Guohong Shao

Gene expression profiling identifies MMP-12 and ADAMDEC1 as potential pathogenic mediators of pulmonary sarcoidosis

RATIONALE Little is known about the genetic regulation of granulomatous inflammation in sarcoidosis. OBJECTIVES To determine if tissue gene array analysis would identify novel genes engaged in inflammation and lung remodeling in patients with sarcoidosis. METHODS Gene expression analysis was performed on tissues obtained from patients with sarcoidosis at the time of diagnosis (untreated) (n = 6) compared with normal lung tissue (n = 6). Expression of select genes was further confirmed in lung tissue from a second series of patients with sarcoidosis and disease-free control subjects (n = 11 per group) by semi-quantitative RT-PCR. Interactive gene networks were identified in patients with sarcoidosis using Ingenuity Pathway Analysis (Ingenuity Systems, Inc., Redwood, CA) software. The expression of proteins corresponding to selected overexpressed genes was determined using fluorokine multiplex analysis, and immunohistochemistry. Selected genes and proteins were then analyzed in bronchoalveolar lavage fluid in an independent series of patients with sarcoidosis (n = 36) and control subjects (n = 12). MEASUREMENTS AND MAIN RESULTS A gene network engaged in Th1-type responses was most significantly overexpressed in the sarcoidosis lung tissues, including genes not previously reported in the context of sarcoidosis (e.g., IL-7). MMP-12 and ADAMDEC1 transcripts were most highly expressed (> 25-fold) in sarcoidosis lung tissues, corresponding with increased protein expression by immunohistochemistry. MMP-12 and ADAMDEC1 gene and protein expression were increased in bronchoalveolar lavage samples from patients with sarcoidosis, correlating with disease severity. CONCLUSIONS Tissue gene expression analyses provide novel insights into the pathogenesis of pulmonary sarcoidosis. MMP-12 and ADAMDEC1 emerge as likely mediators of lung damage and/or remodeling and may serve as markers of disease activity.

Monocyte activation by necrotic cells is promoted by mitochondrial proteins and formyl peptide receptors.

Objective:Necrotic cells evoke potent innate immune responses through unclear mechanisms. The mitochondrial fraction of the cell retains constituents of its bacterial ancestors, including N-formyl peptides, which are potentially immunogenic. Thus, we hypothesized that the mitochondrial fraction of the cell, particularly N-formyl peptides, contributes significantly to the activation of monocytes by necrotic cells. Design:Human peripheral blood monocytes were incubated with necrotic cell fractions and mitochondrial proteins to investigate their potential for immune cell activation. Setting:University Medical Center Research Laboratory. Subjects:Healthy human adults served as blood donors. Measurements and Main Results:Human blood monocyte activation was measured after treatment with cytosolic, nuclear and mitochondrial fractions of necrotic HepG2 cells or necrotic HepG2 cells depleted of N-formyl peptides [Rho(0) cells]. The specific role of the high affinity formyl peptide receptor (FPR) was then tested using specific pharmacologic inhibitors and RNA silencing. The capacity of mitochondrial N-formyl peptides to activate monocytes was confirmed using a synthetic peptide conforming to the N-terminus of mitochondrial nicotinamide adenine dinucleotide subunit 6. The results demonstrated that mitochondrial cell fractions most potently activated monocytes, and interleukin (IL)-8 was selectively released at low-protein concentrations. Mitochondria from Rho(0) cells induced minimal monocyte IL-8 release, and specific pharmacologic inhibitors and RNA-silencing confirmed that FPR contributes significantly to monocyte IL-8 responses to both necrotic cells and mitochondrial proteins. N-formyl peptides alone did not induce monocyte IL-8 release; whereas, the combination of mitochondrial N-formyl peptides and mitochondrial transcription factor A (TFAM) dramatically increased IL-8 release from monocytes. Likewise, high mobility group box 1, the nuclear homolog of TFAM, did not induce monocyte IL-8 release unless combined with mitochondrial N-formyl peptides. Conclusions:Interactions between mitochondrial N-formyl peptides and FPR in the presence of other mitochondrial antigens (e.g., TFAM) contributes significantly to the activation of monocytes by necrotic cells.

Mitochondrial Transcription Factor A Serves as a Danger Signal by Augmenting Plasmacytoid Dendritic Cell Responses to DNA

Plasmacytoid dendritic cells (pDC) are potent APCs known to regulate immune responses to self-Ags, particularly DNA. The mitochondrial fraction of necrotic cells was found to most potently promote human pDC activation, as reflected by type I IFN release, which was dependent upon the presence of mitochondrial DNA and involved TLR9 and receptors for advanced glycation end products. Mitochondrial transcription factor A (TFAM), a highly abundant mitochondrial protein that is functionally and structurally homologous to high mobility group box protein 1, was observed to synergize with CpG-containing oligonucleotide, type A, DNA to promote human pDC activation. pDC type I IFN responses to TFAM and CpG-containing oligonucleotide, type A, DNA indicated their engagement with receptors for advanced glycation end products and TLR9, respectively, and were dependent upon endosomal processing and PI3K, ERK, and NF-κB signaling. Taken together, these results indicate that pDC contribute to sterile immune responses by recognizing the mitochondrial component of necrotic cells and further incriminate TFAM and mitochondrial DNA as likely mediators of pDC activation under these circumstances.

Mitochondrial Transcription Factor A, an Endogenous Danger Signal, Promotes TNFα Release via RAGE- and TLR9-Responsive Plasmacytoid Dendritic Cells

Objective Mitochondrial transcription factor A (TFAM) is normally bound to and remains associated with mitochondrial DNA (mtDNA) when released from damaged cells. We hypothesized that TFAM, bound to mtDNA (or equivalent CpG-enriched DNA), amplifies TNFα release from TLR9-expressing plasmacytoid dendritic cells (pDCs) by engaging RAGE. Materials and Methods Murine Flt3 ligand-expanded splenocytes obtained from C57BL/6 mice were treated with recombinant human TFAM, alone or in combination with CpG-enriched DNA with subsequent TNFα release measured by ELISA. The role of RAGE was determined by pre-treatment with soluble RAGE or heparin or by employing matching RAGE (-/-) splenocytes. TLR9 signaling was evaluated using a specific TLR9-blocking oligonucleotide and by inhibiting endosomal processing, PI3K and NF-κB. Additional studies examined whether heparin sulfate moieties or endothelin converting enzyme-1 (ECE-1)-dependent recycling of endosomal receptors were required for TFAM and CpG DNA recognition. Main Results TFAM augmented splenocyte TNFα release in response to CpGA DNA, which was strongly dependent upon pDCs and regulated by RAGE and TLR9 receptors. Putative TLR9 signaling pathways, including endosomal acidification and signaling through PI3K and NF-κB, were essential for splenocyte TNFα release in response to TFAM+CpGA DNA. Interestingly, TNFα release depended upon endothelin converting enzyme (ECE)-1, which cleaves and presumably activates TLR9 within endosomes. Recognition of the TFAM-CpGA DNA complex was dependent upon heparin sulfate moieties, and recombinant TFAM Box 1 and Box 2 proteins were equivalent in terms of augmenting TNFα release. Conclusions TFAM promoted TNFα release in a splenocyte culture model representing complex cell-cell interactions in vivo with pDCs playing a critical role. To our knowledge, this study is the first to incriminate ECE-1-dependent endosomal cleavage of TLR9 as a critical step in the signaling pathway leading to TNFα release. These findings, and others reported herein, significantly advance our understanding of sterile immune responses triggered by mitochondrial danger signals.

Nicotine Treatment Improves Toll-Like Receptor 2 and Toll-Like Receptor 9 Responsiveness in Active Pulmonary Sarcoidosis

BACKGROUND New evidence links nicotine to the regulation of T cell-mediated inflammation via a 7 nicotinic cholinergic receptor activation, and chronic nicotine exposure (smoking) reduces the incidence of granulomatous diseases. We sought to determine whether nicotine treatment was well tolerated while effectively normalizing immune responses in patients with active pulmonary sarcoidosis. METHODS Consenting adults with symptomatic sarcoidosis (n 5 13) were randomly assigned to receive 12 weeks of nicotine treatment plus conventional therapy or conventional therapy alone. Obtained blood cells were evaluated for their responsiveness to selected Toll-like receptor (TLR) and nucleotide oligomerization domain-like receptor ligands and T cell surface marker expression before and after nicotine treatment. Asymptomatic patients (n 5 6) and disease-free subjects (n 5 6) served as comparative control subjects. Adverse events were monitored for the duration of the study. RESULTS Compared with the asymptomatic group, symptomatic patients had impaired peripheral responses to TLR2, TLR4, and TLR9 ligands (anergy) and reduced peripheral populations of CD4 1 FoxP3 1 regulatory T cells (Tregs). Nicotine treatment was associated with restoration of TLR2 and TLR9 responsiveness, and expansion of Tregs, including the CD4 1 CD25 2 FoxP3 1 phenotype. There were no serious adverse events or signs of nicotine dependency. CONCLUSIONS Nicotine treatment in active pulmonary sarcoidosis was well tolerated and restored peripheral immune responsiveness to TLR2 and TLR9 agonists and expansion of FoxP3 1 Tregs, including a specific “preactivated” (CD25 2 ) phenotype. The immune phenotype of patients with symptomatic sarcoidosis treated with nicotine closely resembled that of asymptomatic patients, supporting the notion that nicotine treatment may be beneficial in this patient population.

Intestinal epithelium is more susceptible to cytopathic injury and altered permeability than the lung epithelium in the context of acute sepsis

Mitochondrial morphology and function are altered in intestinal epithelia during endotoxemia. However, it is unclear whether mitochondrial abnormalities occur in lung epithelial cells during acute sepsis or whether mitochondrial dysfunction corresponds with altered epithelial barrier function. Thus, we hypothesized that the intestinal epithelium is more susceptible to mitochondrial injury than the lung epithelium during acute sepsis and that mitochondrial dysfunction precedes impaired barrier function. Using a resuscitated feline model of Escherichia coli‐induced sepsis, lung and ileal tissues were harvested after 6 h for histological and mitochondrial ultrastructural analyses in septic (n = 6) and time‐matched controls (n = 6). Human lung epithelial cells (HLEC) and Caco‐2 monolayers (n = 5) were exposed to ‘cytomix’ (TNFα: 40 ng/ml, IL‐1β: 20 ng/ml, IFNγ: 10 ng/ml) for 24–72 h, and measurements of transepithelial electrical resistance (TER), epithelial permeability and mitochondrial membrane potential (ΔΨ) were taken. Lung epithelial morphology, mitochondrial ultrastructure and pulmonary gas exchange were unaltered in septic animals compared to matching controls. While histologically intact, ileal epithelia demonstrated marked mitochondrial ultrastructural damage during sepsis. Caco‐2 monolayers treated with cytomix showed a significant decrease in mitochondrial ΔΨ within 24 h, which was associated with a progressive reduction in TER and increased epithelial permeability over the subsequent 48 h. In contrast, mitochondrial ΔΨ and epithelial barrier functions were preserved in HLEC following cytomix. These findings indicate that intestinal epithelium is more susceptible to mitochondrial damage and dysfunction than the lung epithelium in the context of sepsis. Early alterations in mitochondrial function portend subsequent epithelial barrier dysfunction.

At-risk but viable myocardium in a large animal model of non ST-segment elevation acute coronary syndrome: cardiovascular magnetic resonance with ex vivo validation

BackgroundPatients with non-ST-segment elevation acute coronary syndrome (NSTE-ACS) have varying degrees of salvageable myocardium at risk of irreversible injury. We hypothesized that a novel model of NSTE-ACS produces acute myocardial injury, measured by increased T2 cardiovascular magnetic resonance (CMR), without significant necrosis by late gadolinium enhancement (LGE).MethodsIn a canine model, partial coronary stenosis was created and electrodes placed on the epicardium. Myocardial T2, an indicator of at-risk myocardium, was measured pre- and post-tachycardic pacing.ResultsSerum troponin-I (TnI) was not detectable in unoperated sham animals but averaged 1.97 ± 0.72 ng/mL in model animals. Coronary stenosis and pacing produced significantly higher T2 in the affected vs. the remote myocardium (53.2 ± 4.9 vs. 43.6 ± 2.8 ms, p < 0.01) with no evident injury by LGE. Microscopy revealed no significant irreversible cellular injury. Relative respiration rate (RRR) of affected vs. remote myocardial tissue was significantly lower in model vs. sham animals (0.72 ± 0.07 vs. 1.04 ± 0.07, p < 0.001). Lower RRR corresponded to higher final TnI levels (R2 = 0.83, p = 0.004) and changes in CaMKIID and mitochondrial gene expression.ConclusionsA large animal NSTE-ACS model with mild TnI elevation and without ST elevation, similar to the human syndrome, demonstrates signs of acute myocardial injury by T2-CMR without significant irreversible damage. Reduced tissue respiration and associated adaptations of critical metabolic pathways correspond to increased myocardial injury by serum biomarkers in this model. T2-CMR as a biomarker of at-risk but salvageable myocardium warrants further consideration in preclinical and clinical studies of NSTE-ACS.