A peptide derived from phage-display limits psoriasis-like lesions in mice.

BACKGROUND: Psoriasis is a pro-inflammatory disease with unknown etiology, that is characterized by skin inflammation and keratinocytes hyperproliferation. Specific inhibition of inflammation has shown positive effects avoiding the progression of the psoriatic lesions in different animal models of the disease, turning this strategy as a remarkable therapeutic alternative. OBJECTIVE: To screen the effectiveness of a novel IFN-α/ß signalling inhibitor in the development reduction of skin lesions in IMQ and TPA mice models of psoriasis. METHODS: We used a Phage-peptide library for the screening of a peptide with inhibitory effects on the development of psoriasis-like lesions in mice. To evaluate the in vivo effect of the phage-peptides (Phpep3D) and the derived peptide (Pep3D), we administered Phpep3D or Pep3D intradermally in mice with imiquimod (IMQ)-induced psoriasis and 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced psoriasis. We scored the lesions, and we determined the number of neutrophils and the production of some pro-inflammatory cytokines in the lesions. RESULTS: In this work, we describe how the Ph3pepD and Pep3D reduced skin thickness, redness, and acanthosis despite the presence of the psoriasis inducers, IMQ or TPA. We also found that Pep3D reduced the number of GR1+ infiltrated cells and decreased the production of IL-17A and TNFα in the psoriatic skin of mice. In-silico, docking analysis showed that Pep3D may interact with the interferon-alpha receptor, but further analyses should be performed to uncover the mechanism of action of this peptide. CONCLUSION: Our results suggest that Pep3D could be used as a new treatment for psoriasis.

Expression of CRAMP via PGN-TLR-2 and of alpha-defensin-3 via CpG-ODN-TLR-9 in corneal fibroblasts.

AIMS: To search for the induction of the expression of antimicrobial peptides in corneal fibroblasts treated with bacterial components. METHODS: RT-PCR was performed to search for mRNAs expression of antimicrobial peptides and toll-like receptors (TLRs) in murine primary cultures of corneal fibroblast (PCCF) treated with lipopolysaccharide (LPS) from Escherichia coli, peptidoglycan from Staphylococcus aureus, and cytosine-phosphorous-guanine oligonucleotide (CpG-ODN). Cellular activation was blocked with anti-TRL antibodies. RESULTS: LPS did not induce expression of antimicrobial peptide in corneal fibroblasts. Cathelin related antimicrobial peptide (CRAMP) and alpha-defensin 3 were overexpressed in a time and dose dependent manner in corneal fibroblasts treated with peptidoglycan and with CpG-ODN, respectively. CRAMP expression was blocked when PCCF were treated with anti-TLR-2 antibodies. alpha-Defensin 3 was not expressed in NIH murine corneal fibroblasts (which do not express the TLR-9 molecule) treated with CpG-ODN. CONCLUSION: Results suggest that corneal fibroblasts, which are the second cellular barrier of the cornea, can play an important part in the innate immunity of the eye via TLR stimulation.

TLRs and NODs mRNA expression pattern in healthy mouse eye.

AIMS: To look for TLR and NOD mRNA expression in the healthy eye and in other immune privileged and non-immune privileged mouse organs. METHODS: Semiquantitative RT-PCR was performed to look for TLR1-9 and NOD1 and NOD2 mRNA expressions in the whole eye, in the anterior (AP) and posterior (PP) portions of the eye, in corneal fibroblasts (CF) and in ovary, brain, testis, heart, lung, and spleen. RESULTS: All the TLR mRNAs were expressed in the whole eye of Balb/c mice. NIH and C57BL/6 did not express TLR9 and TLR8, respectively. NIH expressed higher levels of TLR1, 2, 3, and 6 than the other strains. C57BL/6 expressed the lowest levels of all TLRs. TLR9, 5, and 4 were the less expressed in all strains. All TLRs were expressed in Balb/c PP and TLR1 was not expressed in AP. In NIH and Balb/c CF the majority of TLRs were overexpressed with LPS. In testis, expression of most TLRs was absent. Non-immune privileged organs expressed most of the TLRs. All the organs expressed NOD1 and NOD2. In PP NOD2 was not expressed. CONCLUSION: TLRs and NODs are expressed in the eye, and could have an important role in the innate immunity.

High levels of IgG class antibodies to recombinant HSP60 kDa of Yersinia enterocolitica in sera of patients with uveitis.

AIMS: To determine the levels of IgG class antibodies to recombinant heat shock protein 60 kDa of Yersinia enterocolitica (rHSP60Ye), Klebsiella pneumoniae (rHSP60Kp), Escherichia coli (rHSP60Ec), Shigella flexneri (rHSP60Sf), and Streptococcus pyogenes (rHSP60Sp) in the serum of patients with HLA-B27 associated acute anterior uveitis (HLA-B27 associated AAU), idiopathic acute anterior uveitis (idiopathic AAU), pars planitis, Vogt-Koyanagi-Harada (VKH), and healthy subjects. METHODS: The genes that code for HSP60Ye, HSP60Kp, HSP60Ec, HSP60Sf, and HSP60Sp were cloned by PCR from genomic DNA. The rHSPs were purified by affinity using a Ni-NTA resin. The serum levels of IgG class antibodies to rHSP60s were determined by ELISA in patients with uveitis (n = 42) and in healthy subjects (n = 25). RESULTS: The majority of patients with uveitis had higher levels of IgG class antibodies to rHSP60Ye compared with levels of healthy subjects (p = 0.01), although these differences were only observed in the HLA-B27 associated AAU (p = 0.005) and in pars planitis patients (p = 0.001). The levels of IgG antibodies to the rHSP60Kp, rHSP60Sf, rHSP60Ec, and rHSP60Sp were similar in patients with uveitis and in healthy subjects (p>0.05). CONCLUSION: The results suggest that HSP60Ye could be involved in the aetiology of HLA-B27 associated AAU and pars planitis.

IgG antibodies to enterobacteria 60 kDa heat shock proteins in the sera of HLA-B27 positive ankylosing spondylitis patients.

OBJECTIVE: To study the association of HLA-B27 and the IgG response to the 60 kDa HSPs of Klebsiella pneumoniae, Yersinia enterocolitica, Shigella flexneri, Escherichia coli and Salmonella typhi. METHODS: IgG against the 60 kDa HSPs of enterobacteria was determined by ELISA in the sera from 49 HLA-B27+ ankylosing spondylitis (AS) patients; 41 HLA-B27+ healthy relatives of AS patients and 101 HLA-B27-unrelated healthy individuals. RESULTS: HLA-B27+ patients and healthy individuals, showed significantly higher IgG antibody levels to the Klebsiella, Yersinia and Salmonella HSPs than HLA-B27- healthy controls. B27+ patients had a significantly higher response to E. coli HSP than the two other groups. IgG response anti-Shigella HSP was similar in the three groups. CONCLUSIONS: There is a relationship between HLA-B27 and the response to HSPs 60 from Klebsiella, Yersinia, Escherichia and Salmonella, that may be important in the initiation of AS.

Cellular immune response to Klebsiella pneumoniae antigens in patients with HLA-B27+ ankylosing spondylitis.

OBJECTIVE: To study the reactivity of peripheral blood mononuclear cells (PBMC) of patients with ankylosing spondylitis (AS) and rheumatoid arthritis (RA) and healthy controls to Klebsiella pneumoniae antigens and to the GroEL-like proteins from K. pneumoniae (HSP60Kp) and Mycobacterium leprae recombinant heat shock protein 65 (rHSP65Ml). METHODS: PBMC of 13 patients with AS and 9 with RA and 10 controls were stimulated in vitro by heat shock induced K. pneumoniae antigens in a cell blot assay, by insolubilized HSP60Kp, by cytosolic proteins (CP) from K. pneumoniae cultivated at 37 degrees C or 45 degrees C, by soluble HSP60Kp, or by rHSP65Ml. RESULTS: In the cell blot assay 7/13 AS and 3/9 RA patients responded to fraction 4, which contains mainly HSP60Kp, and no controls responded (AS vs. controls: p = 0.007). The response to the insolubilized HSP60Kp was positive in 6/13 AS patients but negative in RA patients and controls (p = 0.004). The response to CP45 degrees C was positive in 7/13 AS, in 2/9 RA, and no controls (AS vs controls: p<0.015). Response to the soluble HSP60Kp was found in 7/13 AS and 5/9 RA patients, but no controls (AS vs. controls: p = 0.0075). Response to rHSP65Ml was positive in 3/13 AS, 7/9 RA patients, and 1/10 controls (AS vs RA: p = 0.027; RA vs. controls: p = 0.005; AS vs. controls: nonsignificant). CONCLUSION: In PBMC of the majority of patients with AS and in some with RA, but not in healthy controls, there are cells that proliferate in the presence of HSP60 of K. pneumoniae.

Recognition of B cells epitopes of the Klebsiella pneumoniae GroEL-like protein by HLA-B27 positive subjects.

The presence of antibodies against antigens of K. pneumoniae in HLA-B27 positive patients with ankylosing spondylitis (AS), has been well documented. We have previously reported that sera from HLA-B27 positive subjects react with the K. pneumoniae GroEL-like protein (HSP60Kp) and have higher titers than HLA-B27 negative individuals. We cloned the gene that codes for this protein, determined hydrophilic regions by computer analysis of the predicted amino acid sequence and found that residues 389-397, 360-368 and 282-290, were possible B cell epitopes. To test this prediction, and to determine if the HLA-B27 positive and negative AS patients recognize the same or different epitopes, we truncated the hsp60Kp gene, from the 3; terminal nucleotide, to obtain fragments having or not the predicted epitopes. Four polypeptides of 40, 37, 30 and 18 kDa were obtained and analysed, by ELISA and inhibition of ELISA, for their reactivity with IgG antibodies from three high responders HLA-B27 positive AS patients and three HLA-B27 negative subjects who recognized the rHSP60Kp. Sera from both HLA-B27 positive and negative subjects reacted equally well with rHSP60Kp or with the 40 and 37 kDa peptides, which do not have residues 389-397 and 360-368, respectively, but reactivity was lost with the 30 kDa peptide, which also lacks residues 282-290. Contrary to what we expected, antibodies from HLA-B27 negative and positive individuals recognized the same epitope of the HSP60Kp. Our results indicate that the important epitope for B cells could be the 282-290 region and that the contribution of the two other predicted regions is minimal. We also conclude that the differences in response to the HSP60Kp in HLA-B27 positive AS patients and HLA-B27 negative individuals is not qualitative, but only quantitative.

Antibody response to Klebsiella pneumoniae 60 kDa protein in familial and sporadic ankylosing spondylitis: role of HLA-B27 and characterization as a GroEL-like protein.

OBJECTIVE: To study the antibody response of HLA-B27+ patients with ankylosing spondylitis (AS) and their first degree relatives to the 60 kDa protein of Klebsiella pneumoniae and to characterize this protein. METHODS: Sera from 84 individuals were analyzed by ELISA to determine the titer of antibodies against the 60 kDa protein of K. pneumoniae. Subjects were divided into 3 categories: Group 1: 44 HLA-B27+ AS related individuals (35 patients, 9 healthy controls); Group 2: 28 healthy B27- AS related individuals; and Group 3: 12 healthy B27- non-AS related subjects. The 60 kDa protein of K. pneumoniae was induced at 45 degrees C and purified by electroelution from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was characterized as a GroEL-like heat shock protein (HSP). The recognition of GroEL-like protein was confirmed by immunoblot of 2 dimension electrophoresis. The response to GroEL-like protein from other bacteria and the response to lipopolysaccharide (LPS) was also analyzed by immunoblot. RESULTS: HLA-B27+ individuals (Group 1), independent of their disease status, showed a significant higher response to the 60 kDa protein of K. pneumoniae than HLA-B27- subjects from Groups 2 and 3 (p < 0.0001). This protein was characterized as a HSP of the GroEL family and designated HSP60Kp. The GroEL of other enterobacteria as well as that of Mycobacterium leprae were recognized by HLA-B27+ individuals by immunoblot, whereas HLA-B27- individuals did not. LPS was not recognized by HLA-B27 positive or negative subjects. CONCLUSION: These findings suggest a relationship between HLA-B27 and the response to a GroEL-like protein that could have implications in AS.

Cloning and sequencing of the gene that codes for the Klebsiella pneumoniae GroEL-like protein associated with ankylosing spondylitis.

Ankylosing spondylitis (AS) is a chronic inflammatory disease of the sacroiliac joints and vertebral column of unknown aetiology, but strongly related to the presence of the HLA-B27 antigen. The participation of bacterial infections as triggering factors have also been suggested. We have associated the 60 kDa heat shock protein of Klebsiella pneumoniae (HSP60Kp) with AS since we have previously demonstrated that most of the patients have IgG antibodies and active T cells that recognize preferentially this protein, but we have not yet identified the epitopes involved in the recognition. In order to know the amino acid sequence of HSP60Kp, and to be able to analyse in the future the relevant epitopes; we amplified by PCR and cloned the gene coding for this protein into the SmaI site of pUC19. The nucleotide sequence of the gene was obtained by the Sanger method using both manual and automatic techniques. Amino acid sequence of the HSP60Kp was deduced by translating the nucleotide sequence of the gene. The antigenic analysis of this sequence was compared to the antigenic analysis of the reported sequences of Escherichia coli GroEL and Yersinia enterocolitica HSP60. Using a software to predict HLA class I motifs, the nonapeptide (KRGIDKAVL) residues 117-125 of HSP60Kp showed a much higher affinity for HLA-B27 than the similar nonapeptide of E. coli GroEL and Y. enterocolitica HSP60. The only difference between the three peptides was in position nine. This finding could explain the association of AS only with the HSP60 of Klebsiella pneumoniae. On the other hand, hydrophilicity analysis, which indicates B cell epitopes, showed three similar strongly antigenic regions in the three proteins.

Antibody response to nitrogenase-positive and -negative Klebsiella pneumoniae strains in juvenile-onset ankylosing spondylitis patients and their first degree relatives: lack of differential recognition of the bacterial nitrogenase.

In the search for the pathogenic consequences of the molecular mimicry between the Klebsiella pneumoniae nitrogenase and the HLA-B27 antigen, sera from individuals belonging to 16 kindreds with juvenile-onset ankylosing spondylitis cases, were analyzed for antibodies against nitrogenase-positive and -negative K. pneumoniae whole bacterial extracts. An initial screening for nitrogenase producing K. pneumoniae strains was performed in 31 clinical isolates. The best nitrogenase producing strain was selected as well as a non producing one for immunoblot analysis using sera from 82 subjects, 55 HLA-B27 positive, of which 26 had some clinical manifestations. Even though electrophoretic patterns were different in both strains, there was no distinctive differential recognition of the 30-40 kDa proteins where the nitrogenase subcomponent which shares the sequence QTDRED with the HLA-B27 molecule is located. On the other hand, strong recognition of a protein of 60 kDa (p60Kp) was detected in 75% of HLA-B27 positive tested subjects independently of their clinical status. Studies on the nature of this protein and its participation in the pathogenesis of ankylosing spondylitis are now in progress.