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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

HMGB1 present in necrotic extract induces HIV-1 replication in U1 cells. (a) Western blot of cell supernatants (necrotic extracts) obtained after freeze-thawing cycles of peripheral blood mononuclear cells (PBMC) (30 × 106 cells/mL) from healthy donors: Molecular weight marker (I); supernatants after immune depletion of HMGB1 with nonspecific rabbit polyclonal antibody (II); depletion with anti-HMGB1 antibody −5 μg (III) and 10 μg (IV); necrotic extract loaded 20 μL (V), 10 μL (VI), and 5 μL (VII); 100 ng (VIII) and 75 ng (IX) of recombinant HMGB1; cell debris (X). Numbers to the left depict positions of molecular mass markers (in kDa). (b) Levels of HIV p24 protein in cell culture supernatants after 72 h incubation of U1 cells with necrotic extract (HMGB1 concentration 1 μg/mL): HMGB1-depleted necrotic extract and mock cells. PMA served as a positive control (20 nM). The levels of viral replication were approximately 2-fold higher after stimulation by necrotic extract compared to the mock cells (P = 0.002). Results from three independent experiments in duplicates are presented.
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fig1: HMGB1 present in necrotic extract induces HIV-1 replication in U1 cells. (a) Western blot of cell supernatants (necrotic extracts) obtained after freeze-thawing cycles of peripheral blood mononuclear cells (PBMC) (30 × 106 cells/mL) from healthy donors: Molecular weight marker (I); supernatants after immune depletion of HMGB1 with nonspecific rabbit polyclonal antibody (II); depletion with anti-HMGB1 antibody −5 μg (III) and 10 μg (IV); necrotic extract loaded 20 μL (V), 10 μL (VI), and 5 μL (VII); 100 ng (VIII) and 75 ng (IX) of recombinant HMGB1; cell debris (X). Numbers to the left depict positions of molecular mass markers (in kDa). (b) Levels of HIV p24 protein in cell culture supernatants after 72 h incubation of U1 cells with necrotic extract (HMGB1 concentration 1 μg/mL): HMGB1-depleted necrotic extract and mock cells. PMA served as a positive control (20 nM). The levels of viral replication were approximately 2-fold higher after stimulation by necrotic extract compared to the mock cells (P = 0.002). Results from three independent experiments in duplicates are presented.

Mentions: Necrotic extract or HMGB1was mixed with the TLR-ligands, LPS, CpG-ODN, IL-1β, and flagellin in PBS in different concentrations and incubated for 16 hours at 4°C. Concentrations are given in Figures 1–3. The suboptimal stimulatory concentrations (capable to trigger HIV replication from U1 cells) of necrotic extract as well as LPS, flagellin, CpG-ODN, and Il-1β were estimated in a series of experiments (data not included). Complexes were also mixed and denatured by heating at 95°C for five minutes to verify the stimulatory effect of complex formation on U1 cells.


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)

HMGB1 present in necrotic extract induces HIV-1 replication in U1 cells. (a) Western blot of cell supernatants (necrotic extracts) obtained after freeze-thawing cycles of peripheral blood mononuclear cells (PBMC) (30 × 106 cells/mL) from healthy donors: Molecular weight marker (I); supernatants after immune depletion of HMGB1 with nonspecific rabbit polyclonal antibody (II); depletion with anti-HMGB1 antibody −5 μg (III) and 10 μg (IV); necrotic extract loaded 20 μL (V), 10 μL (VI), and 5 μL (VII); 100 ng (VIII) and 75 ng (IX) of recombinant HMGB1; cell debris (X). Numbers to the left depict positions of molecular mass markers (in kDa). (b) Levels of HIV p24 protein in cell culture supernatants after 72 h incubation of U1 cells with necrotic extract (HMGB1 concentration 1 μg/mL): HMGB1-depleted necrotic extract and mock cells. PMA served as a positive control (20 nM). The levels of viral replication were approximately 2-fold higher after stimulation by necrotic extract compared to the mock cells (P = 0.002). Results from three independent experiments in duplicates are presented.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375154&req=5

fig1: HMGB1 present in necrotic extract induces HIV-1 replication in U1 cells. (a) Western blot of cell supernatants (necrotic extracts) obtained after freeze-thawing cycles of peripheral blood mononuclear cells (PBMC) (30 × 106 cells/mL) from healthy donors: Molecular weight marker (I); supernatants after immune depletion of HMGB1 with nonspecific rabbit polyclonal antibody (II); depletion with anti-HMGB1 antibody −5 μg (III) and 10 μg (IV); necrotic extract loaded 20 μL (V), 10 μL (VI), and 5 μL (VII); 100 ng (VIII) and 75 ng (IX) of recombinant HMGB1; cell debris (X). Numbers to the left depict positions of molecular mass markers (in kDa). (b) Levels of HIV p24 protein in cell culture supernatants after 72 h incubation of U1 cells with necrotic extract (HMGB1 concentration 1 μg/mL): HMGB1-depleted necrotic extract and mock cells. PMA served as a positive control (20 nM). The levels of viral replication were approximately 2-fold higher after stimulation by necrotic extract compared to the mock cells (P = 0.002). Results from three independent experiments in duplicates are presented.
Mentions: Necrotic extract or HMGB1was mixed with the TLR-ligands, LPS, CpG-ODN, IL-1β, and flagellin in PBS in different concentrations and incubated for 16 hours at 4°C. Concentrations are given in Figures 1–3. The suboptimal stimulatory concentrations (capable to trigger HIV replication from U1 cells) of necrotic extract as well as LPS, flagellin, CpG-ODN, and Il-1β were estimated in a series of experiments (data not included). Complexes were also mixed and denatured by heating at 95°C for five minutes to verify the stimulatory effect of complex formation on U1 cells.

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