Emerging Role of HMGB1 in the Pathogenesis of Schistosomiasis Liver Fibrosis

In chronic schistosomiasis, liver fibrosis is linked to portal hypertension, which is a condition associated with high mortality and morbidity. High mobility group box 1 (HMGB1) was originally described as a nuclear protein that functions as a structural co-factor in transcriptional regulation. However, HMGB1 can also be secreted into the extracellular milieu under appropriate signal stimulation. Extracellular HMGB1 acts as a multifunctional cytokine that contributes to infection, injury, inflammation, and immune responses by binding to specific cell-surface receptors. HMGB1 is involved in fibrotic diseases. From a clinical perspective, HMGB1 inhibition may represent a promising therapeutic approach for treating tissue fibrosis. In this study, we demonstrate elevated levels of HMGB1 in the sera in experimental mice or in patients with schistosomiasis. Using immunohistochemistry, we demonstrated that HMGB1 trafficking in the hepatocytes of mice suffering from acute schistosomiasis was inhibited by Glycyrrhizin, a well-known HMGB1 direct inhibitor, as well as by DIC, a novel and potential anti-HMGB1 compound. HMGB1 inhibition led to significant downregulation of IL-6, IL4, IL-5, IL-13, IL-17A, which are involved in the exacerbation of the immune response and liver fibrogenesis. Importantly, infected mice that were treated with DIC or GZR to inhibit HMGB1 pro-inflammatory activity showed a significant increase in survival and a reduction of over 50% in the area of liver fibrosis. Taken together, our findings indicate that HMGB1 is a key mediator of schistosomotic granuloma formation and liver fibrosis and may represent an outstanding target for the treatment of schistosomiasis

CK2 Phosphorylation of Schistosoma mansoni HMGB1 Protein Regulates Its Cellular Traffic and Secretion but Not Its DNA Transactions

parasite resides in mesenteric veins where fecundated female worms lay hundred of eggs daily. Some of the egg antigens are trapped in the liver and induce a vigorous granulomatous response. High Mobility Group Box 1 (HMGB1), a nuclear factor, can also be secreted and act as a cytokine. Schistosome HMGB1 (SmHMGB1) is secreted by the eggs and stimulate the production of key cytokines involved in the pathology of schistosomiasis. Thus, understanding the mechanism of SmHMGB1 release becomes mandatory. Here, we addressed the question of how the nuclear SmHMGB1 can reach the extracellular space. eggs of infected animals and that SmHMGB1 that were localized in the periovular schistosomotic granuloma were phosphorylated.We showed that secretion of SmHMGB1 is regulated by phosphorylation. Moreover, our results suggest that egg-secreted SmHMGB1 may represent a new egg antigen. Therefore, the identification of drugs that specifically target phosphorylation of SmHMGB1 might block its secretion and interfere with the pathogenesis of schistosomiasis

Binding isotherm of HMGB1 to fluorescently labeled linear DNA.

<p>A) FAM-labeled 20-bp dsDNA at a 50 nM concentration was titrated with increasing HMGB1 (black circles) or HMGB1ΔC (red circles) concentrations, and the fluorescence polarization (P) of the fluorescent probe was measured after a 15-min incubation at 25 °C. (a) The binding stoichiometry of HMGB1 or HMGB1ΔC to FAM-labeled dsDNA was calculated. Increasing protein concentrations were added to a solution containing a mixture of 2 μM unlabeled dsDNA and 50 nM FAM-labeled dsDNA; thus, the [Protein]/[DNA] ratio varied from 0 to 15. The polarization values were measured by exciting the probe at 490 nm and reading the FAM-emission fluorescence at 520 nm after a 15-min incubation at 25 °C.</p

Binding of HMGB1 protein to linear dsDNA monitored by fluorescence spectroscopy.

<p>A) Interaction between HMGB1 (black circles) or HMGB1ΔC (red circles) with 20-bp DNA was analyzed by the quenching of the Trp emission fluorescence. Both proteins were kept at 2 μM, and the DNA concentration was varied from 0 to 2 μM. Trp emission spectra were collected after a 15-min incubation at 25 °C. B) Interaction between HMGB1 or HMGB1ΔC with 20-bp DNA, as analyzed by bis-ANS displacement. The protein and bis-ANS concentrations were 0.5 μM and 10 μM, respectively, whereas the DNA concentration varied from 0 to 1.2 μM. The emission spectra of bis-ANS were acquired after a 15-min incubation time at 25 °C. Normalized spectrum areas were calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079572#pone-0079572-g004" target="_blank">Figure 4</a>. Control experiments were performed similarly but in the absence of protein.</p

Thermal denaturation of the HMGB1 protein.

<p>A) The Trp fluorescence emission spectra of HMGB1 (black circles) and HMGB1ΔC (red circles) at each temperature were acquired and converted into CM and α according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079572#eqn1" target="_blank">Equations 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079572#eqn2" target="_blank">2</a>, respectively. The curves were adjusted by sigmoidal fitting, and the T_m was obtained directly from the fitting. B) The CD signal at 222 nm for the HMGB1 and HMGB1ΔC spectra at each temperature was converted into the loss of secondary structure content. The buffer contained 10 mM Tris.HCl at pH 7.2, 50 mM NaCl, 0.5 mM DTT, 0.1 mM EDTA and 5% of glycerol.</p

Influence of low pH on the HMGB1 structure.

<p>A) HMGB1 (black circles) and HMGB1ΔC (red circles) at 5 μM concentration were incubated at different pH values (in citrate/citric acid buffer), and the CM variation (ΔCM) was calculated. Because of the small change in ΔCM, even in a very acidic pH, both proteins were also incubated with Gdn.HCl at pH 2.3 and 5.5 M (black triangle for HMGB1 and red triangle for HMGB1ΔC). B) The secondary structure content of 5 μM HMGB1 at neutral pH (black straight lines) and pH 2.3 (black medium-dashed lines) and of HMGB1ΔC at neutral pH (red straight lines) and pH 2.3 (red medium-dashed lines) was monitored by CD at 20 °C. Spectra were converted to molar ellipticity, as described in the Material & Methods section. C) The interaction of bis-ANS and the proteins was assessed by exciting 10 μM probe in a solution containing 5 μM HMGB1 (black circles) or HMGB1ΔC (red circles) at different pH values after a 1-h incubation at 25 °C. For comparison, HMGB1 and HMGB1ΔC were incubated at pH 2.3 in the presence of 5.5 M Gdn.HCl (closed triangles). Normalized spectrum areas were obtained by dividing the spectrum area value of each pH point by the area value at neutral pH.</p

Denaturation of HMGB1 and HMGB1ΔC as a function of increasing Gdn.HCl concentration.

<p>A) The CM of HMGB1 (black circles) and HMGB1ΔC (red circles) at 5 μM was obtained for each [Gdn.HCl] using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079572#eqn1" target="_blank">Equation 1</a>, as described in the Material and Methods Section. B) Trp fluorescence spectra were obtained and converted to degree of denaturation (α) according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079572#eqn2" target="_blank">Equation 2</a>. The resistance to unfolding can be analyzed by G_1/2, which reflects the concentration necessary to unfold 50% of the protein population and is detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079572#pone-0079572-t001" target="_blank">Table 1</a>. </p

Schematic representation of HMGB1-mediated DNA bending.

<p>A 20-bp oligonucleotide labeled with FAM (green star, F) and TAMRA (orange star, T) fluorophores in the presence of HMGB1 or HMGB1∆C undergoes bending at different angles, measured by the distance between these two fluorophores. Bending angle values were obtained using the two-kinked model. The difference observed in size and color intensity of the fluorophores molecules is proportional to their emission quenching. The acidic tail of HMGB1 and its interaction with other part of the molecule are represented by green and dashed lines, respectively.</p

Structural organization of the human HMB1 protein.

<p>A) Schematic representation of the human HMGB1 structure showing Box A, Box B and the acidic tail motifs. Both boxes are rich in positive amino acid residues (+), whereas the acidic tail is exclusively composed of acidic amino acid residues (-) (residues 186-215). The removal of the acidic tail generated a truncated construct (HMGB1ΔC). B) Two micrograms of HMGB1 and HMGB1ΔC were separately applied onto a 15% SDS-PAGE. In a third lane, 5 μL the pre-stained molecular weight standards (Bio-Rad) were applied. The gel was stained by Coomassie Blue G-250 dye and. C) Western blotting with anti-human HMGB1 to confirm the recombinant protein identity. The 6His-Tag was not removed.</p

Analysis of the secondary and tertiary contents of HMGB1 and HMGB1ΔC by CD and Trp fluorescence spectroscopies.

<p>A) CD spectra of 5 μM HMGB1 (black lines) and HMGB1ΔC (red lines) at 25 °C and neutral pH. Each spectrum was converted to molar ellipticity for proper comparison. B) Normalized Trp fluorescence spectra of 5 μM HMGB1 and HMGB1ΔC in the native state (straight lines) and denatured state with 5.5 M Gdn.HCl (medium-dashed lines). All experiments were performed at 25 °C, and the buffer composition was 10 mM Tris.HCl at pH 7.2, 50 mM NaCl, 0.5 mM DTT, 0.1 mM EDTA and 5% glycerol.</p