Mitochondrial chaperone HSP-60 regulates anti-bacterial immunity via p38 MAP kinase signaling.

Mitochondria play key roles in cellular immunity. How mitochondria contribute to organismal immunity remains poorly understood. Here, we show that HSP-60/HSPD1, a major mitochondrial chaperone, boosts anti-bacterial immunity through the up-regulation of p38 MAP kinase signaling. We first identify 16 evolutionarily conserved mitochondrial components that affect the immunity of Caenorhabditis elegans against pathogenic Pseudomonas aeruginosa (PA14). Among them, the mitochondrial chaperone HSP-60 is necessary and sufficient to increase resistance to PA14. We show that HSP-60 in the intestine and neurons is crucial for the resistance to PA14. We then find that p38 MAP kinase signaling, an evolutionarily conserved anti-bacterial immune pathway, is down-regulated by genetic inhibition of hsp-60, and up-regulated by increased expression of hsp-60 Overexpression of HSPD1, the mammalian ortholog of hsp-60, increases p38 MAP kinase activity in human cells, suggesting an evolutionarily conserved mechanism. Further, cytosol-localized HSP-60 physically binds and stabilizes SEK-1/MAP kinase kinase 3, which in turn up-regulates p38 MAP kinase and increases immunity. Our study suggests that mitochondrial chaperones protect host eukaryotes from pathogenic bacteria by up-regulating cytosolic p38 MAPK signaling.

GroEL protein of the Leptospira spp. interacts with host proteins and induces cytokines secretion on macrophages.

BACKGROUND: Leptospirosis is a zoonotic disease caused by infection with spirochetes from Leptospira genus. It has been classified into at least 17 pathogenic species, with more than 250 serologic variants. This wide distribution may be a result of leptospiral ability to colonize the renal tubules of mammalian hosts, including humans, wildlife, and many domesticated animals. Previous studies showed that the expression of proteins belonging to the microbial heat shock protein (HSP) family is upregulated during infection and also during various stress stimuli. Several proteins of this family are known to have important roles in the infectious processes in other bacteria, but the role of HSPs in Leptospira spp. is poorly understood. In this study, we have evaluated the capacity of the protein GroEL, a member of HSP family, of interacting with host proteins and of stimulating the production of cytokines by macrophages. RESULTS: The binding experiments demonstrated that the recombinant GroEL protein showed interaction with several host components in a dose-dependent manner. It was also observed that GroEL is a surface protein, and it is secreted extracellularly. Moreover, two cytokines (tumor necrosis factor-α and interleukin-6) were produced when macrophages cells were stimulated with this protein. CONCLUSIONS: Our findings showed that GroEL protein may contribute to the adhesion of leptospires to host tissues and stimulate the production of proinflammatory cytokines during infection. These features might indicate an important role of GroEL in the pathogen-host interaction in the leptospirosis.

Cross-protective efficacy of immuno-stimulatory recombinant Brugia malayi protein HSP60 against the Leishmania donovani in BALB/c mice.

Coinfection of Leishmania with bacteria, viruses, protozoans, and nematodes alter the immune system of the host, thereby influencing the disease outcomes. Here, we have determined the immunogenic property and protective efficacy of the cross-reactive molecule HSP60 of filarial parasite B. malayi against the L. donovani in BALB/c mice. Parasitological parameters results showed a significant decrease in the parasite burden (~59%; P < 0.001) and also a substantial increase in the delayed-type hypersensitivity (DTH) response (P < 0.001) in mice immunized with 10 µg of rBmHSP60. Protection against L. donovani in mice immunized with rBmHSP60 resulted from activation of the T cells, which is characterized by higher levels of nitric oxide (NO) production, enhanced cell proliferation, higher levels (expression and release) of IFN- γ, TNF- α, and IL-12, also, higher production of IgG and IgG2a antibodies. This strong Th1 immune response creates an inflammatory domain for L. donovani and protects the host from VL.

Development of IglC and GroEL recombinant vaccines for francisellosis in Nile tilapia, Oreochromis niloticus.

Warm-water piscine francisellosis is a granulomatous bacterial disease caused by Francisella orientalis (Fo). The disease has been detected in a wide range of fish species globally, causing mortalities as high as 90% and significant economic losses. Currently there are no commercially available vaccines and few treatment options exist. In the current study, two novel recombinant vaccines were prepared using diatom-expressed IglC or bacterial-expressed GroEL proteins. The vaccine antigens were emulsified with either nanoparticles or a commercially available oil-based adjuvant. Nile tilapia, Oreochromis niloticus, fingerlings were immunized intracoelomically with the recombinant IglC or GroEL vaccines, diatoms alone or phosphate buffer saline. Approximately 840-degree days post-vaccination, fish were challenged via immersion with 106 CFU/mL of wild-type Fo. Twenty-one days post challenge (dpc), the highest relative percent survival was recorded in the IglC-Montanide group (75%), compared to 53%, 50%, 22%, 19% and 16% in the IglC-nanoparticles, GroEL-Montanide, GroEL-nanoparticles, diatoms-Montanide and diatoms-nanoparticles groups, respectively. Protection correlated with significantly higher specific antibody responses in the IglC-Montanide group. Moreover, a significantly lower bacterial load was detected in spleen samples from the IglC-Montanide survivor tilapia compared to the other experimental groups. This is the first report of recombinant vaccines against piscine francisellosis in tilapia. The Fo vaccines described in our study may facilitate development of a safe, cost-effective and highly protective vaccine against francisellosis in farmed tilapia.

Evaluation of the protective effect of immunization spf DBA/2J mice with selected bacterial, recombinant Hsp60 antigens during Salmonella Enteritidis challenge.

Salmonella Enteritidis is one of the most common causes of food poisoning in humans. Many attempts have been made to develop an effective vaccine against S. Enteritidis for use in poultry, but experiments aimed at the complete elimination of this pathogen from poultry farms have not provided satisfactory results. The development of new generation vaccines against salmonellosis, such as subunit vaccines based on heat shock proteins (HSPs), is strongly justified. The high immunogenicity of Hsp60 isolated from Procaryota, including Salmonella, has been suggested by the presence of IgG anti-Hsp60 antibodies in mice immunized with these proteins. The aim of the studies was to evaluate the protective effects of immunization with recombinant Hsp60 from selected gram-negative bacteria (S. Enteritidis, Escherichia coli, Pasteurella multocida, Histophilus somni) in spf DBA/2 J mice experimentally infected with S. Enteritidis. The study demonstrated that double subcutaneous immunization of mice with a dose of 10 µg rHsp60 induced a specific immune response of IgG antibodies in tested animals. The median lethal dose (LD50) for the murine model spf DBA/2 J orally infected with S. Enteritidis was estimated at 6.84 × 105 cfu/animal. Mice immunized with rHsp60 from gastrointestinal pathogens (S. Enteritidis and E. coli) showed better survival after experimental infection with a 3 × LD50 dose from S. Enteritidis, compared to animals immunized with proteins obtained from respiratory pathogens (P. multocida and H. somni). However, the log-rank analysis did not show significant differences in the survival rates between rHsp60-immunized mice and controls. S. Enteritidis was not isolated any less frequently from internal organs and faeces of rHsp60-immunized mice than from controls. Nevertheless, the level of haptoglobin (but not IL-6) was increased in all mice in which the presence of the pathogen was observed. Bacterial Hsp60 is an interesting candidate for a subunit vaccine, but its use in livestock animals must be further investigated.

Comparison of protective efficacy between two DNA vaccines encoding DnaK and GroEL against fish nocardiosis.

Fish nocardiosis is a chronic granulomatous bacterial disease mainly caused by three pathogenic bacteria, including Nocardia seriolae, N. asteroids and N. salmonicida. Molecular chaperone DnaK and GroEL were identified to be the common antigens of the three pathogenic Nocardia species in our previous studies. To evaluate the immune protective effect of two DNA vaccines encoding DnaK or GroEL against fish nocardiosis, hybrid snakehead were vaccinated and the immune responses induced by these two vaccines were comparatively analyzed. The results suggested it needed at least 7 d to transport DnaK or GroEL gene from injected muscle to head kidney, spleen and liver and stimulate host's immune system for later protection after immunization by DNA vaccines. Additionally, non-specific immunity parameters (serum lysozyme (LYZ), peroxidase (POD), acid phosphatase (ACP), alkaline phosphatase (AKP) and superoxide dismutase (SOD) activities), specific antibody (IgM) production and immune-related genes (MHCIα, MHCIIα, CD4, CD8α, IL-1ß and TNFα) were used to evaluate the immune responses induced in vaccinated hybrid snakehead. It proved that all the above-mentioned immune activities were significantly enhanced after immunization with these two DNA vaccines. The protective efficacy of pcDNA-DnaK and pcDNA-GroEL DNA vaccines, in terms of relative percentage survival (RPS), were 53.01% and 80.71% respectively. It demonstrated that these two DNA vaccines could increase the survival rate of hybrid snakehead against fish nocardiosis, albeit with variations in immunoprotective effects. Taken together, these results indicated that both pcDNA-DnaK and pcDNA-GroEL DNA vaccines could boost the innate, humoral and cellular immune response in hybrid snakehead and show highly protective efficacy against fish nocardiosis, suggesting that DnaK and GroEL were promising vaccine candidates. These findings will promote the development of DNA vaccines against fish nocardiosis in aquaculture.

Long-Term Efficacy and Safety of Immunomodulatory Therapy for Atherosclerosis.

BACKGROUND AND AIMS: The long-term effect of immune tolerance has not been explored so far in atherosclerosis. In the present study, we assessed the effect of mucosal tolerance to a multi antigenic construct expressing three peptides from ApoB, HSP60, and outer membrane protein from Chlamydia pneumonia (AHC) for 30 weeks at every 6-week interval to understand the kinetics of immune modulation in disease progression. The safety profile of the molecule was also evaluated in mice. METHODS: Apobtm2SgyLdlrtm1Her/J mice (5-6 weeks) were orally dosed with multi antigenic construct (AHC) molecule on alternate days, followed by high-fat diet feeding to initiate atherosclerosis. RESULTS: Treated animals showed an efficient reduction in plaque growth and lipid accumulation at 6 weeks (49%, p < 0.01) and 12 weeks (42.3%, p < 0.01) which decreased to 29% (p = 0.0001) at 18 weeks and at later time points. Macrophage accumulation was significantly lower at all time points (53% at 12 weeks to 27% at 30 weeks). Regulatory T cells increased in the spleen following treatment until 12 weeks (week 0 (2.57 ± 0.18 vs. 6.36 ± 0.03, p = 0.02), week 6 (4.52 ± 0.2 vs. 8.87 ± 0.32, p = 0.02), and week 12 (8.74 ± 0.37 vs. 15.4 ± 0.27, p = 0.02)) but showed a decline later. A similar trend was observed with tolerogenic dendritic cells. We observed an increase in antibody levels to low-density lipoprotein and oxidized LDL at later stages. AHC molecule was found to be safe in acute and repeated dose toxicity studies. CONCLUSIONS: Our results suggest that immune tolerance to AHC protein by oral administration is able to provide efficient atheroprotection up to 18 weeks and moderately at later stages. Apart from immune regulatory cells, protective antibodies may also have a role in controlling atherosclerosis.

Novel mutant P277 peptide VP to ameliorate atherogenic side-effects and to preserve anti-diabetic effects in NOD mice.

P277 is a 24 amino-acids peptide, residues 437-460 of human heat shock protein 60 (HSP60). P277 or sequence repeated 6 × P277 was previously found showing potency preventive and therapeutic anti-diabetes functions in NOD mice, but aroused atherosclerosis due to the induction of anti-HSP65 autoantibodies as reported. To determine the intrinsic B epitope sequence, we screened P277 with pepscan method and then proved by detection of sera IgG from peptide fragments vaccinated mouse and rabbits. Results indicated HSP60 443-448 (ALLRCI) is potential intrinsic B epitope sequence of P277. We modified P277 by deleting the former three amino acids of ALLRCI (VP) or replacing these six with alanine (AP). The detection of serum lipid parameter in NOD mice and aorta endothelial damage levels in high-cholesterol diets fed rabbits demonstrated that VP induced higher anti-diabetes efficacy and caused less arteriosclerosis-liked diseases separately. With less TLR2/4 activation of dendritic cells and macrophages, VP treatment reduced Th1 related P277 specific pro-inflammatory cytokines production and increased regulatory immune responses both in vivo and in vitro. These results indicated that optimized VP peptide might serve as a promising candidate for mouse type 1 diabetes therapy.

Effects of oral and subcutaneous administration of HSP60 on myeloid-derived suppressor cells and atherosclerosis in ApoE-/- mice.

HSP60 has been proved to be closely related to atherosclerosis due to its antigenicity. To determine this antigenicity effect, the ApoE-/- mice were fed with western-type diet and HSP60 was administrated orally or subcutaneously (SC) for potential vaccine against atherosclerosis. Here, we observed the ApoE-/- mice with oral HSP60 administration group showed a significant reduction in plaque size at the aortic root; accompanied by increased MSDCs (CD11b+Gr1+) in peripheral blood and spleen which was mostly composed of M-MDSCs (CD11b+LY6G-LY6Chigh), and increased plasma IL-10 and splenic Foxp3, Arg1, iNOS mRNA as well as decreased plasma IFN-γ and splenic T-bet mRNA compared to control group. Surprisingly, ApoE-/- mice with subcutaneous HSP60 administration group showed contrary results and their MDSCs were mostly composed of G-MDSCs (CD11b+LY6G+LY6Clow). As expected, both PBS-oral and PBS-SC groups showed no significant effects on both the immune response and atherosclerotic plaque formation. In contrast, subcutaneous administration of HSP60 causes the opposite response. Thus, we propose the proper method for administering HSP60 as a new immunologic agent for prevention and treatment of atherosclerosis.

SjHSP60 induces CD4+ CD25+ Foxp3+ Tregs via TLR4-Mal-drived production of TGF-ß in macrophages.

CD4+ CD25+ Foxp3+ regulatory T cells (Tregs) play a pivotal role in limiting immunopathological damage to host organs after schistosome infection. Transforming growth factor-ß (TGF-ß) is an essential factor for the periphery conversion of CD4+ CD25- T cells into CD4+ CD25+ Foxp3+ Tregs by inducing the key transcription factor Foxp3. Antigen presenting cells (APCs), which highly express TGF-ß, are involved in parasite antigen-induced Treg conversion in peripheral. However, the mechanisms underlying high TGF-ß induction in APCs by parasite antigens remain to be clarified during schistosome infection. Here, we demonstrated that Schistosoma japonicum stress protein, heat shock protein 60 (SjHSP60), promoted TGF-ß production in macrophages (Mφ). Furthermore, we showed that activation of TLR4-Mal (MyD88 adaptor-like protein) signaling by SjHSP60 is necessary for induction of TGF-ß expression in Mφ, which subsequently promoted Treg induction. Our results not only demonstrate a novel mechanism of TGF-ß production in Mφ for inducing Tregs in mice with schistosomiasis, but also allude to the possibility of targeting parasite stress protein for potential therapeutics.

The evaluation of immunogenic impact of selected bacterial, recombinant Hsp60 antigens in DBA/2J mice.

Heat Shock Proteins (HSP) are highly conserved proteins that are widely spread throughout all organisms. They function in the cytoplasm as chaperones; however, they could be expressed on the cell surface. It has been shown that Hsp60 obtained from gram-negative bacteria are able to stimulate cells of the acquired and innate immune system. The aim of this study was the evaluation of the immunogenic properties of recombinant Hsp60 proteins derived from four common pathogenic bacteria: Escherichia coli, Histophilus somni, Pasteurella multocida and Salmonella Enteritidis. The analysis of the humoral immune response in DBA/2J mice hyperimmunized with selected rHsp60 revealed high levels of IgG rHsp60-antibody with the predominance of the IgG1 subclass, in the reaction with both homologous and heterologous antigens. The presence of IgG2a and IgG2b was also observed; however, no antibodies of subclass IgG3 were detected. The comparison of plasma IgG antibody reactivity of mice immunized with two different doses of rHsp60 (10/20 µg) showed that the lower dose was sufficient to induce a strong humoral response. The reactivity of the IgG rHsp60-antibody with whole bacterial cells showed a significantly higher reaction with H. somni compared with other pathogens. It was demonstrated that the addition of all rHsp60 with polymyxin B to the culture medium stimulated splenocytes isolated from hyperimmunized mice to release IL-1ß and IL-6. As a strong stimulator of the immune system, bacterial-origin Hsp60 seems to be an interesting potential component of subunit vaccines aimed at the development of protection for animals during infections caused by gram-negative bacteria.

Evaluation of immune responses induced by polymeric OMP25-BLS Brucella antigen.

Brucellosis is one the serious infectious diseases caused deleterious health and economic losses. Vaccination with subunit vaccines is the efficient alternative way than live attenuated vaccines against infectious diseases. Herein a new chimeric OMP25-BLS antigen emulsified in Chitosan Nanoparticles was designed and its immune responses were compared with control groups. Also, the role of heat shock protein 60 kDa in combination with OMP25-BLS antigen was assessed. Structural and antigenic features of chimeric antigen were predicted using bioinformatics tools. Moreover, the humoral and cellular immune responses were measured by ELISA in seven different groups. Observations showed rOMP25-BLS structure was highly stable and antigenic. Cytokines analysis showed rOMP25 and rOMP25-BLS + rHSP60 induced higher titer of INF-γ than rHSP60 and rOMP25-BLS. There was no statistically significant difference between positive control group and rOMP25-BLS + rHSP60 in inducing TNF-α (p < .05). Additionally, the highest titer of IL-4 was dedicated to rOMP25 among other immunized treatments, while there were no significant differences between positive control group and other immunized groups with recombinant proteins (p < .05). In addition, rOMP25-BLS and rHSP60 induced higher titer of total antibody compared to other groups. Also, rHSP60 could improve IgG2a to IgG1 ratio when it used in combination with chimeric antigen. Moreover, the lymphocyte proliferation index was higher in chimeric rOMP25-BLS + HSP60 antigen. In conclusion, while rOMP25-BLS chimeric antigen unable to induce efficient cellular response than individual injection of rOMP25, its injection in combination with rHSP60 could improve cellular immunity.

Helicobacter pylori-derived heat shock protein 60 increases the induction of regulatory T-cells associated with persistent infection.

Local Treg responses are involved in Helicobacter pylori-related inflammation and clinical outcomes after infection, and H. pylori-derived HSP60 (HpHSP60) is an important virulence factor associated with gastric carcinogenesis. This study to investigate the role of HpHSP60 in immunosuppression, particularly with regard to whether it could induce the production of Treg cells. For this purpose, human peripheral blood mononuclear cells (PBMCs) were treated with or without HpHSP60 in the presence of an anti-CD3 mAb to determine the effect of HpHSP60 on cell proliferation. In this report, HpHSP60 decreased the expression of CDK4 to significantly arrest the proliferation of mitogen-stimulated T-cells, which correlated with the induction of Treg cells. Moreover, monocytic cells were essential for the induction of HpHSP60-induced Treg cells via the secretion of IL-10 and TGF-ß after treatment with HpHSP60. Blockage of HpHSP60 with specific monoclonal antibodies significantly reduced the colonization of H. pylori and the expression of Treg cells in vivo. Overall, our results suggest that HpHSP60 could act on macrophages to trigger the expression of IL-10 and TGF-ß, thereby leading to an increase in Treg cells and inhibition of T-cell proliferation.

HSP60 and HSP90ß from blunt snout bream, Megalobrama amblycephala: Molecular cloning, characterization, and comparative response to intermittent thermal stress and Aeromonas hydrophila infection.

Heat shock proteins (HSPs) play critical roles in the process of anti-stress and immunity and are implicated in autoimmune diseases. In order to understand the comparative stress responses of HSP60 and HSP90ß under intermittent thermal stress and Aeromonas hydrophila infection, we cloned their full-length cDNAs from Megalobrama amblycephala liver, predicted their secondary and tertiary structure, and examined their tissue-specific expression patterns. The full length of HSP60 and HSP90ß cDNAs indicated that they included all signature sequences of corresponding protein families. They showed high homology to their counterparts in other species, and were consistent with the known classification of fishes based on phylogenetic analysis. HSP60 showed the highest expression in head-kidney, brain, and gill, while HSP90ß presented higher in hindgut, liver, and brain. Significant mRNA expression differences were determined between HSP60 and HSP90ß in tissues of bladder, liver, heart, and gill. During thermal stress and recovery phase, the highest expression of them were observed at the first recovery for 2 d and 1 d, respectively. The expression between them were extremely significant difference during the first recovery and second stress period. After A. hydrophila infection, their expressions were extremely significantly upregulated. The significant upregulation and rapid response indicated that they were sensitive to thermal stress and bacterial challenge. This study demonstrated that HSP60 and HSP90ß might participate in innate immune and environmental responses of M. amblycephala. It indicated that they could be used as biomarkers to test the stress caused by local aquaculture environment.

Oral Tolerization with Mycobacterial Heat Shock Protein 65 Reduces Chronic Experimental Atherosclerosis in Aged Mice.

BACKGROUND: Atherosclerosis is a chronic inflammatory disease of the artery wall where both innate and adaptive immunity play important roles. Modulation of the immune response against the stress protein antigen, heat shock protein (HSP) 60, by administration of mycobacterial HSP65 (mbHSP65) orally and/or nasally shows promising therapeutic results in young animals in the sense of less severe experimental atherosclerosis; however, the case of aged animals with already established atherosclerosis has so far never been investigated. OBJECTIVE: To investigate if mbHSP65 immunization would further accelerate atherosclerotic progression in aged ApoE-/- mice (18 months old) with already long-established atherosclerosis and if these mice could be orally tolerized against mbHSP65. METHODS: Aged wild-type (WT) and ApoE-/- mice (65 weeks) were immunized and/or orally treated with mbHSP65 and then either kept on normal chow or changed to high-cholesterol diet (HCD). Atherosclerosis was assessed by en face analysis and the number of CD4+CD25+FoxP3+ T regulatory cells (Tregs) was assessed by flow cytometry in lymph node and spleen cells. Total cholesterol and triglyceride levels were determined. Soluble mammalian HSP60 and anti-mouse HSP60 (mHSP60) and anti-mbHSP65 antibodies were detected by enzyme-linked immunosorbent assay. RESULTS: As expected, aged WT mice had only minor lesions in the aorta, which did not change under HCD for 14 weeks. Aged ApoE-/- mice already had large complicated plaques, which increased in size under HCD. mbHSP65 immunization led to a significant aggravation of atherosclerosis in both WT and ApoE-/- mice irrespective of the nature of their diet. This increase was accompanied by increased titers of both anti-mHSP60 and anti-mbHSP65 antibodies in the circulation. The increased plaque formation could be significantly diminished with oral mbHSP65 tolerization. An increased number of Tregs and lower or unchanged levels of cholesterol and triglycerides were associated with the reduced size of aortal lesions. CONCLUSION: Oral tolerization against mbHSP65 could be used both to prevent and to treat chronic atherosclerosis in aged individuals.

Immune Responses Induced by HSP60 DNA Vaccine against Toxoplasma gondii Infection in Kunming Mice.

Toxoplasma gondii can infect all the vertebrates including human, and leads to serious toxoplasmosis and considerable veterinary problems. T. gondii heat shock protein 60 (HSP60) is associated with the activation of antigen presenting cells by inducing initial immune responses and releasing inflammatory cytokines. It might be a potential DNA vaccine candidate for this parasite. A pVAX-HSP60 DNA vaccine was constructed and immune responses was evaluated in Kunming mice in this study. Our data indicated that the innate and adaptive immune responses was elicited by successive immunizations with pVAX-HSP60 DNA, showing apparent increases of CD3e+CD4+ and CD3e+CD8a+ T cells in spleen tissues of the HSP60 DNA-immunized mice (24.70±1.23% and 10.90±0.89%, P<0.05) and higher levels of specific antibodies in sera. Furthermore, the survival period of the immunized mice (10.53±4.78 day) were significantly prolonged during the acute T. gondii infection. Decrease of brain cysts was significant in the experimental group during the chronic infection (P<0.01). Taken together, TgHSP60 DNA can be as a vaccine candidate to prevent the acute and chronic T. gondii infections.

Autoimmunity to HSP60 during diet induced obesity in mice.

Adaptive immunity has been implicated in adipose tissue inflammation, obesity and its adverse metabolic consequences. No obesity-related autoantigen has yet been identified, although heat shock protein 60 (HSP60) has been implicated in other autoimmune diseases. We investigated whether feeding a high-fat diet to C57BL/6J mice would cause autoimmunity to HSP60 and whether immunomodulation with peptides from HSP60 would reverse the resulting obesity or metabolic dysfunction. Obese mice had higher circulating levels of HSP60 associated with increased T-lymphocyte proliferation responses and the emergence of circulating IgG1 and IgG2c antibody levels against HSP60. Treatment with escalating doses of a mixture of three proven immunomodulatory HSP60 peptides did not reduce weight but completely reversed the increase in VLDL/LDL levels and partially reversed the glucose intolerance in obese mice. Obese mice mount an autoimmune response to HSP60, which partly underlies the resulting metabolic disturbances.

Behçet Disease serum is immunoreactive to neurofilament medium which share common epitopes to bacterial HSP-65, a putative trigger.

Autoimmune and dysimmune inflammatory mechanisms on a genetically susceptible background are implicated in the etiology of Behçet's Disease (BD). Heat-shock protein-65 (HSP-65) derived from Streptococcus sanguinis was proposed as a triggering factor based on its homology with human HSP-60. However, none of the autoantigens identified so far in sera from BD share common epitopes with bacterial HSP-65 or has a high prevalence. Here, we report that sera from BD patients are immunoreactive against filamentous neuronal processes in the mouse brain, retina and scrotal skin in great majority of patients. By using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and peptide mass fingerprinting, Western blotting and peptide blocking experiments, we have identified neurofilament medium (NF-M) as the probable antigen for the serologic response observed. Clustal Omega analyses detected significant structural homology between the human NF-M and bacterial HSP-65 corresponding to amino acids 111-126, 213-232 and 304-363 of mycobacterial HSP-65, which were previously identified to induce proliferation of lymphocytes obtained from BD patients. We also found that sera immunoreactive against NF-M cross-reacted with bacterial HSP-65. These findings suggest that NF-M may be involved in autoimmunity in BD due to its molecular mimicry with bacterial HSP-65.

Evaluation of the relationship between salivary concentration of anti-heat shock protein immunoglobulin and clinical manifestations of Behçet's disease.

OBJECTIVE: This study investigated the relationship between the concentration of anti-heat shock protein (HSP) 60 antibody in resting saliva and the severity of Behçet's disease (BD). METHOD: Sixty-five patients diagnosed with BD at Tokyo Medical and Dental University Hospital were enrolled in this study. Based on clinical severity scores, patients were categorized as having mild, moderate, or severe BD. Periodontal status was evaluated with the Community Periodontal Index (CPI), and anti-HSP60 antibody concentrations in resting saliva were measured with an enzyme-linked immunosorbent assay. RESULTS: The mean antibody concentration in patients in the moderate group was significantly higher than concentrations in the mild and severe groups. No significant difference was found between the mild and severe groups. Gingival inflammation, identified with the CPI, was associated with a higher antibody concentration. The antibody concentration in patients who had stomatitis for more than 2 weeks was significantly higher than in those with stomatitis for less than 2 weeks. The antibody concentration in patients who had taken colchicine was significantly lower than that in subjects who had not. CONCLUSION: These results suggest that the concentration of anti-HSP60 antibody in resting saliva may be effective as a non-invasive indicator for the diagnosis (screening) and prognostication of BD.

A Humanized Monoclonal Antibody against Heat Shock Protein 60 Suppresses Murine Arthritis and Colitis and Skews the Cytokine Balance toward an Anti-Inflammatory Response.

We have previously shown that naturally occurring as well as acquired Abs against the Mycobacterium tuberculosis heat shock protein (HSP)65 protect against the induction of murine autoimmune inflammatory arthritis. In the present work, we have studied the anti-inflammatory effect of prozumab, a humanized anti-HSP mAb in murine inflammatory arthritis and colitis, and its effects on cytokine secretion. Prozumab was shown to bind to HSP60, the highly conserved mammalian homolog of the bacterial protein, and it was found to be effective in protecting and suppressing autoimmune arthritis in the models of adjuvant arthritis and collagen-induced arthritis in rats and mice, respectively, as well as in acute hapten-mediated colitis and chronic, spontaneous colitis models. Mechanistically, prozumab induces IL-10 secretion from naive human PBMCs and suppresses the secretion of IFN-γ and IL-6 from anti-CD3-activated human PBMCs. These findings make prozumab a promising potential drug for treating human rheumatoid arthritis and inflammatory bowel disease, as well as a wide range of autoimmune inflammatory diseases.