Lipopolysaccharide and IL-1β coordinate a synergy on cytokine production by upregulating MyD88 expression in human gingival fibroblasts

Both lipopolysaccharide (LPS) and interleukin (IL)-1β activate the MyD88-dependent signaling pathways to stimulate proinflammatory cytokine expression. However, it remains unknown how LPS and IL-1β interact with each other to coordinate the stimulation. In this study, we sought to investigate the interaction between LPS and IL-1β on MyD88-dependent signaling pathways in human gingival fibroblasts (HGFs). Results showed that LPS derived from Porphyromonas gingivalis (Pg LPS) and IL-1β cooperatively stimulated mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NFκB) signaling pathways, and subsequent expression of proinflammatory cytokine expression. Furthermore, our results showed that Pg LPS and IL-1β exerted a synergy on MyD88 expression and knockdown of MyD88 expression by small interfering RNA diminished the synergistic effect of Pg LPS and IL-1β on IL-6 expression, suggesting that upregulation of MyD88 is involved in the coordinated stimulation by Pg LPS and IL-1β of proinflammatory cytokine expression. Finally, our results showed that pharmacological inhibitors for MAPK and NFκB significantly reduced IL-6 secretion stimulated by Pg LPS and IL-1β, indicating that the MyD88-dependent MAPK and NFκB signaling pathways are essential for the upregulation of proinflammatory cytokine expression by Pg LPS and IL-1β. Taken together, this study showed that LPS and IL-1β coordinate a synergy on cytokine production by upregulating MyD88 expression in HGFs.     

A retrospective evaluation of critically ill patients infected with H1N1 influenza A virus in Bursa,Turkey, during the 2009–2010 pandemic

Background

H1N1 influenza A virus infections were first reported in April 2009 and spread rapidly, resulting in mortality worldwide. The aim of this study was to evaluate patients with H1N1 infection treated in the intensive care unit (ICU) in Bursa, Turkey.

Methods

Demographic characteristics, clinical features, and outcome relating to H1N1 infection were retrospectively analysed in patients treated in the ICU.

Results

Twenty-three cases of H1N1 infection were treated in the ICU. The mean age of patients was 37 years range: (17–82). Fifteen patients were female (65.2%). The mean Acute Physiology and Chronic Health Evaluation (APACHE) II score was 19 range: (5–39). The most common symptoms were dyspnea (73.9%), fever (69.6%), and cough (60.9%). Mechanical ventilation was required for all patients. Oseltamivir and antibiotics were administered to all patients. Six (26.1%) patients died. APACHE II scores were higher in the deceased 28.5 range: [16–39] vs. 14 range: [5–28] in survivors; p = 0.013).

Conclusion

When compared to the literature, the demographic, epidemiological, and clinical characteristics were similar in the cases we encountered. The mortality rate was high despite the use of appropriate treatment. We believe that the high mortality is related to higher APACHE II scores. The H1N1 virus should be considered in community acquired pneumonia, especially in younger patients presenting with severe pneumonia.     

Strain- and host species-specific inflammasome activation,IL-1β release,and cell death in macrophages infected with uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC) is the main etiological agent of urinary tract infections (UTIs). Little is known about interactions between UPEC and the inflammasome, a key innate immune pathway. Here we show that UPEC strains CFT073 and UTI89 trigger inflammasome activation and lytic cell death in human macrophages. Several other UPEC strains, including two multidrug-resistant ST131 isolates, did not kill macrophages. In mouse macrophages, UTI89 triggered cell death only at a high multiplicity of infection, and CFT073-mediated inflammasome responses were completely NLRP3-dependent. Surprisingly, CFT073- and UTI89-mediated responses only partially depended on NLRP3 in human macrophages. In these cells, NLRP3 was required for interleukin-1β (IL-1β) maturation, but contributed only marginally to cell death. Similarly, caspase-1 inhibition did not block cell death in human macrophages. In keeping with such differences, the pore-forming toxin α-hemolysin mediated a substantial proportion of CFT073-triggered IL-1β secretion in mouse but not human macrophages. There was also a more substantial α-hemolysin-independent cell death response in human vs. mouse macrophages. Thus, in mouse macrophages, CFT073-triggered inflammasome responses are completely NLRP3-dependent, and largely α-hemolysin-dependent. In contrast, UPEC activates an NLRP3-independent cell death pathway and an α-hemolysin-independent IL-1β secretion pathway in human macrophages. This has important implications for understanding UTI in humans.     

A new begomovirus–betasatellite complex is associated with chilli leaf curl disease in Sri Lanka

Leaf curl disease of chilli (LCDC) is a major constraint in production of chilli in the Indian subcontinent. The objective of this study was to identify the begomovirus species occurring in chilli in Sri Lanka, where the LCDC was initially recorded in 1938. The virus samples were collected from the North Central Province, the major chilli growing region in Sri Lanka with a history of epidemic prevalence of LCDC. The virus could be readily transmitted by Bemisia tabaci to chilli, tomato and tobacco, where vein clearing followed by leaf curl developed. The genome analysis of two isolates obtained from two distantly located fields showing 100 % LCDC, revealed that the DNA-A genome (2754 nucleotides) shared 89.5 % sequence identity with each other and 68.80–84.40 % sequence identity with the other begomoviruses occurring in the Indian subcontinent. The closest identity (84.40 %) of the virus isolates was with Tomato leaf curl Sri Lanka virus (ToLCLKV). The results support that a new begomovirus species is affecting chilli in Sri Lanka and the name Chilli leaf curl Sri Lanka virus (ChiLCSLV) is proposed. Recombination analysis indicated that ChiLCSLV was a recombinant virus potentially originated from the begomoviruses prevailing in southern India and Sri Lanka. The genome of betasatellite associated with the two isolates consisted of 1366 and 1371 nucleotides and shared 95.2 % sequence identity with each other and 41.50–73.70 % sequence identity with the other betasatellite species. The results suggest that a new begomovirus betasatellite, Chilli leaf curl Sri Lanka betasatellite is associated with LCDC in Sri Lanka. This study demonstrates a new species of begomovirus and betasatellite complex is occurring in chilli in Sri Lanka and further shows that diverse begomovirus species are affecting chilli production in the Indian subcontinent.     

Dipyrithione inhibits IFN-γ-induced JAK/STAT1 signaling pathway activation and IP-10/CXCL10 expression in RAW264.7 cells

Objective  

This study investigates the effects of dipyrithione (PTS2) on the expression of IP-10/CXCL10, which has been observed in a wide variety of chronic inflammatory disorders and autoimmune conditions.     

A novel 1,25-dihydroxyvitamin D–activin A pathway in human alveolar macrophages is dysfunctional in patients with pulmonary alveolar proteinosis (PAP)

We have shown that activin A, a cytokine implicated in regulating B-cell proliferation, is severely deficient in alveolar macrophages from patients with pulmonary alveolar proteinosis (PAP), an autoimmune disorder characterized by surfactant accumulation and neutralizing autoantibodies to granulocyte–macrophage colony stimulating factor. Mechanisms of activin regulation in alveolar macrophages are not well understood. Based on previous gene array results from PAP bronchoalveolar lavage cells suggesting deficiencies in vitamin D target genes, and on recent evidence of vitamin D receptor elements (VDREs) in the human activin A gene promoter, we investigated the effects of 1,25-dihydroxyvitamin D (vitamin D₃) on activin A expression in alveolar macrophages from healthy individuals and PAP patients. Activin A expression was stimulated by LPS in cultures of either healthy control or PAP alveolar macrophages; in contrast, vitamin D₃ increased activin A only in healthy controls but not in PAP. Compared to healthy controls, freshly obtained (uncultured) PAP alveolar macrophages displayed healthy intrinsic vitamin D receptor expression but deficient expression of vitamin D target genes, cathelicidin and thioredoxin interacting protein. PAP patients also demonstrated a relative insufficiency of circulating vitamin D. Investigation of activin A in murine alveolar macrophages confirmed a lack of functional response to vitamin D as anticipated since murine activin A does not contain VDREs. Results suggest that mechanisms of activin A deficiency in PAP alveolar macrophages may involve dysregulation of a novel species-specific vitamin D–activin A pathway.     

MD-2 regulates LPS-induced NLRP3 inflammasome activation and IL-1beta secretion by a MyD88/NF-κB-dependent pathway in alveolar macrophages cell line

Myeloid differentiation protein 2 (MD-2) is required in the recognition of lipopolysaccharide (LPS) by toll-like receptor 4 (TLR4), and participates in LPS-induced alveolar macrophage (AM) inflammation during acute lung injury (ALI). Activation of the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome aggravates inflammation in LPS-induced ALI. However, there is currently little known about the relationship between MD-2 signaling and the NLRP3 inflammasome. This study showed that NLRP3 expression, IL-1beta (IL-1β) secretion, and pyroptosis were up-regulated after LPS stimulation in the NR8383 AM cell-line. MD-2 gene knock-down reduced LPS-induced mRNA and protein expression of NLRP3 and IL-1β secretion in NR8383 cells, and inhibited the MyD88/NF-κB signaling pathway. Conversely, over-expression of MD-2 not only heightened NLRP3, MyD88, and NF-κB p65 protein expression, it also aggravated the LPS-induced inflammatory response. Furthermore, the NF-κB inhibitor SN50 had a beneficial role in decreasing NLRP3 and caspase-1 mRNA and protein expression. The observations suggest that MD-2 helps to regulate LPS-induced NLRP3 inflammasome activation and the inflammatory response in NR8383 cells, and likely does so by affecting MyD88/NF-κB signaling.     

Matrix-Assisted Laser Desorption Ionization–Time of Flight and Comparative Genomic Analysis of M-18 Group A Streptococcus Strains Associated with an Acute Rheumatic Fever Outbreak in Northeast Italy in 2012 and 2013

Acute rheumatic fever (ARF) is a postsuppurative sequela caused by Streptococcus pyogenes infections affecting school-age children. We describe here the occurrence of an ARF outbreak that occurred in Bologna province, northeastern Italy, between November 2012 and May 2013. Molecular analysis revealed that ARF-related group A Streptococcus (GAS) strains belonged to the M-18 serotype, including subtypes emm18.29 and emm18.32. All M-18 GAS strains shared the same antigenic profile, including SpeA, SpeB, SpeC, SpeL, SpeM, and SmeZ. Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) analysis revealed that M-18 GAS strains grouped separately from other serotypes, suggesting a different S. pyogenes lineage. Single nucleotide polymorphisms and phylogenetic analysis based on whole-genome sequencing showed that emm18.29 and emm18.32 GAS strains clustered in two distinct groups, highlighting genetic variations between these subtypes. Comparative analysis revealed a similar genome architecture between emm18.29 and emm18.32 strains that differed from noninvasive emm18.0 strains. The major sources of differences between M-18 genomes were attributable to the prophage elements. Prophage regions contained several virulence factors that could have contributed to the pathogenic potential of emm18.29 and emm18.32 strains. Notably, phage ΦSPBO.1 carried erythrogenic toxin A gene (speA1) in six ARF-related M-18 GAS strains but not in emm18.0 strains. In addition, a phage-encoded hyaluronidase gene (hylP.2) presented different variants among M-18 GAS strains by showing internal deletions located in the α-helical and TSβH regions. In conclusion, our study yielded insights into the genome structure of M-18 GAS strains responsible for the ARF outbreak in Italy, thus expanding our knowledge of this serotype.     

Clusterin induces matrix metalloproteinase-9 expression via ERK1/2 and PI3K/Akt/NF-κB pathways in monocytes/macrophages

Most solid tumor tissues possess a significant population of macrophages, which are known to be closely linked with tumor progression and metastasis. Clusterin has been reported to be overexpressed in various tumors and to have a tumor-promoting role. As clusterin induction and macrophage infiltration occur concurrently at the tumor site, it raises a possibility that clusterin may regulate the function of macrophages via facilitating ECM remodeling. Here, we demonstrate for the first time the expression of MMP-9 by clusterin in human primary monocytes as well as human and murine macrophage cell lines, THP-1, and Raw264.7. MMP-9 expression was accompanied by increased enzymatic activity, as revealed by gelatin zymography. The MMP-9 activity promoted by clusterin was found to be dependent on the activation of ERK1/2 and PI3K/Akt but not p38 or JNK pathways. Inhibition of PI3K activity did not affect the activation of ERK1/2 and vice versa, indicating that the two pathways were independently operated to stimulate MMP-9 activity. Moreover, clusterin facilitated nuclear translocation of NF-κB p65 along with IκB-α degradation and phosphorylation, which was critical for MMP-9 expression. As NF-κB is a central regulator of inflammation, clusterin may provide a molecular link between inflammation and cancer via up-regulating NF-κB and MMP-9. Collectively, these data highlight a novel role of clusterin as a stimulator for MMP-9 expression in macrophages, which may contribute to the tissue reorganization by serving as a modulator for ECM degradation.     

Small hepatitis B surface antigen interacts with and modulates enoyl–coenzyme A hydratase expression in hepatoma cells

Enoyl–coenzyme A hydratase (ECHS1) is a key enzyme in the metabolism of fatty acids in mitochondria. We previously reported that hepatitis B surface antigen (HBsAg) interacted with ECHS1 in a yeast two-hybrid system. In the current study, we further examined their interaction by using GST pull-down and co-immunoprecipitation assays. The results confirmed that small hepatitis B surface antigen (SHBs) interacted with ECHS1. Furthermore, confocal imaging showed that SHBs and ECHS1 co-localized in HepG2 cells. To clarify the biological function of the interaction, human hepatoma cell lines that transiently and stably expressed SHBs were generated. The expression of SHBs led to a significant decrease in ECHS1 protein levels. ECHS1 protein levels were reduced to 48.44 ± 7.12 % in Huh7 cells transiently expressing SHBs, and to 54.97 ± 3.54 % in HepG2 cells stably expressing SHBs. In conclusion, our findings suggest that SHBs interacts with ECHS1 and regulates ECHS1 protein levels in hepatoma cells.     

Aβ induces its own prion protein N-terminal fragment (PrPN1)–mediated neutralization in amorphous aggregates

Plasma membrane cellular prion protein (PrP^C) is a high-affinity receptor for toxic soluble amyloid-β (Aβ) oligomers that mediates synaptic dysfunction. Secreted forms of PrP^C resulting from PrP^C α-cleavage (PrPN1) or shedding (shed PrP^C) display neuroprotective activity in neuronal cultures and in mouse models of Aβ-induced neuronal dysfunction. In vitro, recombinant PrPN1 and PrP inhibit Aβ fibrillization. However, the mechanism by which PrPN1 and shed PrP^C neutralize Aβ oligomers is unclear, and evidence of such neuroprotective activity in Alzheimer's disease (AD) patients is lacking. Here, we show that PrPN1 association with Aβ causes a conformational change resulting in the formation of amorphous and insoluble aggregates that are not compatible with the assembly of Aβs. Using postmortem brain tissues of AD patients, we were able to coimmunoprecipitate Aβ with PrP^C molecules and observed a coaggregation of Aβ and PrPN1 in the guanidine-extractable fraction presumably representing insoluble amyloid plaques. Furthermore, PrP^C α-cleavage is increased in AD brains, and we noticed a significant positive correlation between the levels of α-cleavage and of guanidine-extractable Aβ. These data strongly support the hypothesis that PrP^C α-cleavage is an endogenous neuroprotective mechanism in AD and support the development of PrP^C-derived peptides as therapeutic molecules for AD.