Ethanol-triggered Lipophagy Requires SQSTM1 in AML12 Hepatic Cells.

Ethanol-induced hepatic lipophagy plays an important cytoprotective role against liver injury, but its mechanism is not fully determined. In the present study, ethanol-induced lipophagy was studied in an immortalized mouse hepatocyte line, AML12. We found that ethanol treatment elevated lipid content in these cells, which could be regulated by autophagy. To determine the potential mechanism, we investigated the role of a key adaptor molecule SQSTM1/p62. SQSTM1 can bind to LC3 on autophagosomes and ubiquitinated molecules on cargos, thus facilitating the autophagic engulfment of the cargo. We found that both LC3 and SQSTM1 could colocalize with lipid droplets (LDs) following ethanol treatment. Colocalization of LC3 with LDs was significantly inhibited by SQSTM1 knockdown, which also reduced ethanol-induced lipid elevation. In addition, increased ubiquitin signals were found to colocalize with SQSTM1 on LDs in response to ethanol. Moreover, the SQSTM1 signal was colocalized with that of perilipin1, a major protein on LDs. Finally, perilipin1 knockdown significantly altered ethanol-induced lipophagy. Taken together, these data support a model in which autophagosomes were directed to the LDs via SQSTM1, which bound to ubiquitinated proteins, possibly including perilipin 1, on LDs. This study provides a potential mechanistic explanation to how ethanol induces lipophagy in hepatocytes.

Mechanisms of Action of Vitamin D as Supplemental Therapy for Pneumocystis Pneumonia.

The combination of trimethoprim and sulfamethoxazole (TMP-SMX) is the most effective regimen for therapy of Pneumocystis pneumonia (PCP). As many patients with PCP are allergic or do not respond to it, efforts have been devoted to develop alternative therapies for PCP. We have found that the combination of vitamin D3 (VitD3) (300 IU/kg/day) and primaquine (PMQ) (5 mg/kg/day) was as effective as TMP-SMX for therapy of PCP. In this study, we investigated the mechanisms by which vitamin D enhances the efficacy of PMQ. C57BL/6 mice were immunosuppressed by CD4+ cell depletion, infected with Pneumocystismurina for 8 weeks, and then treated for 9 days with the combination of VitD3 and PMQ (VitD3-PMQ) or with TMP-SMX or PMQ to serve as controls. The results showed that vitamin D supplementation increased the number of CD11c+ cells, suppressed the production of proinflammatory cytokines (tumor necrosis factor alpha [TNF-α], gamma interferon [IFN-γ], and interleukin-6 [IL-6]) and inducible nitric oxide synthase (iNOS), and enhanced the expression of genes related to antioxidation (glutathione reductase and glutamate-cysteine ligase modifier subunit), antimicrobial peptides (cathelicidin), and autophagy (ATG5 and beclin-1). These results suggest that the main action of vitamin D is enhancing the ability of the host to defend against Pneumocystis infection.

Regulation of Collagen V Expression and Epithelial-Mesenchymal Transition by miR-185 and miR-186 during Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis is a devastating disease, with no good diagnostic biomarker and limited treatment options. Previous studies suggest that collagen V overexpression and collagen V-mediated immune response play roles in the pathogenesis of idiopathic pulmonary fibrosis. This study aimed to identify dysregulated miRNA-related collagen V overexpression during idiopathic pulmonary fibrosis. We found that the expression levels of miR-185 and miR-186 were decreased in the lungs of idiopathic pulmonary fibrosis patients. The levels of miR-185 and miR-186 were not correlated with disease severity of idiopathic pulmonary fibrosis. The direct regulation of COL5A1 by miR-185 and miR-186 was confirmed by a luciferase reporter assay. Furthermore, mimics of miR-185 and miR-186 blocked transforming growth factor-ß-induced collagen V overexpression and alleviated transforming growth factor-ß-induced epithelial-mesenchymal transition in A549 cells and HCC827 cells. Our findings suggest that attenuated expression of miR-185 and miR-186 may be responsible for collagen V overexpression during idiopathic pulmonary fibrosis, and these miRNAs may serve as pathogenesis-related biomarkers and treatment targets.

Evaluation of two new enzyme immunoassay reagents for diagnosis of histoplasmosis in a cohort of clinically characterized patients.

The performance characteristics of the recently available analyte-specific reagent based enzyme immunoassay (ASR-EIA) and in vitro diagnostic (IVD) kit for urine Histoplasma antigen detection were evaluated in a cohort of 50 clinically characterized patients with histoplasmosis and 50 control patients. Overall sensitivity and specificity of the ASR-EIA were significantly improved compared with those of the IVD kit (sensitivity 72% vs. 22%, P<.001, specificity 98% vs. 84%, P = .014). Fourteen specimens from patients with clinically characterized histoplasmosis (five with pulmonary histoplasmosis and nine with progressive disseminated histoplasmosis) were falsely negative by ASR-EIA. All 10 specimens from patients with severe symptoms of progressive disseminated histoplasmosis were positive by ASR-EIA, although the average reading value of these 10 specimens was not significantly different from that of others with positive results. Compared to the MiraVista antigen assay, both the IVD kit and the ASR-EIA were significantly less sensitive in detecting Histoplasma antigen in the urine of patients with histoplasmosis. The ASR-EIA and MiraVista assay had comparable specificity. In conclusion, the ASR-EIA has improved performance compared with the IVD kit in the detection of Histoplasma antigen in the urine. However, users should be aware of the potential for false negative results using the currently recommended cutoff value.

Myeloid-derived suppressor cells impair alveolar macrophages through PD-1 receptor ligation during Pneumocystis pneumonia.

Myeloid-derived suppressor cells (MDSCs) were recently found to accumulate in the lungs during Pneumocystis pneumonia (PcP). Adoptive transfer of these cells caused lung damage in recipient mice, suggesting that MDSC accumulation is a mechanism of pathogenesis in PcP. In this study, the phagocytic activity of alveolar macrophages (AMs) was found to decrease by 40% when they were incubated with MDSCs from Pneumocystis-infected mice compared to those incubated with Gr-1(+) cells from the bone marrow of uninfected mice. The expression of the PU.1 gene in AMs incubated with MDSCs also was decreased. This PU.1 downregulation was due mainly to decreased histone 3 acetylation and increased DNA methylation caused by MDSCs. MDSCs were found to express high levels of PD-L1, and alveolar macrophages (AMs) were found to express high levels of PD-1 during PcP. Furthermore, PD-1 expression in AMs from uninfected mice was increased by 18-fold when they were incubated with MDSCs compared to those incubated with Gr-1(+) cells from the bone marrow of uninfected mice. The adverse effects of MDSCs on AMs were diminished when the MDSCs were pretreated with anti-PD-L1 antibody, suggesting that MDSCs disable AMs through PD-1/PD-L1 ligation during PcP.

Accumulation of myeloid-derived suppressor cells in the lungs during Pneumocystis pneumonia.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of hematopoietic precursors with the ability to adversely affect host immunity. They have been shown to accumulate in pathological conditions, such as cancer and some microbial diseases. In the mouse and rat models of Pneumocystis pneumonia (PcP), we found a distinct population of cells with MDSC-like morphology in the bronchoalveolar lavage (BAL) fluid, constituting up to 50% of the total cells in BAL fluid. These cells were not seen in the BAL fluid from normal animals or from Pneumocystis-infected animals that had been successfully treated for PcP with a combination of trimethoprim and sulfamethoxazole. With flow cytometry, these cells were found to express the characteristic MDSC surface markers Gr-1 and CD11b in mice or CD11bc and His48 in rats. Using reverse transcription-PCR, we demonstrated that these cells produced high levels of arginase-1 and inducible nitric oxide synthase (iNOS) mRNA. These cells were shown to suppress CD4(+) T-cell proliferation in response to stimulation by anti-CD3 and anti-CD28 antibodies. Adoptive transfer of these cells to normal mice caused lung damage, as indicated by elevated levels of albumin and lactate dehydrogenase in the BAL fluid. These experiments provide evidence of the presence of MDSCs in the lungs during PcP. Further studies on the roles of MDSCs in PcP are warranted in order to develop treatment strategies which can reduce the number of MDSCs and the damage caused by these cells.

Sphingolipid-mediated inhibition of apoptotic cell clearance by alveolar macrophages.

A decreased clearance of apoptotic cells (efferocytosis) by alveolar macrophages (AM) may contribute to inflammation in emphysema. The up-regulation of ceramides in response to cigarette smoking (CS) has been linked to AM accumulation and increased detection of apoptotic alveolar epithelial and endothelial cells in lung parenchyma. We hypothesized that ceramides inhibit the AM phagocytosis of apoptotic cells. Release of endogenous ceramides via sphingomyelinase or exogenous ceramide treatments dose-dependently impaired apoptotic Jurkat cell phagocytosis by primary rat or human AM, irrespective of the molecular species of ceramide. Similarly, in vivo augmentation of lung ceramides via intratracheal instillation in rats significantly decreased the engulfment of instilled target apoptotic thymocytes by resident AM. The mechanism of ceramide-induced efferocytosis impairment was dependent on generation of sphingosine via ceramidase. Sphingosine treatment recapitulated the effects of ceramide, dose-dependently inhibiting apoptotic cell clearance. The effect of ceramide on efferocytosis was associated with decreased membrane ruffle formation and attenuated Rac1 plasma membrane recruitment. Constitutively active Rac1 overexpression rescued AM efferocytosis against the effects of ceramide. CS exposure significantly increased AM ceramides and recapitulated the effect of ceramides on Rac1 membrane recruitment in a sphingosine-dependent manner. Importantly, CS profoundly inhibited AM efferocytosis via ceramide-dependent sphingosine production. These results suggest that excessive lung ceramides may amplify lung injury in emphysema by causing both apoptosis of structural cells and inhibition of their clearance by AM.

Downregulation of PU.1 leads to decreased expression of Dectin-1 in alveolar macrophages during Pneumocystis pneumonia.

Dectin-1 is an important macrophage phagocytic receptor recognizing fungal beta-glucans. In this study, the mRNA levels of the Dectin-1 gene were found to be decreased by 61% in alveolar macrophages (AMs) from Pneumocystis-infected mice. The expression of Dectin-1 protein on the surface of these cells was also significantly decreased. By fluorescence in situ hybridization, mRNA expression levels of the transcription factor PU.1 were also found to be significantly reduced in AMs from Pneumocystis-infected mice. Electrophoretic mobility shift assay showed that PU.1 protein bound Dectin-1 gene promoter. With a luciferase reporter gene driven by the Dectin-1 gene promoter, the expression of the PU.1 gene in NIH 3T3 cells was found to enhance the luciferase activity in a dose-dependent manner. PU.1 expression knockdown by small interfering RNA (siRNA) caused a 63% decrease in Dectin-1 mRNA level and 40% decrease in protein level in AMs. Results of this study indicate that downregulation of PU.1 during Pneumocystis pneumonia leads to decreased expression of Dectin-1 in AMs.

Effects of decreased calmodulin protein on the survival mechanisms of alveolar macrophages during Pneumocystis pneumonia.

Pneumocystis infection causes increased intracellular levels of reactive oxygen species (ROS) and the subsequent apoptosis of alveolar macrophages (Amø). Assessments of key prosurvival molecules in Amø and bronchoalveolar lavage fluids from infected rats and mice showed low levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) and reduced activation of phosphoinositide-3 kinase (PI-3K). Ubiquitous calcium-sensing protein calmodulin protein and mRNA levels were also reduced in Amø during Pneumocystis pneumonia (Pcp). Calmodulin has been implicated in control of GM-CSF production and PI-3K activation in other immune cell types. Experiments to determine the control of GM-CSF and PI-3K by calmodulin in Amø showed that GM-CSF expression and PI-3K activation could not be induced when calmodulin was inhibited. Calmodulin inhibition also led to increased levels of ROS and apoptosis in cells exposed to bronchoalveolar lavage fluids from infected animals. Supplementation of Amø with exogenous calmodulin increased survival signaling via GM-CSF and PI-3K and reduced ROS and apoptosis. These data support the hypotheses that calmodulin levels at least partially control survival signaling in Amø and that restoration of GM-CSF or PI-3K signaling will improve host response to the organism.

Decreased inflammatory response in Toll-like receptor 2 knockout mice is associated with exacerbated Pneumocystis pneumonia.

Pneumocystis pneumonia (PcP) is marked by substantial inflammatory damage to the lung. We have found that Toll-like receptor 2 (TLR2) mediates macrophage inflammatory responses to Pneumocystis and hypothesized that TLR2 deficiency would lead to less severe inflammation and milder lung injury during PcP. Histopathology examination showed that TLR2-/- mice with PcP indeed exhibited milder pulmonary inflammation. TLR2-/- mouse lungs contained less TNF-alpha and displayed lower levels of NF-kappaB activation during PcP. However, TLR2-/- mice with PcP displayed increased severity in symptoms and organism burden. The increased organism burden is likely due to defects in protective mechanisms in TLR2-/- mice. mRNA levels of the inducible nitric oxide synthase and NADPH oxidase p47phox, as well as nitric oxide levels in the lungs, were decreased in TLR2-/- PcP mice. Taken together, this study shows that TLR2-mediated inflammatory responses contribute to a certain degree to the clearance of Pneumocystis organism in mice.

Suppression of alveolar macrophage apoptosis prolongs survival of rats and mice with pneumocystis pneumonia.

The number of alveolar macrophages is decreased in patients or animals with Pneumocystis pneumonia (Pcp). This loss of alveolar macrophages is in part due to apoptosis caused by Pneumocystis infection. The mechanism of apoptosis induction is unknown. Cell-free bronchoalveolar lavage fluids from Pneumocystis-infected rats or mice have the ability to induce apoptosis in normal alveolar macrophages. To characterize the mechanisms by which apoptosis proceeds in alveolar macrophages during Pcp, specific caspase inhibitors are tested for their ability to suppress the apoptosis. In vitro induction of apoptosis can be inhibited by the caspase-9 inhibitor (Z-LEHD-FMK) but not by the inhibitor to caspase-8 or -10. The caspase-9 inhibitor can also inhibit apoptosis of alveolar macrophages in vivo when it is intranasally instilled into dexamethasone-immunosuppressed, Pneumocystis-infected rats or L3T4 cell-depleted, Pneumocystis-infected mice. The number of alveolar macrophages rebounds in caspase-9 inhibitor-treated Pcp animals. Phagocytic activity of alveolar macrophages in treated animals is also recovered, and organism burden in these animals is reduced. Administration of caspase-9 inhibitor also clears the exudate that normally fills the alveoli during Pcp and decreases lung inflammation. Furthermore, caspase-9-treated Pcp animals survive for the entire 70-day period of the study, whereas nontreated Pcp animals die 40-60 days after initiation of infection. Depletion of recovered alveolar macrophages by intranasal administration of clodronate-containing liposomes in caspase-9 inhibitor-treated animals abrogates the effects of the inhibitor. Together, these results indicate that immunomodulation of the host response may be an alternative to current treatments for Pcp.

Toll-like receptor 2 mediates alveolar macrophage response to Pneumocystis murina.

The innate immune response to Pneumocystis infection is not well understood. In this study, normal C57BL/6 mouse alveolar macrophages were found to respond to Pneumocystis murina organisms through Toll-like receptor 2 (TLR2), leading to the nuclear translocation of NF-kappaB and the production of proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) and chemokine macrophage inflammatory protein 2 (MIP-2). P. murina stimulation of normal alveolar macrophages from C57BL/6 mice resulted in increased TLR2 transcription but not increased TLR4 transcription. In gain-of-function studies with HEK293 cells expressing TLR2 or TLR4, only TLR2 was found to stimulate an NF-kappaB response to P. murina. TNF-alpha and MIP-2 production in response to P. murina by mouse alveolar macrophages was inhibited by a monoclonal antibody that specifically blocked the ligand-binding ability of TLR2. Alveolar macrophages from TLR2 knockout (TLR2-/-) mice showed little increase in TNF-alpha and MIP-2 mRNA levels upon P. murina stimulation. An in vivo study showed that TLR2-/- mice challenged with P. murina had reduced cytokine responses. These results indicate that TLR2 plays a major role in the innate immune response to P. murina.

Justicidin A decreases the level of cytosolic Ku70 leading to apoptosis in human colorectal cancer cells.

The natural product justicidin A, an arylnaphthalide lignan isolated from Justicia procumbens, significantly inhibited the growth of human colorectal cancer cells HT-29 and HCT 116 at day 6 post-treatment. Further study revealed that justicidin A-treated HT-29 and HCT 116 colorectal cancer cells died of apoptosis. Justicidin A treatment caused DNA fragmentation and an increase in phosphatidylserine exposure of the cells. The number of cells in the sub-G1 phase was also increased upon justicidin A treatment. Caspase-9 but not caspase-8 was activated, suggesting that justicidin A treatment damaged mitochondria. The mitochondrial membrane potential was altered and cytochrome c and Smac were released from mitochondria to the cytoplasm upon justicidin A treatment. The level of Ku70 in the cytoplasm was decreased, but that of Bax in mitochondria was increased by justicidin A. Since Ku70 normally binds and sequesters Bax, these results suggest that justicidin A decreases the level of Ku70 leading to translocation of Bax from the cytosol to mitochondria to induce apoptosis. Oral administration of justicidin A was shown to suppress the growth of HT-29 cells transplanted into NOD-SCID mice, suggesting chemotherapeutic potential of justicidin A on colorectal cancer cells.

Evidence for the requirement of T cell costimulation in the pathogenesis of natural Pneumocystis carinii pulmonary infection.

Pneumocystis carinii pneumonia (PCP) is a frequent and serious opportunistic infection in immunocompromized patients. Although the pathogenesis of PCP-mediated lung injury is poorly understood, a central involvement of host inflammatory responses has been implicated. We have found that while the loss of specific T cell costimulatory signals increases susceptibility to the spontaneous pneumocystis infection, PCP-induced pulmonary injury (and subsequent morbidity and mortality) involves other intact costimulatory pathways. Mice that are genetically deficient for the costimulatory receptor CD154 (CD154 knockout (ko) mice) spontaneously developed PCP, consistent with the increased susceptibility of X-linked hyper IgM syndrome patients (caused by CD154 gene mutations) to P. carinii infection. In these mice PCP was manifested by progressive weight loss, dyspnea and death. In contrast, CD154 ko mice also genetically lacking ICAM1 (CD154 koxICAM1 ko) or CD28 (CD154 koxCD28 ko) costimulatory receptors had later onset of weight loss and significantly prolonged survival. Although onset of infection and age-matched P. carinii organism burden were equivalent, the CD154 single knockout mice had evidence of greater pulmonary inflammation vs. the double ko's. These findings suggest that costimulation-dependent T cell-mediated inflammation plays an important role in both susceptibility to and pathogenesis of PCP, and may identify potential molecular targets for novel immunomodulatory treatment approaches.