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Impact of HMGB1/TLR Ligand Complexes on HIV-1 Replication: Possible Role for Flagellin during HIV-1 Infection.

Nowak P, Abdurahman S, Lindkvist A, Troseid M, Sönnerborg A - Int J Microbiol (2012)

Bottom Line: Moreover, the stimulatory effect of necrotic extract was inhibited by depletion of HMGB1.Elevated levels of anti-flagellin antibodies were present in plasma from HIV-1-infected patients and significantly decreased during 2 years of antiretroviral therapy.Conclusions.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, Institution of Medicine, Karolinska University Hospital and Karolinska Institutet, 14186 Stockholm, Sweden.

ABSTRACT
Objective. We hypothesized that HMGB1 in complex with bacterial components, such as flagellin, CpG-ODN, and LPS, promotes HIV-1 replication. Furthermore, we studied the levels of antiflagellin antibodies during HIV-1-infection. Methods. Chronically HIV-1-infected U1 cells were stimulated with necrotic extract/recombinant HMGB1 in complex with TLR ligands or alone. HIV-1 replication was estimated by p24 antigen in culture supernatants 48-72 hours after stimulation. The presence of systemic anti-flagellin IgG was determined in 51 HIV-1-infected patients and 19 controls by immunoblotting or in-house ELISA. Results. Flagellin, LPS, and CpG-ODN induced stronger HIV-1 replication when incubated together with necrotic extract or recombinant HMGB1 than activation by any of the compounds alone. Moreover, the stimulatory effect of necrotic extract was inhibited by depletion of HMGB1. Elevated levels of anti-flagellin antibodies were present in plasma from HIV-1-infected patients and significantly decreased during 2 years of antiretroviral therapy. Conclusions. Our findings implicate a possible role of HGMB1-bacterial complexes, as a consequence of microbial translocation and cell necrosis, for immune activation in HIV-1 pathogenesis. We propose that flagellin is an important microbial product, that modulates viral replication and induces adaptive immune responses in vivo.

No MeSH data available.


Related in: MedlinePlus

HIV-1-infected patients exhibit elevated levels of flagellin-specific antibodies. (a) Approximately 1.8 μg of recombinant flagellin was twofold serially diluted (4 series) and resolved on 10–20% SDS-PAGE gel, transferred onto a nitrocellulose membrane, and detected with immunoblot assay. Serum from HIV-1-infected or control subjects diluted 1 : 1000 was used as a primary antibody. Each panel is a separate immunoblot of the same recombinant flagellin detected with serum from HIV-1 patients (HIV 1, HIV 2, HIV 3, HIV 4) or control subjects (CS 1, CS 2). To confirm equal sample loading and protein transfer, immunoblotted membranes from HIV 1 and CS 2 were stripped off and reprobed with monoclonal antibody directed against flagellin (blots in the lower panel). The experiments were performed using sera from ten HIV-1-infected patients and four control subjects. These presented data are representative for all immunoblots. The position of recombinant flagellin protein is indicated with an arrow to the right. Numbers to the left depict positions of molecular mass markers (in kDa). (b) Recombinant flagellin (column 1) and lysates of flagellated or aflagellate E. coli (columns 3 and 4) were subjected to SDS-PAGE and immunoblotted using serum from HIV infected patient (column 1 and 3, diluted 1 : 1000) as primary antibody or polyclonal anti-flagellin antibody (columns 2 and 4, diluted 1 : 1000). The membrane in column 1 was stripped off and reprobed with polyclonal anti-flagellin antibody (column 2). The numbers above the figures indicate the amount of sample loaded in each well (in μL). MM, magic marker (Invitrogen); F+: flagellated; F−: aflagellate whole bacterial lysate; Pkl: polyclonal; Ab: antibody. Numbers to the left depict positions of molecular mass markers (in kDa).
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fig4: HIV-1-infected patients exhibit elevated levels of flagellin-specific antibodies. (a) Approximately 1.8 μg of recombinant flagellin was twofold serially diluted (4 series) and resolved on 10–20% SDS-PAGE gel, transferred onto a nitrocellulose membrane, and detected with immunoblot assay. Serum from HIV-1-infected or control subjects diluted 1 : 1000 was used as a primary antibody. Each panel is a separate immunoblot of the same recombinant flagellin detected with serum from HIV-1 patients (HIV 1, HIV 2, HIV 3, HIV 4) or control subjects (CS 1, CS 2). To confirm equal sample loading and protein transfer, immunoblotted membranes from HIV 1 and CS 2 were stripped off and reprobed with monoclonal antibody directed against flagellin (blots in the lower panel). The experiments were performed using sera from ten HIV-1-infected patients and four control subjects. These presented data are representative for all immunoblots. The position of recombinant flagellin protein is indicated with an arrow to the right. Numbers to the left depict positions of molecular mass markers (in kDa). (b) Recombinant flagellin (column 1) and lysates of flagellated or aflagellate E. coli (columns 3 and 4) were subjected to SDS-PAGE and immunoblotted using serum from HIV infected patient (column 1 and 3, diluted 1 : 1000) as primary antibody or polyclonal anti-flagellin antibody (columns 2 and 4, diluted 1 : 1000). The membrane in column 1 was stripped off and reprobed with polyclonal anti-flagellin antibody (column 2). The numbers above the figures indicate the amount of sample loaded in each well (in μL). MM, magic marker (Invitrogen); F+: flagellated; F−: aflagellate whole bacterial lysate; Pkl: polyclonal; Ab: antibody. Numbers to the left depict positions of molecular mass markers (in kDa).

Mentions: Encouraged by the in vitro data we aimed to evaluate whether flagellin is a potentially important antigen in vivo during HIV-1 infection. Therefore, serum samples from HIV-1-infected patients and control subjects were used to measure the level of flagellin-specific antibodies by Western blot analysis. When diluted 1 : 1000, all of the sera samples from the HIV-1-infected patients analyzed exhibited easily detectable bands that recognized the first two dilutions of flagellin derived from S. typhimurium (Figure 4(a), upper panels). A relative increase in flagellin-specific IgG in HIV patients was observed if serum samples were diluted 1 : 500. In contrast, in only one control subject (CS#2) flagellin was detected faintly at the highest dilution (Figure 4(a), lower panels). Although semiquantitative analysis of detected bands was not performed, the levels of flagellin-specific IgG observed were in all cases strikingly elevated in HIV-1-infected patients. Similar pattern of anti-flagellin IgG was observed when plasma instead of sera was used. In order to address the specificity of the flagellin IgG, we subjected the bacterial lysates from flagellated and aflagellate E. coli to protein separation on the SDS-PAGE gel. The Western blotting with HIV-1 serum (as a primary antibody) showed similar pattern as when the polyclonal anti-flagellin antibody was used confirming that the specificity of the antibodies was not limited to the recombinant protein (Figure 4(b)).


Impact of HMGB1/TLR Ligand Complexes on HIV-1 Replication: Possible Role for Flagellin during HIV-1 Infection.

Nowak P, Abdurahman S, Lindkvist A, Troseid M, Sönnerborg A - Int J Microbiol (2012)

HIV-1-infected patients exhibit elevated levels of flagellin-specific antibodies. (a) Approximately 1.8 μg of recombinant flagellin was twofold serially diluted (4 series) and resolved on 10–20% SDS-PAGE gel, transferred onto a nitrocellulose membrane, and detected with immunoblot assay. Serum from HIV-1-infected or control subjects diluted 1 : 1000 was used as a primary antibody. Each panel is a separate immunoblot of the same recombinant flagellin detected with serum from HIV-1 patients (HIV 1, HIV 2, HIV 3, HIV 4) or control subjects (CS 1, CS 2). To confirm equal sample loading and protein transfer, immunoblotted membranes from HIV 1 and CS 2 were stripped off and reprobed with monoclonal antibody directed against flagellin (blots in the lower panel). The experiments were performed using sera from ten HIV-1-infected patients and four control subjects. These presented data are representative for all immunoblots. The position of recombinant flagellin protein is indicated with an arrow to the right. Numbers to the left depict positions of molecular mass markers (in kDa). (b) Recombinant flagellin (column 1) and lysates of flagellated or aflagellate E. coli (columns 3 and 4) were subjected to SDS-PAGE and immunoblotted using serum from HIV infected patient (column 1 and 3, diluted 1 : 1000) as primary antibody or polyclonal anti-flagellin antibody (columns 2 and 4, diluted 1 : 1000). The membrane in column 1 was stripped off and reprobed with polyclonal anti-flagellin antibody (column 2). The numbers above the figures indicate the amount of sample loaded in each well (in μL). MM, magic marker (Invitrogen); F+: flagellated; F−: aflagellate whole bacterial lysate; Pkl: polyclonal; Ab: antibody. Numbers to the left depict positions of molecular mass markers (in kDa).
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig4: HIV-1-infected patients exhibit elevated levels of flagellin-specific antibodies. (a) Approximately 1.8 μg of recombinant flagellin was twofold serially diluted (4 series) and resolved on 10–20% SDS-PAGE gel, transferred onto a nitrocellulose membrane, and detected with immunoblot assay. Serum from HIV-1-infected or control subjects diluted 1 : 1000 was used as a primary antibody. Each panel is a separate immunoblot of the same recombinant flagellin detected with serum from HIV-1 patients (HIV 1, HIV 2, HIV 3, HIV 4) or control subjects (CS 1, CS 2). To confirm equal sample loading and protein transfer, immunoblotted membranes from HIV 1 and CS 2 were stripped off and reprobed with monoclonal antibody directed against flagellin (blots in the lower panel). The experiments were performed using sera from ten HIV-1-infected patients and four control subjects. These presented data are representative for all immunoblots. The position of recombinant flagellin protein is indicated with an arrow to the right. Numbers to the left depict positions of molecular mass markers (in kDa). (b) Recombinant flagellin (column 1) and lysates of flagellated or aflagellate E. coli (columns 3 and 4) were subjected to SDS-PAGE and immunoblotted using serum from HIV infected patient (column 1 and 3, diluted 1 : 1000) as primary antibody or polyclonal anti-flagellin antibody (columns 2 and 4, diluted 1 : 1000). The membrane in column 1 was stripped off and reprobed with polyclonal anti-flagellin antibody (column 2). The numbers above the figures indicate the amount of sample loaded in each well (in μL). MM, magic marker (Invitrogen); F+: flagellated; F−: aflagellate whole bacterial lysate; Pkl: polyclonal; Ab: antibody. Numbers to the left depict positions of molecular mass markers (in kDa).
Mentions: Encouraged by the in vitro data we aimed to evaluate whether flagellin is a potentially important antigen in vivo during HIV-1 infection. Therefore, serum samples from HIV-1-infected patients and control subjects were used to measure the level of flagellin-specific antibodies by Western blot analysis. When diluted 1 : 1000, all of the sera samples from the HIV-1-infected patients analyzed exhibited easily detectable bands that recognized the first two dilutions of flagellin derived from S. typhimurium (Figure 4(a), upper panels). A relative increase in flagellin-specific IgG in HIV patients was observed if serum samples were diluted 1 : 500. In contrast, in only one control subject (CS#2) flagellin was detected faintly at the highest dilution (Figure 4(a), lower panels). Although semiquantitative analysis of detected bands was not performed, the levels of flagellin-specific IgG observed were in all cases strikingly elevated in HIV-1-infected patients. Similar pattern of anti-flagellin IgG was observed when plasma instead of sera was used. In order to address the specificity of the flagellin IgG, we subjected the bacterial lysates from flagellated and aflagellate E. coli to protein separation on the SDS-PAGE gel. The Western blotting with HIV-1 serum (as a primary antibody) showed similar pattern as when the polyclonal anti-flagellin antibody was used confirming that the specificity of the antibodies was not limited to the recombinant protein (Figure 4(b)).

Bottom Line: Moreover, the stimulatory effect of necrotic extract was inhibited by depletion of HMGB1.Elevated levels of anti-flagellin antibodies were present in plasma from HIV-1-infected patients and significantly decreased during 2 years of antiretroviral therapy.Conclusions.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, Institution of Medicine, Karolinska University Hospital and Karolinska Institutet, 14186 Stockholm, Sweden.

ABSTRACT
Objective. We hypothesized that HMGB1 in complex with bacterial components, such as flagellin, CpG-ODN, and LPS, promotes HIV-1 replication. Furthermore, we studied the levels of antiflagellin antibodies during HIV-1-infection. Methods. Chronically HIV-1-infected U1 cells were stimulated with necrotic extract/recombinant HMGB1 in complex with TLR ligands or alone. HIV-1 replication was estimated by p24 antigen in culture supernatants 48-72 hours after stimulation. The presence of systemic anti-flagellin IgG was determined in 51 HIV-1-infected patients and 19 controls by immunoblotting or in-house ELISA. Results. Flagellin, LPS, and CpG-ODN induced stronger HIV-1 replication when incubated together with necrotic extract or recombinant HMGB1 than activation by any of the compounds alone. Moreover, the stimulatory effect of necrotic extract was inhibited by depletion of HMGB1. Elevated levels of anti-flagellin antibodies were present in plasma from HIV-1-infected patients and significantly decreased during 2 years of antiretroviral therapy. Conclusions. Our findings implicate a possible role of HGMB1-bacterial complexes, as a consequence of microbial translocation and cell necrosis, for immune activation in HIV-1 pathogenesis. We propose that flagellin is an important microbial product, that modulates viral replication and induces adaptive immune responses in vivo.

No MeSH data available.


Related in: MedlinePlus