Macrophage migration inhibitory factor is critical for dengue NS1-induced endothelial glycocalyx degradation and hyperpermeability

<div><p>Vascular leakage is one of the salient characteristics of severe dengue. Nonstructural protein 1 (NS1) of dengue virus (DENV) can stimulate endothelial cells to secrete endothelial hyperpermeability factor, macrophage migration inhibitory factor (MIF), and the glycocalyx degradation factor heparanase 1 (HPA-1). However, it is unclear whether MIF is directly involved in NS1-induced glycocalyx degradation. In this study, we observed that among NS1, MIF and glycocalyx degradation-related molecules, the HPA-1, metalloproteinase 9 (MMP-9) and syndecan 1 (CD138) serum levels were all increased in dengue patients, and only NS1 and MIF showed a positive correlation with the CD138 level in severe patients. To further characterize and clarify the relationship between MIF and CD138, we used recombinant NS1 to stimulate human cells <i>in vitro</i> and challenge mice <i>in vivo</i>. Our tabulated results suggested that NS1 stimulation could induce human endothelial cells to secrete HPA-1 and immune cells to secrete MMP-9, resulting in endothelial glycocalyx degradation and hyperpermeability. Moreover, HPA-1, MMP-9, and CD138 secretion after NS1 stimulation was blocked by MIF inhibitors or antibodies both <i>in vitro</i> and in mice. Taken together, these results suggest that MIF directly engages in dengue NS1-induced glycocalyx degradation and that targeting MIF may represent a possible therapeutic approach for preventing dengue-induced vascular leakage.</p></div

DENV NS1-induced HPA-1 secretion leading to glycocalyx degradation and hyperpermeability is MIF dependent.

<p><b>(A)</b> HUVECs were treated with PBS or 20 μg/ml NS1 recombinant proteins for the indicated time, followed by collection of the supernatants for CD138 detection by ELISA. (n = 4) <b>(B)</b> HUVECs were treated with PBS, 20 μg/ml NS1, or 20 μg/ml NS1 mixed with 10 μg/ml anti-NS1 antibodies (2E8 or DN5C6). After 24 h of incubation, the culture medium was collected, and the concentration of CD138 was determined by ELISA. (n = 3) <b>(C)</b> HUVECs seeded as monolayers in upper Transwell chambers were treated with PBS, 20 μg/ml NS1 or 20 μg/ml NS1 mixed with DMSO or the indicated concentration of OGT 2115. After 24 h, endothelial permeability was determined by a Transwell permeability assay, as described in the Materials and Methods section. (n = 3) <b>(D)</b> HUVECs were treated with PBS or 20 μg/ml NS1 with or without 5 μM OGT 2115. After 24 h, the cell culture medium was collected, and the CD138 concentration was measured by ELISA. (n = 5) <b>(E)</b> HUVECs were treated with PBS or 20 μg/ml NS1 with or without 100 μM p425, 50 μM ISO-1, or 10 μg/ml anti-MIF antibodies, as indicated. After 24 h, the cell culture medium was collected, and the HPA-1 concentration was measured by ELISA. (n = 3) <b>(F)</b> HUVECs were treated with PBS or 20 μg/ml NS1 with or without p425, ISO-1 or anti-MIF antibodies. After 24 h, the cell culture medium was collected, and the CD138 concentration was measured by ELISA. (n = 5) <b>(G)</b> HUVECs were treated with PBS or NS1 (20 μg/ml) with or without anti-MIF polyclonal antibodies (10 μg/ml) for 24 h. The distribution of HPA-1 (red) and CD138 (green) was assessed by staining with specific antibodies. Sialic acid expression on HUVECs monolayers was assessed by staining with WGA-FITC (green). *P<0.05, **P<0.01, ***P<0.001; ns, not significant; unpaired t-test (panel A), Kruskal-Wallis ANOVA (panel B, C, D, E and F).</p

The correlations of serum NS1, MIF, HPA-1, MMP-9 and CD138 levels in severe dengue patients.

<p>The correlations of the concentrations of <b>(A)</b> NS1, <b>(B)</b> MIF, <b>(C)</b> HPA-1 and <b>(D)</b> MMP-9 and CD138 in the same group of severe dengue patients were plotted. Linear regressions were analyzed using nonparametric correlation test (panel A, B, C and D).</p

NS1 induces MMP-9 secretion from human leukocytes but not HUVECs.

<p><b>(A)</b> HUVECs were treated with PBS or NS1 for the indicated times; then, the supernatants were collected for MMP-9 detection by ELISA. (n = 3) <b>(B)</b> Isolated human leukocytes (WBCs) and <b>(D)</b> PMA-activated THP-1 cells were treated with PBS or NS1, and the culture medium was collected at the indicated times. The concentration of MMP-9 in the culture medium was determined by ELISA. (n = 4) <b>(C)</b> Isolated WBCs were treated with PBS, NS1, or NS1 mixed with anti-NS1 antibodies for 24 h, and the concentration of MMP-9 in the culture medium was determined by ELISA. (n = 4) <b>(E)</b> After PMA activation, THP-1 and primary isolated WBCs were subjected to the desired treatment. After 24 h, the cell culture supernatants were collected. An MMP antibody array that detects various MMPs and TIMPs was used to assess the major subclass of MMPs induced by NS1. THP-1 cells or WBCs were stimulated with PBS or NS1; then, the culture supernatants were collected and analyzed for extracellular matrix proteins. Membranes of the human MMP antibody array were probed with the supernatant collected from bovine serum albumin (BSA)-treated THP-1 cells, BSA-treated WBCs, 20 μg/ml NS1-treated THP-1 cells or NS1-treated WBCs. The quantification of MMPs array membranes was analyzed by ImageJ. PC, positive control; NC, negative control. <b>(F)</b> After treatment with 20 μg/ml NS1 for 24 h, the 5X-concentrated WBCs and 10X-concentrated THP-1 supernatants were analyzed by electrophoresis with a 7.5% acrylamide gel containing gelatin. The gel was stained with Coomassie blue to reveal the white bands corresponding to the proteolysis of gelatin by MMPs. S/N, supernatant; *P<0.05, **P<0.01, ***P<0.001; unpaired t-test (panel B and D), Kruskal-Wallis ANOVA (panel C).</p

The serum concentrations of NS1, HPA-1, MMP-9, CD138 and MIF in healthy donors and dengue patients.

<p>The serum concentrations of <b>(A)</b> NS1 <b>(B)</b> HPA-1, <b>(C)</b> MMP-9, <b>(D)</b> CD138 and <b>(E)</b> MIF in healthy donors and dengue patients were compared as indicated. *P<0.05; **P<0.01; ***P<0.001; ns: not significant; ANOVA with Dunn's test for multiple comparisons (panel A, D,and E), Tukey's multiple comparisons test (panel B and C).</p

DENV NS1-induced MMP-9 secretion from THP-1 cells causes endothelial hyperpermeability and glycocalyx degradation.

<p><b>(A)</b> PMA-activated THP-1 cells were incubated with NS1; the supernatants were collected at the indicated times and incubated with HUVECs for 24 h. The endothelial permeability was determined by a Transwell permeability assay (n = 3), as described in the Materials and Methods section. <b>(B) (C)</b> PMA-activated THP-1 cells were incubated with NS1 for 24 h, and the resulting supernatants were collected. Fresh RPMI 1640 or NS1-treated THP-1 cell culture supernatants were incubated with HUVECs, with or without the indicated concentrations of <b>(B)</b> SB-3CT or <b>(C)</b> MMP-9 inhibitor I. After 24 h of incubation, endothelial permeability was determined by Transwell permeability assay. (n = 3) <b>(D)</b> NS1-treated THP-1 cell-conditioned medium was collected at the indicated times and incubated with HUVECs for 24 h. The concentration of CD138 in the supernatant was determined by ELISA. (n = 3) <b>(E) (F)</b> Fresh RPMI 1640 or NS1-treated THP-1 cell culture supernatant was mixed with or without the indicated concentrations of <b>(E)</b> SB-3CT or <b>(F)</b> MMP-9 inhibitor I and incubated with HUVECs for 24 h. The concentration of CD138 in the supernatant was determined by ELISA (n = 3). S/N, supernatant; **P<0.01, ***P<0.001; unpaired t-test (panel A and D), Kruskal-Wallis ANOVA (panel B, C, E and F).</p

MIF inhibition attenuates DENV NS1-induced glycocalyx degradation in mice.

<p><b>(A)</b> Before the injection of PBS or NS1, the blood of 8- to 12-week-old BALB/c mice (n = 3) was collected by orbital sinus sampling with 10% citrate. Next, the mice were intravenously injected with 50 μg of NS1 or 100 μl of PBS. Blood samples were collected from the mice immediately after injection and every 24 h thereafter until 120 h. The plasma concentrations of NS1, MIF, HPA-1, and MMP-9 were measured by ELISA. Arrow, injection time point. <b>(B)</b> BALB/c mice received two subcutaneous injections of PBS, NS1, or recombinant E or prM at the same location within 24 h. One day after the second injection, the mice were sacrificed, and a series of skin tissue sections were hybridized with anti-α-SMA and anti-CD138 antibodies and stained with DAB (brown). <b>(C-E)</b> BALB/c mice (n = 5) were intraperitoneally injected with BSA or NS1 with or without an MIF inhibitor (ISO-1), and the peritoneal lavage fluid was collected 24 h after the injection. The concentrations of <b>(C)</b> HPA-1, <b>(D)</b> MMP-9 and <b>(E)</b> CD138 in the peritoneal lavage fluid were measured by ELISA. *P<0.05, **P<0.01, ***P<0.001; unpaired t-test (panel A), Kruskal-Wallis ANOVA (panel C, D and E).</p

NS1-induced MMP-9 secretion from THP-1 cells is regulated by MIF.

<p><b>(A)</b> PMA-activated THP-1 cells were treated with PBS, NS1, or NS1 with p425 for 24 h, and the concentration of MMP-9 in the supernatant was determined by ELISA (n = 5). <b>(B)</b> The MMP-9 levels in the supernatants of PMA-activated THP-1-shLuc and THP-1-shMIF cell cultures were detected by ELISA after incubation with or without NS1 for 24 h. (n = 3) <b>(C)</b> PMA-activated THP-1-shLuc and THP-1-shMIF cells were incubated with PBS or NS1 for 24 h; then, the supernatant was collected and incubated with HUVEC monolayers grown in upper Transwell chambers. After 24 h of incubation, endothelial permeability was determined using streptavidin-HRP and TMB. (n = 6) <b>(D)</b> The supernatant of THP-1-stimulated HUVEC cultures was collected, and the CD138 concentration was determined by ELISA (n = 5). *P<0.05, **P<0.01, ***P<0.001; ns, not significant; Kruskal-Wallis ANOVA (panel A), unpaired t-test (panel B, C and D).</p

The proposed mechanisms of DENV NS1-induced endothelial glycocalyx degradation.

<p>Circulating DENV NS1 may bind to endothelial cells via TLR4 or other molecules to stimulate the secretion of MIF, which in turn elevates the protein level of active HPA-1, causing it to digest glycocalyx on the intravascular luminal surface of endothelial cells. On the other hand, NS1 can also bind to TLR4 on leukocytes to secrete MMP-9, causing glycocalyx degradation. Even though the effector molecules causing glycocalyx degradation (HPA-1 and MMP-9) are different, MIF is part of both HPA-1 secretion by endothelial cells and MMP-9 secretion by leukocytes.</p

Antibody Profiling of Dengue Severities Using Flavivirus Protein Microarrays

Dengue is a viral disease transmitted by Aedes aegypti mosquitoes. According to the World Health Organization, about half of the world’s population is at risk of dengue. There are four serotypes of the dengue virus. After infection with one serotype, it will be immune to such a serotype. However, subsequent infection with other serotypes will increase the risk of severe outcomes, e.g., dengue hemorrhagic fever, dengue shock syndrome, and even death. Since severe dengue is challenging to predict and lacks molecular markers, we aim to build a multiplexed Flavivirus protein microarray (Flaviarray) that includes all of the common Flaviviruses to profile the humoral immunity and cross-reactivity in the dengue patients with different outcomes. The Flaviarrays we fabricated contained 17 Flavivirus antigens with high reproducibility (R-square = 0.96) and low detection limits (172–214 pg). We collected serums from healthy subjects (n = 36) and dengue patients within 7 days after symptom onset (mild dengue (n = 21), hospitalized nonsevere dengue (n = 29), and severe dengue (n = 36)). After profiling the serum antibodies using Flaviarrays, we found that patients with severe dengue showed higher IgG levels against multiple Flavivirus antigens. With logistic regression, we found groups of markers with high performance in distinguishing dengue patients from healthy controls as well as hospitalized from mild cases (AUC > 0.9). We further reported some single markers that were suitable to separate dengue patients from healthy controls (AUC > 0.9) and hospitalized from mild outcomes (AUC > 0.8). Together, Flaviarray is a valuable tool to profile antibody specificities, uncover novel markers for decision-making, and shed some light on early preventions and treatments