Neutrophils are classically defined as terminally differentiated, short-lived cells; however, neutrophils can be long-lived with phenotypic plasticity. During inflammation, a subset of neutrophils ...transdifferentiate into a population called neutrophil-DC hybrids (PMN-DCs) having properties of both neutrophils and dendritic cells. While these cells ubiquitously appear during inflammation, the role of PMN-DCs in disease remains poorly understood. We observed the differentiation of PMN-DCs in pre-clinical murine models of fungal infection: blastomycosis, aspergillosis and candidiasis. Using reporter strains of fungal viability, we found that PMN-DCs associate with fungal cells and kill them more efficiently than undifferentiated canonical neutrophils. During pulmonary blastomycosis, PMN-DCs comprised less than 1% of leukocytes yet contributed up to 15% of the fungal killing. PMN-DCs displayed higher expression of pattern recognition receptors, greater phagocytosis, and heightened production of reactive oxygen species compared to canonical neutrophils. PMN-DCs also displayed prominent NETosis. To further study PMN-DC function, we exploited a granulocyte/macrophage progenitor (GMP) cell line, generated PMN-DCs to over 90% purity, and used them for adoptive transfer and antigen presentation studies. Adoptively transferred PMN-DCs from the GMP line enhanced protection against systemic infection in vivo. PMN-DCs pulsed with antigen activated fungal calnexin-specific transgenic T cells in vitro and in vivo, promoting the production of interferon-γ and interleukin-17 in these CD4+ T cells. Through direct fungal killing and induction of adaptive immunity, PMN-DCs are potent effectors of antifungal immunity and thereby represent innovative cell therapeutic targets in treating life-threatening fungal infections.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
To contribute to complete the knowledge of the underlying mechanisms of action involved in air pollution particulate matter (PM)-induced cytotoxicity, an aerosol was collected in Dunkerque, a French ...seaside City heavily industrialized. In this work, we focused our attention on its physical and chemical characteristics, its cytotoxicity, and its role in the induction of the volatile organic compound (VOC) and/or polycyclic aromatic hydrocarbon (PAH)-metabolizing enzymes in human lung epithelial cells (A549). Size distribution showed that 92.15% of the collected PM were PM
2.5 and the specific surface area was 1
m
2/g. Inorganic (i.e. Fe, Al, Ca, Na, K, Mg, Pb, etc.) and organic (i.e. VOC, PAH, etc.) chemicals were found in collected PM, revealing that much of them derived from wind-borne dust from the industrial complex and the heavy motor vehicle traffic. The thermal desorption study indicated that organic chemicals were not only adsorbed onto the surface but also highly incrusted in the structure of PM. The lethal concentrations at 10% and 50% of collected PM were 23.72
μg/mL (or 6.33
μg/cm
2) and 118.60
μg/mL (or 31.63
μg/cm
2), respectively. The VOC and/or PAH-coated onto PM induced significant increases in mRNA expressions of cytochrome P450 (
cyp)
1a1,
cyp2e1,
cyp2f1,
nadph quinone oxydo-
reductase-1, and
glutathione s-transferase-pi 1, versus controls. Hence, we concluded that the metabolic activation of the very low doses of VOC and/or PAH-coated onto the inorganic condensation nuclei from Dunkerque City's PM is one of the underlying mechanisms of action closely involved in its cytotoxicity in human lung epithelial cells.
Studies of Rat and Human Retinas Predict a Role for the Polyol Pathway in Human Diabetic Retinopathy
Zeina Dagher ,
Yong Seek Park ,
Veronica Asnaghi ,
Todd Hoehn ,
Chiara Gerhardinger and
Mara ...Lorenzi
Department of Ophthalmology, Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts
Address correspondence and reprint requests to Mara Lorenzi, MD, Harvard Medical School, Schepens Eye Research Institute,
20 Staniford St., Boston, MA 02114. E-mail: lorenzi{at}vision.eri.harvard.edu
Abstract
The polyol (sorbitol) pathway of glucose metabolism is activated in many cell types when intracellular glucose concentrations
are high, and it can generate cellular stress through several mechanisms. The role of the polyol pathway in the pathogenesis
of diabetic retinopathy has remained uncertain, in part because it has been examined preferentially in galactose-induced retinopathy
and in part because inhibition studies may not have achieved full blockade of the pathway. Having observed that the streptozotocin-induced
diabetic rat accurately models many cellular processes characteristic of human diabetic retinopathy, we tested in the diabetic
rat if documented inhibition of the polyol pathway prevents a sequence of retinal vascular abnormalities also present in human
diabetes. An inhibitor of aldose reductase, the rate-limiting enzyme in the pathway, prevented the early activation of complement
in the wall of retinal vessels and the decreased levels of complement inhibitors in diabetic rats, as well as the later apoptosis
of vascular pericytes and endothelial cells and the development of acellular capillaries. Both rat and human retinal endothelial
cells showed aldose reductase immunoreactivity, and human retinas exposed to high glucose in organ culture increased the production
of sorbitol by a degree similar to that observed in the rat. Excess aldose reductase activity can be a mechanism for human
diabetic retinopathy.
DMEM, Dulbecco’s modified Eagle’s medium
mAb, monoclonal antibody
MAC, membrane attack complex
NSE, neuron-specific enolase, PECAM-1, platelet endothelial cell adhesion molecule-1
RIPA, radioimmunoprecipitation assay
TUNEL, transferase-mediated dUPT nick-end labeling
vWf, von Willebrand factor
Footnotes
Y.S.P. and V.A. contributed equally to this study.
M.L. has received an honorarium from Pfizer.
Accepted May 26, 2004.
Received January 22, 2004.
DIABETES
Microbes have developed their own specific strategies to cope with reactive oxygen species (ROS). Catalase, a heme‐containing tetramer expressed in a broad range of aerobic fungi, shows remarkable ...efficiency in degrading hydrogen peroxide (H2O2) for fungal survival and host invasion. Here, it is demonstrated that catalase inactivation by blue light renders fungal cells highly susceptible to ROS attack. To confirm catalase as a major molecular target of blue light, wild type Candida albicans are systematically compared with a catalase‐deficient mutant strain regarding their susceptibility to ROS through 410 nm treatment. Upon testing a wide range of fungal species, it is found that intracellular catalase can be effectively and universally inactivated by 410 nm blue light. It is also found that photoinactivation of catalase in combination with ROS‐generating agents is highly effective in total eradication of various fungal species, including multiple Candida auris strains, the causative agent of the global fungal epidemic. In addition, photoinactivation of catalase is shown to facilitate macrophage killing of intracellular Candida albicans. The antifungal efficacy of catalase photoinactivation is further validated using a C. albicans‐induced mouse model of skin abrasion. Taken together, the findings offer a novel catalase‐photoinactivation approach to address multidrug‐resistant Candida infections.
Most of the fungal microbes have evolved to scavenge H2O2 through expression of catalase. Here, it is found that catalase from most of pathogenic fungi (Candida auris included) can be inactivated by blue light, especially at 410 nm. Photoinactivation of catalase sensitizes these fungal cells highly susceptible to H2O2‐producing agents and immune cells.
Macrophages play a critical role in the elimination of fungal pathogens. They are sensed
cell surface pattern-recognition receptors and are phagocytosed into newly formed organelles called ...phagosomes. Phagosomes mature through the recruitment of proteins and lysosomes, resulting in addition of proteolytic enzymes and acidification of the microenvironment. Our earlier studies demonstrated an essential role of Dectin-1-dependent activation of spleen tyrosine kinase (Syk) in the maturation of fungal containing phagosomes. The absence of Syk activity interrupted phago-lysosomal fusion resulting in arrest at an early phagosome stage. In this study, we sought to define the contribution of Syk to the control of phagocytosed live
in primary macrophages. To accurately measure intracellular yeast division, we designed a carboxyfluorescein succinimidyl ester (CFSE) yeast division assay in which bright fluorescent parent cells give rise to dim daughter cells. The CFSE-labeling of
did not affect the growth rate of the yeast. Following incubation with macrophages, internalized CFSE-labeled
were retrieved by cellular lysis, tagged using ConA-647, and the amount of residual CFSE fluorescence was assessed by flow cytometry.
remained undivided (CFSE bright) for up to 18 h in co-culture with primary macrophages. Treatment of macrophages with R406, a specific Syk inhibitor, resulted in loss of intracellular control of
with initiation of division within 4 h. Delayed Syk inhibition after 8 h was less effective indicating that Syk is critically required at early stages of macrophage-fungal interaction. In conclusion, we demonstrate a new method of tracking division of
using CFSE labeling. Our results suggest that early Syk activation is essential for macrophage control of phagocytosed
.
Itis generally accepted that endothelial cells generate most of their ATP byanaerobic glycolysis and that very little ATP is derived from the oxidation offatty acids or glucose. Previously, we have ...reported that, in cultured humanumbilical vein endothelial cells (HUVECs), activation of AMP-activated proteinkinase (AMPK) by the cell-permeable activator 5-aminoimidazole-4-carboximideriboside (AICAR) is associated with an increase in the oxidation of H-palmitate. In the present study, experimentscarried out with cultured HUVECs revealed the following(1) AICAR-inducedincreases in palmitate oxidation during a 2-hour incubation are associatedwith a decrease in the concentration of malonyl coenzyme A (CoA) (an inhibitorof carnitine palmitoyl transferase 1), which temporally parallels the increasein AMPK activity and a decrease in the activity of acetyl CoA carboxylase(ACC). (2) AICAR does not stimulate either palmitate oxidation when carnitineis omitted from the medium or oxidation of the medium-chain fatty acidoctanoate. (3) When intracellular lipid pools are prelabeled with H-palmitate, the measured rate of palmitateoxidation is 3-fold higher, and in the presence of AICAR, it accounts fornearly 40% of calculated ATP generation. (4) Incubation of HUVECs in aglucose-free medium for 2 hours causes the same changes in AMPK, ACC, malonylCoA, and palmitate oxidation as does AICAR. (5) Under all conditions studied,the contribution of glucose oxidation to ATP production is minimal. Theresults indicate that the AMPK-ACC-malonyl CoA-carnitine palmitoyl transferase1 mechanism plays a key role in the physiological regulation of fatty acidoxidation in HUVECs. They also indicate that HUVECs oxidize fatty acids fromboth intracellular and extracellular sources, and that when this is taken intoaccount, fatty acids can be a major substrate for ATP generation. Finally,they suggest that AMPK is likely to be a major factor in modulating theresponse of the endothelium to stresses that alter its energystate.
Neutrophils are the most abundant white blood cell in the body and are key participants in the defense against fungal infections. Fungal infections occur often in patients with cirrhosis and are ...associated with increased 30‐day and 90‐day mortality. Previous studies have shown that specific neutrophil functions are abnormal in patients with cirrhosis, although the extent of neutrophil dysfunction is not well understood. We tested the ability of neutrophils from 21 hospitalized patients with cirrhosis and 23 healthy control patients to kill Candida albicans, a common fungal pathogen in patients with cirrhosis. Using an assay, we also measured the ability of neutrophils to coordinate multicellular, synchronized control of C. albicans hyphae through a process known as swarming. We found that neutrophils from patients with cirrhosis have significantly decreased fungicidal capacity compared with healthy control neutrophils (53% vs. 74%, P < 0.0001) and diminished ability to control hyphal growth normalized as a ratio to healthy control (0.22 vs. 0.65, P < 0.0001). Moreover, serum from patients with cirrhosis decreases the ability of healthy control neutrophils to kill C. albicans (from 60% to 41%, P < 0.003). Circulating concentration of the inflammatory cytokines tumor necrosis factor α, interleukin‐6, and interleukin‐8 were found to be significantly elevated in patients with cirrhosis compared to healthy controls. Following pretreatment with granulocyte‐colony stimulating factor and granulocyte‐macrophage colony‐stimulating factor, neutrophil function was restored to almost that of healthy controls. Conclusion: Our data establish profound neutrophil dysfunction against, and altered swarming to, C. albicans in patients with cirrhosis. This dysfunction can be partially reversed with cytokine augmentation ex vivo.
Neutrophils from patients with cirrhosis are defective in coordinated swarming response to the human fungal pathogen, Candida albicans.
Exposure to urban airborne particulate matter (PM) has been associated with adverse health effects. In this work, we focused our attention on the capacity of air pollution PM to induce cytotoxic, ...oxidative stress, and inflammatory responses in human epithelial lung cells (L132) in culture. PM were collected in Dunkerque, a French seaside city, and their physical and chemical characteristics were carried out. Their size distribution showed that 92.15% of the PM were equal or smaller than 2.5 and their specific surface area was 1
m
2/g. Inorganic (i.e. Fe, Al, Ca, Na, K, Mg, Pb, etc.) and organic (i.e. VOC, PAH, etc.) chemicals were found in PM. Physical and chemical properties of Dunkerque City’s PM suggested that much of the collected PM derived from wind-borne dust from the industrial complex and the heavy motor vehicle traffic. Their cytotoxicity, as evaluated by survival rate determination, lactate dehydrogenase activity, and mitochondrial dehydrogenase activity showed concentration and time-dependent effects in L132 cells (LC10
=
18.84
μg
PM/ml; LC50
=
75.36
μg
PM/ml). Moreover, in PM-exposed L132 cells, there were concentration- and time-dependent changes in lipid peroxidation, superoxide dismutase activity, 8-hydroxy-2′-deoxyguanosine formation, and poly(ADP-ribosyl)ation, on the one hand, and in tumor necrosis factor-alpha secretion, inducible nitric oxide synthase activity, and nitric oxide release, on the other hand. Taken together, these findings suggested that oxidative stress and inflammatory responses proceeded cytotoxicity in PM-exposed L132 cells.
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