Phthalate drives splenic inflammatory response via activating HSP60/TLR4/NLRP3 signaling axis-dependent pyroptosis.

As the most produced phthalate, di-(2-ethylhexyl) phthalate (DEHP) is a widely environmental pollutant primarily used as a plasticizer, which cause the harmful effects on human health. However, the impact of DEHP on spleen and its underlying mechanisms are still unclear. Pyroptosis is a novel form of cell death induced by activating NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasomes and implicated in pathogenesis of numerous inflammatory diseases. The current study aimed to explore the impact of DEHP on immune inflammatory response in mouse spleen. In this study, the male ICR mice were treated with DEHP (200 mg/kg) for 28 days. Here, DEHP exposure caused abnormal pathohistological and ultrastructural changes, accompanied by inflammatory cells infiltration in mouse spleen. DEHP exposure arouse heat shock response that involves increase of heat shock proteins 60 (HSP60) expression. DEHP also elevated the expressions of toll-like receptor 4 (TLR4) and myeloid differentiation protein 88 (MyD88) proteins, as well as the activation of NF-κB pathway. Moreover, DEHP promoted NLRP3 inflammasome activation and triggered NLRP3 inflammasome-induced pyroptosis. Mechanistically, DEHP drives splenic inflammatory response via activating HSP60/TLR4/NLRP3 signaling axis-dependent pyroptosis. Our findings reveal that targeting HSP60-mediated TLR4/NLRP3 signaling axis may be a promising strategy for inflammatory diseases treatment.

[Diosgenin alleviates NAFLD induced by a high-fat diet in rats via mTOR/SREBP-1c/HSP60/MCAD/SCAD signaling pathway].

This study aims to observe the effects of diosgenin on the expression of mammalian target of rapamycin(mTOR), sterol regulatory element-binding protein-1c(SREBP-1c), heat shock protein 60(HSP60), medium-chain acyl-CoA dehydrogenase(MCAD), and short-chain acyl-CoA dehydrogenase(SCAD) in the liver tissue of the rat model of non-alcoholic fatty liver disease(NAFLD) and explore the mechanism of diosgenin in alleviating NAFLD. Forty male SD rats were randomized into five groups: a control group, a model group, low-(150 mg·kg~(-1)·d~(-1)) and high-dose(300 mg·kg~(-1)·d~(-1)) diosgenin groups, and a simvastatin(4 mg·kg~(-1)·d~(-1)) group. The rats in the control group were fed with a normal diet, while those in the other four groups were fed with a high-fat diet. After feeding for 8 weeks, the body weight of rats in the high-fat diet groups increased significantly. After that, the rats were administrated with the corresponding dose of diosgenin or simvastatin by gavage every day for 8 weeks. The levels of triglyceride(TG), total cholesterol(TC), alanine transaminase(ALT), and aspartate transaminase(AST) in the serum were determined by the biochemical method. The levels of TG and TC in the liver were measured by the enzyme method. Oil-red O staining was employed to detect the lipid accumulation, and hematoxylin-eosin(HE) staining to detect the pathological changes in the liver tissue. The mRNA and protein levels of mTOR, SREBP-1c, HSP60, MCAD, and SCAD in the liver tissue of rats were determined by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR) and Western blot, respectively. Compared with the control group, the model group showed increased body weight, food uptake, liver index, TG, TC, ALT, and AST levels in the serum, TG and TC levels in the liver, lipid deposition in the liver, obvious hepatic steatosis, up-regulated mRNA and protein expression levels of mTOR and SREBP-1c, and down-regulated mRNA and protein expression levels of HSP60, MCAD, and SCAD. Compared with the model group, the rats in each treatment group showed obviously decreased body weight, food uptake, liver index, TG, TC, ALT, and AST levels in the serum, TG and TC levels in the liver, lessened lipid deposition in the liver, ameliorated hepatic steatosis, down-regulated mRNA and protein le-vels of mTOR and SREBP-1c, and up-regulated mRNA and protein levels of HSP60, MCAD, and SCAD. The high-dose diosgenin outperformed the low-dose diosgenin and simvastatin. Diosgenin may prevent and treat NAFLD by inhibiting the expression of mTOR and SREBP-1c and promoting the expression of HSP60, MCAD, and SCAD to reduce lipid synthesis, improving mitochondrial function, and promoting fatty acid ß oxidation in the liver.

Surface-exposed chaperonin 60 derived from Propionibacterium freudenreichii MJ2 inhibits adipogenesis by decreasing the expression of C/EBPα/PPARγ.

Recent studies have shown that the health benefits of probiotics are not limited to those offered by living bacteria. It was reported that both live and killed cells of Propionibacterium freudenreichii MJ2 (MJ2) isolated from raw milk showed antiobesity activity in 3T3-L1 cells and high-fat diet-induced obese mice. This study was aimed at identifying the active component(s) responsible for the antiadipogenic activity of MJ2. Cell wall, surface protein, and cytoplasmic fractions of MJ2 were investigated for their inhibitory effects on adipogenesis in 3T3-L1 cells. Adipocytes treated with the surface protein fraction showed significantly lower lipid accumulation. Using the MASCOT algorithm following LC-MS/MS analysis, 131 surface proteins were identified and they were principally classified into three categories (network clusters related to ribosomes, carbon metabolism, and chaperones). Among them, chaperonin 60 (Cpn60) was selected as a potential candidate protein. Cpn60 inhibited lipid accumulation and adipogenesis during the early period of differentiation (days 0-2) and decreased expression of genes related to adipogenesis (Pparg and Cebpa) and lipogenesis (Fas and Scd1). The expression of Gata2/3, which suppresses adipogenesis, significantly increased in Cpn60-treated cells. Moreover, the nuclear translocation of C/EBPß was inhibited by Cpn60 treatment. In conclusion, Cpn60, a surface protein in MJ2, shows antiadipogenic activity by reducing the expression of C/EBPß through the upregulation of Gata2/3 expression followed by downregulation of Pparg and Cebpa expression.

Evaluation and Characterization of the Insecticidal Activity and Synergistic Effects of Different GroEL Proteins from Bacteria Associated with Entomopathogenic Nematodes on Galleria mellonella.

GroEL is a chaperonin that helps other proteins fold correctly. However, alternative activities, such as acting as an insect toxin, have also been discovered. This work evaluates the chaperonin and insecticidal activity of different GroEL proteins from entomopathogenic nematodes on G. mellonella. The ability to synergize with the ExoA toxin of Pseudomonas aeruginosa was also investigated. The GroELXn protein showed the highest insecticidal activity among the different GroELs. In addition, it was able to significantly activate the phenoloxidase system of the target insects. This could tell us about the mechanism by which it exerts its toxicity on insects. GroEL proteins can enhance the toxic activity of the ExoA toxin, which could be related to its chaperonin activity. However, there is a significant difference in the synergistic effect that is more related to its alternative activity as an insecticidal toxin.

A peptide derived from HSP60 reduces proinflammatory cytokines and soluble mediators: a therapeutic approach to inflammation.

Cytokines are secretion proteins that mediate and regulate immunity and inflammation. They are crucial in the progress of acute inflammatory diseases and autoimmunity. In fact, the inhibition of proinflammatory cytokines has been widely tested in the treatment of rheumatoid arthritis (RA). Some of these inhibitors have been used in the treatment of COVID-19 patients to improve survival rates. However, controlling the extent of inflammation with cytokine inhibitors is still a challenge because these molecules are redundant and pleiotropic. Here we review a novel therapeutic approach based on the use of the HSP60-derived Altered Peptide Ligand (APL) designed for RA and repositioned for the treatment of COVID-19 patients with hyperinflammation. HSP60 is a molecular chaperone found in all cells. It is involved in a wide diversity of cellular events including protein folding and trafficking. HSP60 concentration increases during cellular stress, for example inflammation. This protein has a dual role in immunity. Some HSP60-derived soluble epitopes induce inflammation, while others are immunoregulatory. Our HSP60-derived APL decreases the concentration of cytokines and induces the increase of FOXP3+ regulatory T cells (Treg) in various experimental systems. Furthermore, it decreases several cytokines and soluble mediators that are raised in RA, as well as decreases the excessive inflammatory response induced by SARS-CoV-2. This approach can be extended to other inflammatory diseases.

Quercetin May Improve Fat Graft Survival by Promoting Fat Browning Peripherally.

BACKGROUND: Adipose browning occurs after white fat transfer. But its location and effects on fat graft survival remains controversial. This study was performed to locate the browning of fat grafts, and to explore the effects of quercetin on fat graft browning and fat graft survival. METHODS: Human fat granules were injected into the subcutaneous layer of 12 nude mice. Control group was injected with fat granules and 10% of normal saline, while quercetin group was injected with fat granules and 10% of quercetin. The graft samples (n = 6 for each group) were obtained in weeks 2, 4, 8 and 12. Weight retention rate of the grafts was calculated. Gene and protein expression of mitochondrial markers (silent information regulator 1, SIRT1; heat shock protein 60, HSP60), browning marker (uncoupling protein 1, UCP1), peroxisome proliferator-activated receptor-γ (PPAR-γ), vascular endothelial growth factor A (VEGF-A) were evaluated. Hematoxylin and eosin staining and anti-UCP1 staining were performed. RESULTS: Clusters of small multilocular beige adipocytes were observed in the periphery of fat grafts. Compared with control group, quercetin group had a higher weight retention rate, a higher gene/protein expression of SIRT1, HSP60, UCP1, PPAR-γ and VEGF-A, and a higher occurrence of peripheral adipose browning. CONCLUSIONS: Peripherally located adipose browning occurred after white fat transfer. It can be enhanced by the addition of quercetin through promoting mitochondrial function of fat cells, and may be one of the mechanisms that quercetin improves fat graft survival. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

A peptide derived from chaperonin 60.1, IRL201104, inhibits LPS-induced acute lung inflammation.

Chaperonin 60.1 (Cpn60.1) is a protein derived from Mycobacterium tuberculosis that has been shown, along with its peptide fragment IRL201104, to have beneficial effects in models of allergic inflammation. To further investigate the anti-inflammatory properties of Cpn60.1 and IRL201104, we have investigated these molecules in a model of nonallergic lung inflammation. Mice were treated with Cpn60.1 (0.5-5,000 ng/kg) or IRL201104 (0.00025-2.5 ng/kg), immediately before intranasal instillation of bacterial lipopolysaccharide (LPS). Cytokine levels and cell numbers in mouse bronchoalveolar lavage (BAL) fluid were measured 4 h after LPS administration. In some experiments, mice were depleted of lung-resident phagocytes. Cells from BAL fluid were analyzed for inflammasome function. Human umbilical vein endothelial cells (HUVECs) were analyzed for adhesion molecule expression. Human neutrophils were analyzed for integrin expression, chemotaxis, and cell polarization. Cpn60.1 and IRL201104 significantly inhibited neutrophil migration into the airways, independently of route of administration. This effect of the peptide was absent in TLR4 and annexin A1 knockout mice. Intravital microscopy revealed that IRL201104 reduced leukocyte adhesion and migration into inflamed tissues. However, IRL201104 did not significantly affect adhesion molecule expression in HUVECs or integrin expression, chemotaxis, or polarization of human neutrophils at the studied concentrations. In phagocyte-depleted animals, the anti-inflammatory effect of IRL201104 was not significant. IRL201104 significantly reduced IL-1ß and NLRP3 expression and increased A20 expression in BAL cells. This study shows that Cpn60.1 and IRL201104 potently inhibit LPS-induced neutrophil infiltration in mouse lungs by a mechanism dependent on tissue-resident phagocytes and to a much lesser extent, the proresolving factor annexin A1.

Lactobacillus stress protein GroEL prevents colonic inflammation.

BACKGROUND: We previously showed that supernatants of Lactobacillus biofilms induced an anti-inflammatory response by affecting the secretion of macrophage-derived cytokines, which was abrogated upon immunodepletion of the stress protein GroEL. METHODS: We purified GroEL from L. reuteri and analysed its anti-inflammatory properties in vitro in human macrophages isolated from buffy coats, ex vivo in explants from human biopsies and in vivo in a mouse model of DSS induced intestinal inflammation. As a control, we used GroEL purified (LPS-free) from E. coli. RESULTS: We found that L. reuteri GroEL (but not E. coli GroEL) inhibited pro-inflammatory M1-like macrophages markers, and favored M2-like markers. Consequently, L. reuteri GroEL inhibited pro-inflammatory cytokines (TNFα, IL-1ß, IFNγ) while favouring an anti-inflammatory secretome. In colon tissues from human biopsies, L. reuteri GroEL was also able to decrease markers of inflammation and apoptosis (caspase 3) induced by LPS. In mice, we found that rectal administration of L. reuteri GroEL (but not E. coli GroEL) inhibited all signs of haemorrhagic colitis induced by DSS including intestinal mucosa degradation, rectal bleeding and weight loss. It also decreased intestinal production of inflammatory cytokines (such as IFNγ) while increasing anti-inflammatory IL-10 and IL-13. These effects were suppressed when animals were immunodepleted in macrophages. From a mechanistic point of view, the effect of L. reuteri GroEL seemed to involve TLR4, since it was lost in TRL4-/- mice, and the activation of a non-canonical TLR4 pathway. CONCLUSIONS: L. reuteri GroEL, by affecting macrophage inflammatory features, deserves to be explored as an alternative to probiotics.

Oral administration of Hsp65-producing Lactococcus lactis attenuates allergic asthma in a murine model.

AIMS: Allergic asthma is a chronic inflammatory lung disease characterized by a Th2-type immune response pattern. The development of nonspecific immunotherapy is one of the primary goals for the control of this disease. METHODS AND RESULTS: In this study, we evaluated the therapeutic effects of Lactococcus lactis-producing mycobacterial heat shock protein 65 (LLHsp65) in an ovalbumin (OVA)-induced allergic asthma model. OVA-challenged BALB/c mice were orally administrated with LLHsp65 for 10 consecutive days. The results demonstrate that LLhsp65 attenuates critical features of allergic inflammation, like airway hyperresponsiveness and mucus production. Likewise, the treatment decreases the pulmonary eosinophilia and the serum level of OVA-specific IgE. In addition to deviating immune responses towards Th1-cytokine profile, increase regulatory T cells, and cytokine levels, such as IL-6 and IL-10. CONCLUSIONS: Our results reveal that the mucosal immunotherapy of LLHsp65 significantly reduces the overall burden of airway allergic inflammation, suggesting a promising therapeutic strategy for allergic asthma treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: This research reveals new perspectives on nonspecific immunotherapy based on the delivery of recombinant proteins by lactic acid bacteria to treat of allergic disorders.

Mycobacterial Hsp65 antigen delivered by invasive Lactococcus lactis reduces intestinal inflammation and fibrosis in TNBS-induced chronic colitis model.

Intestinal fibrosis associated with Crohn's disease (CD), which a common and serious complication of inflammatory bowel diseases. In this context, heat shock proteins (HSPs) might serve as an alternative treatment because these antigens play important roles in the regulation of effector T cells. We thus evaluated the anti-inflammatory and antifibrotic capacities of an invasive and Hsp65-producing strain-Lactococcus lactis NCDO2118 FnBPA+ (pXYCYT:Hsp65)-in chronic intestinal inflammation to assess its potential as an alternative therapeutic strategy against fibrotic CD. Experimental colitis was induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS) in BALB/c mice, and the mice were treated orally with L. lactis NCDO2118 FnBPA+ (pXYCYT:Hsp65) via intragastric gavage. The oral administration of this strain significantly attenuated the severity of inflammation and intestinal fibrosis in mice (p < 0.05). These results are mainly justified by reductions in the levels of the pro-fibrotic cytokines IL-13 and TGF-ß and increases in the concentration of the regulatory cytokine IL-10. The L. lactis NCDO2118 FnBPA+ (pXYCYT:Hsp65) strain contributed to reductions in the severity of inflammatory damage in chronic experimental CD, and these findings confirm the effectiveness of this new antifibrotic strategy based on the delivery of therapeutic proteins to inside cells of the host intestinal mucosa.

In silico design of a vaccine candidate based on autotransporters and HSP against the causal agent of shigellosis, Shigella flexneri.

Shigellosis is a diarrheal disease that causes high mortality every year, especially in children, elderly and immunocompromised patients. Recently, resistance strains to antibiotic therapy are in the rise and the World Health Organization prioritizes the development of a safe vaccine against the most common causal agent of shigellosis, Shigella flexneri. This pathogen uses autotransporter proteins such as SigA, Pic and Sap to increase virulence and some of them have been described as highly immunogenic proteins. In this study, we used immune-informatics analysis to identify the most antigenic epitope as a vaccine candidate on three passenger domains of auto-transporter proteins encoded on the pathogenic island SHI-1, to induce immunity against S. flexneri. Epitope identification was done using various servers such as Bepipred, Bcepred, nHLAPRED, NetMHCII, Rankpep and IEDB and the final selection was done based on its antigenicity using the VaxiJen server. Moreover, to enhance immunity, the GroEL adjuvant was added to the final construct as a Toll-like receptor 2 (TLR2) agonist. On the other hand, to predict the tertiary structure, the I-TASSER server was used, and the best model was structurally validated using the ProSA-web software and the Ramachandran plot. Subsequently, the model was refined and used for docking and molecular dynamics analyses with TLR2, which demonstrated an appropriate and stable interaction. In summary, a potential subunit vaccine candidate, that contains B and T cell epitopes with proper physicochemical properties was designed. This multiepitope vaccine is expected to elicit robust humoral and cellular immune responses and vest protective immunity against S. flexneri.

Modulation of allergic inflammation in the lung by a peptide derived from Mycobacteria tuberculosis chaperonin 60.1.

BACKGROUND: We have previously demonstrated that Mycobacteria tuberculosis chaperonin 60.1 inhibits leucocyte diapedesis and bronchial hyperresponsiveness in a murine model of allergic lung inflammation. METHODS: In the present study, we have investigated the effect of a shorter peptide sequence derived from Cpn 60.1, named IRL201104, on allergic lung inflammation induced by ovalbumin (OVA) in mice and by house dust mite (HDM) in guinea pigs, as well as investigating the action of IRL201104 on human cells in vitro. RESULTS: Pre-treatment of mice or guinea pigs with IRL201104 inhibits the infiltration of eosinophils to the lung, cytokine release, and in guinea pig skin, inhibits allergen-induced vascular permeability. The protective effect of intranasal IRL201104 against OVA-induced eosinophilia persisted for up to 20 days post-treatment. Moreover, OVA-sensitized mice treated intranasally with 20 ng/kg of IRL201104 show a significant increase in the expression of the anti-inflammatory molecule ubiquitin A20 and significant inhibition of the activation of NF-κB in lung tissue. Our results also show that A20 expression was significantly reduced in blood leucocytes and ASM obtained from patients with asthma compared to cells obtained from healthy subjects which were restored after incubation with IRL201104 in vitro, when added alone, or in combination with LPS or TNF-α in ASM. CONCLUSIONS: Our results suggest that a peptide derived from mycobacterial Cpn60.1 has a long-lasting anti-inflammatory and immunomodulatory activity which may help explain some of the protective effects of TB against allergic diseases.

Human Molecular Chaperone Hsp60 and Its Apical Domain Suppress Amyloid Fibril Formation of α-Synuclein.

Heat shock proteins play roles in assisting other proteins to fold correctly and in preventing the aggregation and accumulation of proteins in misfolded conformations. However, the process of aging significantly degrades this ability to maintain protein homeostasis. Consequently, proteins with incorrect conformations are prone to aggregate and accumulate in cells, and this aberrant aggregation of misfolded proteins may trigger various neurodegenerative diseases, such as Parkinson's disease. Here, we investigated the possibilities of suppressing α-synuclein aggregation by using a mutant form of human chaperonin Hsp60, and a derivative of the isolated apical domain of Hsp60 (Hsp60 AD(Cys)). In vitro measurements were used to detect the effects of chaperonin on amyloid fibril formation, and interactions between Hsp60 proteins and α-synuclein were probed by quartz crystal microbalance analysis. The ability of Hsp60 AD(Cys) to suppress α-synuclein intracellular aggregation and cytotoxicity was also demonstrated. We show that Hsp60 mutant and Hsp60 AD(Cys) both effectively suppress α-synuclein amyloid fibril formation, and also demonstrate for the first time the ability of Hsp60 AD(Cys) to function as a mini-chaperone inside cells. These results highlight the possibility of using Hsp60 AD as a method of prevention and treatment of neurodegenerative diseases.

Inhibition of Aß1-42 Fibrillation by Chaperonins: Human Hsp60 Is a Stronger Inhibitor than Its Bacterial Homologue GroEL.

Alzheimer's disease is a chronic neurodegenerative disease characterized by the accumulation of pathological aggregates of amyloid beta peptide. Many efforts have been focused on understanding peptide aggregation pathways and on identification of molecules able to inhibit aggregation in order to find an effective therapy. As a result, interest in neuroprotective proteins, such as molecular chaperones, has increased as their normal function is to assist in protein folding or to facilitate the disaggregation and/or clearance of abnormal aggregate proteins. Using biophysical techniques, we evaluated the effects of two chaperones, human Hsp60 and bacterial GroEL, on the fibrillogenesis of Aß1-42. Both chaperonins interfere with Aß1-42 aggregation, but the effect of Hsp60 is more significant and correlates with its more pronounced flexibility and stronger interaction with ANS, an indicator of hydrophobic regions. Dose-dependent ThT fluorescence kinetics and SAXS experiments reveal that Hsp60 does not change the nature of the molecular processes stochastically leading to the formation of seeds, but strongly delays them by recognition of hydrophobic sites of some peptide species crucial for triggering amyloid formation. Hsp60 reduces the initial chaotic heterogeneity of Aß1-42 sample at high concentration regimes. The understanding of chaperone action in counteracting pathological aggregation could be a starting point for potential new therapeutic strategies against neurodegenerative diseases.

Ca2+/Calmodulin-dependent kinase II delta B is essential for cardiomyocyte hypertrophy and complement gene expression after LPS and HSP60 stimulation in vitro.

Inflammation plays an important role in the development of cardiovascular diseases (CVDs), suggesting that the immune system is a target of therapeutic interventions used for treating CVDs. This study evaluated mechanisms underlying inflammatory response and cardiomyocyte hypertrophy associated with bacterial lipopolysaccharide (LPS)- or heat shock protein 60 (HSP60)-induced Toll-like receptor (TLR) stimulation and the effect of a small interfering RNA (siRNA) against Ca2+/calmodulin-dependent kinase II delta B (CaMKIIδB) on these outcomes. Our results showed that treatment with HSP60 or LPS (TLR agonists) induced cardiomyocyte hypertrophy and complement system C3 and factor B gene expression. In vitro silencing of CaMKIIδB prevented complement gene transcription and cardiomyocyte hypertrophy associated with TLR 2/4 activation but did not prevent the increase in interleukin-6 and tumor necrosis factor-alfa gene expression in primary cultured cardiomyocytes. Moreover, CaMKIIδB silencing attenuated nuclear factor-kappa B expression. These findings supported the hypothesis that CaMKIIδB acts as a link between inflammation and cardiac hypertrophy. Furthermore, the present study is the first to show that extracellular HSP60 activated complement gene expression through CaMKIIδB. Our results indicated that a stress stimulus induced by LPS or HSP60 treatment promoted cardiomyocyte hypertrophy and initiated an inflammatory response through the complement system. However, CaMKIIδB silencing prevented the cardiomyocyte hypertrophy independent of inflammatory response induced by LPS or HSP60 treatment.

Dual-targeting GroEL/ES chaperonin and protein tyrosine phosphatase B (PtpB) inhibitors: A polypharmacology strategy for treating Mycobacterium tuberculosis infections.

Current treatments for Mycobacterium tuberculosis infections require long and complicated regimens that can lead to patient non-compliance, increasing incidences of antibiotic-resistant strains, and lack of efficacy against latent stages of disease. Thus, new therapeutics are needed to improve tuberculosis standard of care. One strategy is to target protein homeostasis pathways by inhibiting molecular chaperones such as GroEL/ES (HSP60/10) chaperonin systems. M. tuberculosis has two GroEL homologs: GroEL1 is not essential but is important for cytokine-dependent granuloma formation, while GroEL2 is essential for survival and likely functions as the canonical housekeeping chaperonin for folding proteins. Another strategy is to target the protein tyrosine phosphatase B (PtpB) virulence factor that M. tuberculosis secretes into host cells to help evade immune responses. In the present study, we have identified a series of GroEL/ES inhibitors that inhibit M. tuberculosis growth in liquid culture and biochemical function of PtpB in vitro. With further optimization, such dual-targeting GroEL/ES and PtpB inhibitors could be effective against all stages of tuberculosis - actively replicating bacteria, bacteria evading host cell immune responses, and granuloma formation in latent disease - which would be a significant advance to augment current therapeutics that primarily target actively replicating bacteria.

GroEL, a novel vaccine candidate of piscine Streptococcus agalactiae identified by immunoproteome.

Streptococcus agalactiae is the major etiological agent of streptococcosis, which is responsible for huge economic losses in fishery, particularly in tilapia (Oreochromis niloticus) aquaculture. A research priority to control streptococcosis is to develop vaccines, so we sought to figure out the immunogenic proteins of S. agalactiae and screen the vaccine candidates for streptococcosis in the present study. Immunoproteomics, a technique involving two-dimensional gel electrophoresis (2-DE) followed by immunoblotting and mass spectrometry (MS), was employed to investigate the immunogenic proteins of S. agalactiae THN0901. Whole-cell soluble proteins were separated using 2-DE, and the immunogenic proteins were detected by western blotting using rabbit anti-S. agalactiae sera. A total of 17 immunoreactive spots on the soluble protein profile, corresponding to 15 different proteins, were identified by MALDI-TOF/TOF MS. Among the immunogenic proteins, GroEL attracted our attention as it was demonstrated to be immunogenic and protective against other streptococci. Nevertheless, to date, there have been no published reports on the immunogenicity and protective efficacy of GroEL against piscine S. agalactiae. Therefore, recombinant GroEL (rGroEL) was expressed in Escherichia coli BL21 (DE3) and purified by affinity chromatography. Immunization of tilapia with rGroEL resulted in an increase in antibody titers and conferred protection against S. agalactiae, with the relative percentage survival of 68.61 ±â€¯7.39%. The immunoproteome in the present study narrows the scope of vaccine candidates, and the evaluation of GroEL immunogenicity and protective efficacy shows that GroEL forms an ideal candidate molecule in subunit vaccine against S. agalactiae.

Ca2+/Calmodulin-dependent kinase II delta B is essential for cardiomyocyte hypertrophy and complement gene expression after LPS and HSP60 stimulation in vitro

Inflammation plays an important role in the development of cardiovascular diseases (CVDs), suggesting that the immune system is a target of therapeutic interventions used for treating CVDs. This study evaluated mechanisms underlying inflammatory response and cardiomyocyte hypertrophy associated with bacterial lipopolysaccharide (LPS)- or heat shock protein 60 (HSP60)-induced Toll-like receptor (TLR) stimulation and the effect of a small interfering RNA (siRNA) against Ca2+/calmodulin-dependent kinase II delta B (CaMKIIδB) on these outcomes. Our results showed that treatment with HSP60 or LPS (TLR agonists) induced cardiomyocyte hypertrophy and complement system C3 and factor B gene expression. In vitro silencing of CaMKIIδB prevented complement gene transcription and cardiomyocyte hypertrophy associated with TLR 2/4 activation but did not prevent the increase in interleukin-6 and tumor necrosis factor-alfa gene expression in primary cultured cardiomyocytes. Moreover, CaMKIIδB silencing attenuated nuclear factor-kappa B expression. These findings supported the hypothesis that CaMKIIδB acts as a link between inflammation and cardiac hypertrophy. Furthermore, the present study is the first to show that extracellular HSP60 activated complement gene expression through CaMKIIδB. Our results indicated that a stress stimulus induced by LPS or HSP60 treatment promoted cardiomyocyte hypertrophy and initiated an inflammatory response through the complement system. However, CaMKIIδB silencing prevented the cardiomyocyte hypertrophy independent of inflammatory response induced by LPS or HSP60 treatment.

New DiaP277 analogue shifts DCs to tolerogenic, and modulates NF-Kß1 to suppress autoreactive T lymphocytes in the type 1 diabetic mice.

Therapeutic efficacy of P277 against type 1 diabetes was extensively investigated and clinically evidenced. Clinical trials Phases I and II concluded promising results, while the data of P277 immunogenicity in Phase III trials represented weak responses that led to abolish medical use. But, a therapeutic performance of P277 cannot be forgotten. So, in order to exploit its therapeutic benefits and improve its immunogenicity, we developed a new analogue VP to optimize therapeutic efficacy and enhancing immunosuppressive modulations. However, new analogue was purified, and then used to immunize diabetic NOD mice to investigate antidiabetic effects through modulation of immunological status. So, DCs immune responses, relative TLRs, MyD88, and NF-Kß1 mRNA expression on DCs and splenocytes under VP effect were tested. Circulating and intracellular cytokines were also evaluated at treated and non-treated mice. Splenic T lymphocytes proliferation (Th1 and Treg cells) were also determined. Results revealed that VP significantly down regulates DCs maturation through TLR2, TLR4, and MyD88 pathways. It also shifts DCs to a tolerogenic polarization through NF-Kß1 pathway that mediates Th1 immunosuppression and enhances iTreg expanding in type1diabetes mice. Meanwhile, we noticed that VP significantly enhances iTreg CD25 + FoxP3+ proliferation. In conclusion, VP showed promising immune potential to modulate immune regulatory responses and shifts DCs to suppress autoreactive Th1 cells which ameliorated immunosuppressive potency in the type1 diabetic mice.

HSP60 critically regulates endogenous IL-1ß production in activated microglia by stimulating NLRP3 inflammasome pathway.

BACKGROUND: Interleukin-1ß (IL-1ß) is one of the most important cytokine secreted by activated microglia as it orchestrates the vicious cycle of inflammation by inducing the expression of various other pro-inflammatory cytokines along with its own production. Microglia-mediated IL-1ß production is a tightly regulated mechanism which involves the activation of nucleotide-binding oligomerization domain leucine-rich repeat and pyrin domain-containing 3 (NLRP3) inflammasome pathway. Our previous study suggests the critical role of heat shock protein 60 (HSP60) in IL-1ß-induced inflammation in microglia through TLR4-p38 MAPK axis. However, whether HSP60 regulates endogenous IL-1ß production is not known. Therefore, to probe the underlying mechanism, we elucidate the role of HSP60 in endogenous IL-1ß production. METHODS: We used in vitro (N9 murine microglial cells) and in vivo (BALB/c mouse) models for our study. HSP60 overexpression and knockdown experiment was done to elucidate the role of HSP60 in endogenous IL-1ß production by microglia. Western blotting and quantitative real-time PCR was performed using N9 cells and BALB/c mice brain, to analyze various proteins and transcript levels. Reactive oxygen species levels and mitochondrial membrane depolarization in N9 cells were analyzed by flow cytometry. We also performed caspase-1 activity assay and enzyme-linked immunosorbent assay to assess caspase-1 activity and IL-1ß production, respectively. RESULTS: HSP60 induces the phosphorylation and nuclear localization of NF-κB both in vitro and in vivo. It also induces perturbation in mitochondrial membrane potential and enhances reactive oxygen species (ROS) generation in microglia. HSP60 further activates NLRP3 inflammasome by elevating NLRP3 expression both at RNA and protein levels. Furthermore, HSP60 enhances caspase-1 activity and increases IL-1ß secretion by microglia. Knockdown of HSP60 reduces the IL-1ß-induced production of IL-1ß both in vitro and in vivo. Also, we have shown for the first time that knockdown of HSP60 leads to decreased IL-1ß production during Japanese encephalitis virus (JEV) infection, which eventually leads to decreased inflammation and increased survival of JEV-infected mice. CONCLUSION: HSP60 mediates microglial IL-1ß production by regulating NLRP3 inflammasome pathway and reduction of HSP60 leads to reduction of inflammation in JEV infection.