Activation of the pyrin inflammasome by intracellular Burkholderia cenocepacia

Burkholderia cenocepacia is an opportunistic pathogen that causes chronic infection and induces progressive respiratory inflammation in cystic fibrosis patients. Recognition of bacteria by mononuclear cells generally results in the activation of caspase-1 and processing of IL-1β, a major proinflammatory cytokine. In this study, we report that human pyrin is required to detect intracellular B. cenocepacia leading to IL-1β processing and release. This inflammatory response involves the host adapter molecule ASC and the bacterial type VI secretion system (T6SS). Human monocytes and THP-1 cells stably expressing either small interfering RNA against pyrin or YFP-pyrin and ASC (YFP-ASC) were infected with B. cenocepacia and analyzed for inflammasome activation. B. cenocepacia efficiently activates the inflammasome and IL-1β release in monocytes and THP-1. Suppression of pyrin levels in monocytes and THP-1 cells reduced caspase-1 activation and IL-1β release in response to B. cenocepacia challenge. In contrast, overexpression of pyrin or ASC induced a robust IL-1β response to B. cenocepacia, which correlated with enhanced host cell death. Inflammasome activation was significantly reduced in cells infected with T6SS-defective mutants of B. cenocepacia, suggesting that the inflammatory reaction is likely induced by an as yet uncharacterized effector(s) of the T6SS. Together, we show for the first time, to our knowledge, that in human mononuclear cells infected with B. cenocepacia, pyrin associates with caspase-1 and ASC forming an inflammasome that upregulates mononuclear cell IL-1β processing and release.     

Hyaline cartilage tissue is formed through the co-culture of passaged human chondrocytes and primary bovine chondrocytes

To circumvent the problem of a sufficient number of cells for cartilage engineering, the authors previously developed a two-stage culture system to redifferentiate monolayer culture-expanded dedifferentiated human articular chondrocytes by co-culture with primary bovine chondrocytes (bP0). The aim of this study was to analyze the composition of the cartilage tissue formed in stage 1 and compare it with bP0 grown alone to determine the optimal length of the co-culture stage of the system. Biochemical data show that extracellular matrix accumulation was evident after 2 weeks of co-culture, which was 1 week behind the bP0 control culture. By 3 to 4 weeks, the amounts of accumulated proteoglycans and collagens were comparable. Expression of chondrogenic genes, Sox 9, aggrecan, and collagen type II, was also at similar levels by week 3 of culture. Immunohistochemical staining of both co-culture and control tissues showed accumulation of type II collagen, aggrecan, biglycan, decorin, and chondroitin sulfate in appropriate zonal distributions. These data indicate that co-cultured cells form cartilaginous tissue that starts to resemble that formed by bP0 after 3 weeks, suggesting that the optimal time to terminate the co-culture stage, isolate the now redifferentiated cells, and start stage 2 is just after 3 weeks.     

Airway Epithelial Expression of TLR5 Is Downregulated in Healthy Smokers and Smokers with Chronic Obstructive Pulmonary Disease

The TLRs are important components of the respiratory epithelium host innate defense, enabling the airway surface to recognize and respond to a variety of insults in inhaled air. On the basis of the knowledge that smokers are more susceptible to pulmonary infection and that the airway epithelium of smokers with chronic obstructive pulmonary disease (COPD) is characterized by bacterial colonization and acute exacerbation of airway infections, we assessed whether smoking alters expression of TLRs in human small airway epithelium, the primary site of smoking-induced disease. Microarrays were used to survey the TLR family gene expression in small airway (10th to 12th order) epithelium from healthy nonsmokers (n = 60), healthy smokers (n = 73), and smokers with COPD (n = 36). Using the criteria of detection call of present (P call) ≥50%, 6 of 10 TLRs (TLRs 1-5 and 8) were expressed. Compared with nonsmokers, the most striking change was for TLR5, which was downregulated in healthy smokers (1.4-fold, p < 10(-10)) and smokers with COPD (1.6-fold, p < 10(-11)). TaqMan RT-PCR confirmed these observations. Bronchial biopsy immunofluorescence studies showed that TLR5 was expressed mainly on the apical side of the epithelium and was decreased in healthy smokers and smokers with COPD. In vitro, the level of TLR5 downstream genes, IL-6 and IL-8, was highly induced by flagellin in TLR5 high-expressing cells compared with TLR5 low-expressing cells. In the context that TLR5 functions to recognize pathogens and activate innate immune responses, the smoking-induced downregulation of TLR5 may contribute to smoking-related susceptibility to airway infection, at least for flagellated bacteria.     

Protein energy malnutrition impairs homeostatic proliferation of memory CD8 T cells

Nutrition is a critical but poorly understood determinant of immunity. There is abundant epidemiological evidence linking protein malnutrition to impaired vaccine efficacy and increased susceptibility to infections; yet, the role of dietary protein in immune memory homeostasis remains poorly understood. In this study, we show that protein-energy malnutrition induced in mice by low-protein (LP) feeding has a detrimental impact on CD8 memory. Relative to adequate protein (AP)-fed controls, LP feeding in lymphocytic choriomeningitis virus (LCMV)-immune mice resulted in a 2-fold decrease in LCMV-specific CD8 memory T cells. Adoptive transfer of memory cells, labeled with a division tracking dye, from AP mice into naive LP or AP mice demonstrated that protein-energy malnutrition caused profound defects in homeostatic proliferation. Remarkably, this defect occurred despite the lymphopenic environment in LP hosts. Whereas Ag-specific memory cells in LP and AP hosts were phenotypically similar, memory cells in LP hosts were markedly less responsive to polyinosinic-polycytidylic acid-induced acute proliferative signals. Furthermore, upon recall, memory cells in LP hosts displayed reduced proliferation and protection from challenge with LCMV-clone 13, resulting in impaired viral clearance in the liver. The findings show a metabolic requirement of dietary protein in sustaining functional CD8 memory and suggest that interventions to optimize dietary protein intake may improve vaccine efficacy in malnourished individuals.     

HIV-1 proteins preferentially activate anti-inflammatory M2-type macrophages

HIV-1 proteins, including Tat, gp120, and Nef, activate macrophages (MΦ), which is consistent with the fact that HIV-1 infection is characterized by sustained immune activation. Meanwhile, MΦ are functionally classified into two types: proinflammatory M1-MΦ and anti-inflammatory M2-MΦ. We show that HIV-1 proteins, particularly Nef, preferentially activate M2-MΦ. Extracellular Tat, gp120, and Nef activated MAPK and NF-κB pathways in human peripheral blood monocyte-derived MΦ. However, the activation was marked in M-CSF-derived M2-MΦ but not GM-CSF-derived M1-MΦ. Nef was the most potent activator, and its signaling activation was comparable to that by TNF-α. Indeed, Nef was internalized more rapidly by M2-MΦ than by M1-MΦ. The myristoylation and proline-rich motif of Nef were responsible for the observed signaling activation. Consistent with the activation of MAPK/NF-κB pathways, Nef stimulated the production of a number of proinflammatory cytokines/chemokines by M2-MΦ. However, Nef reduced the expression of CD163 and phagocytosis, the characteristic markers of M2-MΦ, indicating that Nef drives an M2-like to M1-like phenotypic shift. Because the differentiation of most tissue MΦ depends on M-CSF and its receptor, which is the essential axis for the anti-inflammatory M2-MΦ phenotype, the current study reveals an efficient mechanism by which HIV-1 proteins, such as Nef, induce the proinflammatory MΦ.     

YajL, the prokaryotic homolog of the Parkinsonism-associated protein DJ-1, protects cells against protein sulfenylation

YajL is the closest Escherichia coli homolog of the Parkinsonism-associated protein DJ-1, a multifunctional oxidative stress response protein whose biochemical function remains unclear. We recently described the oxidative-stress-dependent aggregation of proteins in yajL mutants and the oxidative-stress-dependent formation of mixed disulfides between YajL and members of the thiol proteome. We report here that yajL mutants display increased protein sulfenic acids levels and that formation of mixed disulfides between YajL and its protein substrates in vivo is inhibited by the sulfenic acid reactant dimedone, suggesting that YajL preferentially forms disulfides with sulfenylated proteins. YajL (but not YajL(C106A)) also forms mixed disulfides in vitro with the sulfenylated form of bovine serum albumin. The YajL-serum albumin disulfides can be subsequently reduced by glutathione or dihydrolipoic acid. We also show that DJ-1 can form mixed disulfides with sulfenylated E. coli proteins and with sulfenylated serum albumin. These results suggest that YajL and possibly DJ-1 function as covalent chaperones involved in the detection of sulfenylated proteins by forming mixed disulfides with them and that these disulfides are subsequently reduced by low-molecular-weight thiols.     

ZBTB32 Is an Early Repressor of the CIITA and MHC Class II Gene Expression during B Cell Differentiation to Plasma Cells

CIITA and MHC class II expression is silenced during the differentiation of B cells to plasma cells. When B cell differentiation is carried out ex vivo, CIITA silencing occurs rapidly, but the factors contributing to this event are not known. ZBTB32, also known as repressor of GATA3, was identified as an early repressor of CIITA in an ex vivo plasma cell differentiation model. ZBTB32 activity occurred at a time when B lymphocyte-induced maturation protein-1 (Blimp-1), the regulator of plasma cell fate and suppressor of CIITA, was minimally induced. Ectopic expression of ZBTB32 suppressed CIITA and I-A gene expression in B cells. Short hairpin RNA depletion of ZBTB32 in a plasma cell line resulted in re-expression of CIITA and I-A. Compared with conditional Blimp-1 knockout and wild-type B cells, B cells from ZBTB32/ROG-knockout mice displayed delayed kinetics in silencing CIITA during ex vivo plasma cell differentiation. ZBTB32 was found to bind to the CIITA gene, suggesting that ZBTB32 directly regulates CIITA. Lastly, ZBTB32 and Blimp-1 coimmunoprecipitated, suggesting that the two repressors may ultimately function together to silence CIITA expression. These results introduce ZBTB32 as a novel regulator of MHC-II gene expression and a potential regulatory partner of Blimp-1 in repressing gene expression.     

Piperine suppresses cerebral ischemia–reperfusion-induced inflammation through the repression of COX-2, NOS-2, and NF-κB in middle cerebral artery occlusion rat model

The pathophysiological mechanisms leading to neuronal injury in middle cerebral artery occlusion (MCAO) model of cerebral stroke are complex and multifactorial that form the bases of behavioral deficits and inflammation mediated damage. The present study demonstrates the effect of piperine pretreatment (10 mg/kg b wt, once daily p.o. for 15 days) on cerebral ischemia-induced inflammation in male Wistar rats. The right middle cerebral artery was occluded for 2 h followed by reperfusion for 22 h. A maximum infarct volume (57.80 %) was observed in ischemic MCAO group. However, piperine administration prior to ischemia showed a significant reduction in infarct volume (28.29 %; p < 0.05) and neuronal loss (12.72 %; p < 0.01). As a result of piperine pretreatment, a significant improvement in behavioral outputs of MCAO rats (p < 0.05-0.01) was observed. Piperine successfully reduced the level of proinflammatory cytokines IL-1β, IL-6 and TNF-α, in ischemic group (p < 0.01). Ischemic group brain has shown edematous morphology with vacuolated architecture and pyknotic nuclei in H & E staining which was successfully ameliorated by piperine administration. Moreover, piperine also succeeded in lowering the expression of COX-2, NOS-2, and NF-κB (p < 0.01). Both cytosolic and nuclear NF-κB were down-regulated in ischemic group pre-administered with piperine (p < 0.01). The present study suggests that piperine is able to salvage the ischemic penumbral zone neurons by virtue of its anti-inflammatory property, thereby limiting ischemic cell death.     

Flaxseed Oil as a Neuroprotective Agent on Lead Acetate-Induced Monoamineric Alterations and Neurotoxicity in Rats

Lead remains a considerable occupational and public health problem, which is known to cause a number of adverse effects in both man and animals. Here, the neuroprotective effect of flaxseed oil (1,000 mg/kg) on lead acetate (20 mg/kg) induced alternation in monoamines and brain oxidative stress was examined in rats. The levels of lead, dopamine (DA), norepinephrine (NE), serotonin (5-HT), lipid peroxidation, nitrite/nitrate (NO), and glutathione (GSH) were determined; also, the activity of acetylcholinesterase (AChE) and Na(+)-K(+)-ATPase were estimated on different brain regions of adult male albino rats. The level of lead was markedly elevated in different brain regions of rats. This leads to enhancement of lipid peroxidation and NO production in brain with concomitant reduction in AChE activity and GSH level. In addition, the levels of DA, NE, and 5-HT were decreased in the brain. These findings were associated with BAX over expression. Treatment of rats with flaxseed oil induced a marked improvement in most of the studied parameters as well as the immunohistochemistry features. These data indicated that dietary flaxseed oil provide protection against lead-induced oxidative stress and neurotoxic effects.