% of 18-month CCL2(-/-)CX3CR1(GFP/GFP) mice. Fluorescein angiography revealed no choroidal neovascularisation in these mice. Patches of retinal pigment epithelium (RPE) and photoreceptor damage were detected in 30% and 50% of 12- and 18-month CCL2(-/-)CX3CR1(GFP/GFP) mice respectively, but not in wild-type mice. All CCL2(-/-)CX3CR1(GFP/GFP) mice exposed to extra-light (~800lux, 6 h/day, 6 months) developed patches of retinal atrophy, and only 20-25% of WT mice which underwent the same light treatment developed atrophic lesions. In addition, synaptophysin expression was detected in the outer nucler layer (ONL) of area related to photoreceptor loss in CCL2(-/-)CX3CR1(GFP/GFP) mice. Markedly increased rhodopsin but reduced cone arrestin expression was observed in retinal outer layers in aged CCL2(-/-)CX3CR1(GFP/GFP) mice. GABA expression was reduced in the inner retina of aged CCL2(-/-)CX3CR1(GFP/GFP) mice. Significantly increased Müller glial and microglial activation was observed in CCL2(-/-)CX3CR1(GFP/GFP) mice compared to age-matched WT mice. Macrophages from CCL2(-/-)CX3CR1(GFP/GFP) mice were less phagocytic, but expressed higher levels of iNOS, IL-1ß, IL-12 and TNF-a under hypoxia conditions. Our results suggest that the deletions of CCL2 and CX3CR1 predispose mice to age- and light-mediated retinal damage. The CCL2/CX3CR1 deficient mouse may thus serve as a model for age-related atrophic degeneration of the RPE, including the dry type of macular degeneration, geographic atrophy.

The role of WNT singling activation and its blockage in the focal retinal lesion of Ccl2-/-/Cx3cr1-/- mice with and without rd8 background

Purpose: The canonical Wnt signaling is activated by retinal injury. Under disease conditions, the Wnt mediates inflammatory responses. Inflammation has been detected in age-related macular degeneration (AMD) retinas and Ccl2-/-/Cx3cr1-/- (DKO) mice with or without rd8 background, a model with progressive AMD-like lesions including focal photoreceptor/RPE degeneration and A2E accumulation. We evaluated the effects of Wnt-β-catenin activation and an antibody against LRP6, the co-receptor of Wnt on these two models.<br/> <br/>Methods: anti-LRP6 antibody (2F1, 1 μl of 5 μg/μL) was intravitreally injected into the right eyes in 3 separate experiments (DKOrd8, N=35; DKO, N=10). The left eyes were injected with mouse IgG as controls. Fundoscopy was taken before injection and sequentially monthly after injection. Two months after injection, light-adapted ERG responses were recorded; then the eyes were harvested for histopathology, the determination of retinal A2E, and molecular analysis. The microarray of ocular mRNA of 92 Wnt genes was compared between the treated and the control eyes. The phosphorylated types of LRP6 and β-catenin and endogenous forms of the proteins were assayed by Western blotting.<br/> <br/>Results: For DKOrd8 mice, the fundus showed a slower progression or alleviation of retinal lesions in the right eyes as compared to the left eyes. Among 35 pairs of eyes, 26 (74.3%) were improved, 7 (20%) stayed the same and 2 (5.7%) remained progressing. Histology confirmed the clinical observation. Light-adapted ERG of the treated eyes exhibited larger amplitudes compared to control eyes (n=6), with greater improvements under UV light stimulus. There was a significantly lower A2E in the treated eyes compared to controls. Microarray of 92 Wnt genes expression pattern was similar in both eyes. Western blotting indicated local administration of 2F1 antibody to suppress the activation of Wnt pathway in the retina. For DKO mice, the treatment improved ERG but less effect on RPE degeneration.<br/> <br/>Conclusions: The canonical Wnt signaling plays a role in the focal retina lesion of both DKOrd8 and DKO mice; and intravitreal anti-LRP6 antibody might be neuroprotective via deactivation of canonical Wnt pathway.<br/>

Para-inflammation-mediated retinal recruitment of bone marrow-derived myeloid cells following whole-body irradiation is CCL2 dependent

Previous studies have shown that following whole-body irradiation bone marrow (BM)-derived cells can migrate into the central nervous system, including the retina, to give rise to microglia-like cells. The detailed mechanism, however, remains elusive. We show in this study that a single-dose whole-body ?-ray irradiation (8 Gy) induced subclinical damage (i.e., DNA damage) in the neuronal retina, which is accompanied by a low-grade chronic inflammation, para-inflammation, characterized by upregulated expression of chemokines (CCL2, CXCL12, and CX3CL1) and complement components (C4 and CFH), and microglial activation. The upregulation of chemokines CCL2 and CXCL12 and complement C4 lasted for more than 160 days, whereas the expression of CX3CL1 and CFH was upregulated for 2 weeks. Both resident microglia and BM-derived phagocytes displayed mild activation in the neuronal retina following irradiation. When BM cells from CX3CR1gfp/+ mice or CX3CR1gfp/gfp mice were transplanted to wild-type C57BL/6 mice, more than 90% of resident CD11b+ cells were replaced by donor-derived GFP+ cells after 6 months. However, when transplanting CX3CR1gfp/+ BM cells into CCL2-deficient mice, only 20% of retinal CD11b+ cells were replaced by donor-derived cells at 6 month. Our results suggest that the neuronal retina suffers from a chronic stress following whole-body irradiation, and a para-inflammatory response is initiated, presumably to rectify the insults and maintain homeostasis. The recruitment of BM-derived myeloid cells is a part of the para-inflammatory response and is CCL2 but not CX3CL1 dependent. © 2012 Wiley Periodicals, Inc.

Interactions of Burkholderia cenocepacia and other Burkholderia cepacia complex bacteria with epithelial and phagocytic cells

Burkholderia cenocepacia is a member of the B. cepacia complex (Bcc), a group of opportunistic bacteria that infect the airways of patients with cystic fibrosis (CF) and are extraordinarily resistant to almost all clinically useful antibiotics. Infections in CF patients with Bcc bacteria generally lead to a more rapid decline in lung function, and in some cases to the 'cepacia syndrome', a virtually deadly exacerbation of the lung infection with systemic manifestations. These characteristics of Bcc bacteria contribute to higher morbidity and mortality in infected CF patients. In the last 10 years considerable progress has been made in understanding the interactions between Bcc bacteria and mammalian host cells. Bcc isolates can survive either intracellularly within eukaryotic cells or extracellularly in host tissues. They survive within phagocytes and respiratory epithelial cells, and they have the ability to breach the respiratory epithelium layer. Survival and persistence of Bcc bacteria within host cells and tissues are believed to play a key role in pulmonary infection and to contribute to the persistent inflammation observed in patients with CF. This review summarizes recent findings concerning the interaction between Bcc bacteria and epithelial and phagocytic cells.

Nontypeable Haemophilus influenzae clearance by alveolar macrophages is impaired by exposure to cigarette smoke

Nontypeable Haemophilus influenzae (NTHI) is an opportunistic gram-negative pathogen that causes respiratory infections and is associated with progression of respiratory diseases. Cigarette smoke is a main risk factor for development of respiratory infections and chronic respiratory diseases. Glucocorticoids, which are anti-inflammatory drugs, are still the most common therapy for these diseases. Alveolar macrophages are professional phagocytes that reside in the lung and are responsible for clearing infections by the action of their phagolysosomal machinery and promotion of local inflammation. In this study, we dissected the interaction between NTHI and alveolar macrophages and the effect of cigarette smoke on this interaction. We showed that alveolar macrophages clear NTHI infections by adhesion, phagocytosis, and phagolysosomal processing of the pathogen. Bacterial uptake requires host actin polymerization, the integrity of plasma membrane lipid rafts, and activation of the phosphatidylinositol 3-kinase (PI3K) signaling cascade. Parallel to bacterial clearance, macrophages secrete tumor necrosis factor alpha (TNF-alpha) upon NTHI infection. In contrast, exposure to cigarette smoke extract (CSE) impaired alveolar macrophage phagocytosis, although NTHI-induced TNF-alpha secretion was not abrogated. Mechanistically, our data showed that CSE reduced PI3K signaling activation triggered by NTHI. Treatment of CSE-exposed cells with the glucocorticoid dexamethasone reduced the amount of TNF-alpha secreted upon NTHI infection but did not compensate for CSE-dependent phagocytic impairment. The deleterious effect of cigarette smoke was observed in macrophage cell lines and in human alveolar macrophages obtained from smokers and from patients with chronic obstructive pulmonary disease.