Dengue Virus Directly Stimulates Polyclonal B Cell Activation

<div><p>Dengue infection is associated to vigorous inflammatory response, to a high frequency of activated B cells, and to increased levels of circulating cross-reactive antibodies. We investigated whether direct infection of B cells would promote activation by culturing primary human B lymphocytes from healthy donors with DENV <i>in vitro</i>. B cells were susceptible, but poorly permissive to infection. Even though, primary B cells cultured with DENV induced substantial IgM secretion, which is a hallmark of polyclonal B cell activation. Notably, DENV induced the activation of B cells obtained from either DENV immune or DENV naïve donors, suggesting that it was not dependent on DENV-specific secondary/memory response. B cell stimulation was dependent on activation of MAPK and CD81. B cells cultured with DENV also secreted IL-6 and presented increased expression of CD86 and HLA-DR, which might contribute to B lymphocyte co-stimulatory function. Indeed, PBMCs, but not isolated B cells, secreted high amounts of IgG upon DENV culture, suggesting that interaction with other cell types <i>in vivo</i> might promote Ig isotype switching and IgG secretion from different B cell clones. These findings suggest that activation signaling pathways triggered by DENV interaction with non-specific receptors on B cells might contribute to the exacerbated response observed in dengue patients.</p></div

DENV-induced B cell activation depends on CD81 activation.

<p>B lymphocytes were mock-treated or cultured with DENV2 (MOI = 1) in the presence or absence of anti-CD81 neutralizing antibody. <b>A)</b> At 12 days post infection, the supernatants were harvested and IgM levels were measured by ELISA. Data are representative of four independent experiments. Statistical analysis were performed and p values are indicated in the figures. <b>B)</b> The cells were harvested after different time points and DENV RNA levels were evaluated by qRT-PCR. Data are representative of three independent experiments. <b>C)</b> After 48h, the cells were harvested and the expression of ERK, p38 and JNK MAPK, phosphorylated (phospho) or not (unphospho) were analyzed in the cell lysates by western blotting, as indicated. The bars indicate the ratio between the analyzed phosphorylated protein and the corresponding unphosphorylated one; dots represent individual data.</p

Activation of MAPK is essential for DENV-induced B cell activation.

<p>Mock or DENV-treated cells were cultured in the presence or absence of ERK (PD98059), p38 (SB203580) and JNK (SP600125) inhibitors. <b>A)</b> After 12 days culture, supernatants were harvested and IgM levels were measured by ELISA. <b>B)</b> After 48h, culture supernatants were harvested and IL-6 levels were measured by ELISA. Data are representative of at least four independent experiments. Statistical analysis were performed and p values are indicated in the figures.</p

DENV-induced IgM secretion does not depend on TLR4 activation.

<p>B lymphocytes were mock-treated or cultured with DENV2 (MOI = 1) in the presence or absence of anti-TLR4 neutralizing antibody. LPS (10μg/ml; Sigma Aldrich) was used as positive control. At 12 days post infection, the supernatants were harvested and IgM levels were measured by ELISA. B cell cultures which showed increased IgM secretion in response to LPS (LPS responders) were graphed separately from the ones that didn’t show (LPS nonresponders). Data are representative of four independent experiments. Statistical analysis were performed and p values are indicated in the figures.</p

Purified B cells cultured with dengue virus showed increased expression of costimulatory molecules.

<p>B lymphocytes were mock-treated or cultured with DENV2 (MOI = 1) for the indicated time points and the expression of CD86 (A) or HLA-DR (B) in CD19+ cells were evaluated by flow cytometry. Each point indicate an individual donor. Statistical analysis were performed and p values are indicated in the figures.</p

B lymphocytes are susceptible, but poorly permissive to DENV infection.

<p>A-B) Purified B lymphocytes were mock-treated or cultured with DENV2 (MOI = 1) for the indicated time points. Cell lysates (A) and supernatants (B) were harvested and virus RNA was measured by quantitative real time RT-PCR. Each line indicate an individual donor after normalization by subtracting the input values. C) C6/36 cell line was incubated with DENV2 (MOI = 1) or with supernatants obtained from B cells previously cultured with DENV. After the indicated time points the cells were harvested and virus RNA was measured by qRT-PCR. D) Purified B cells were mock-treated or incubated with DENV2. After 48h, the cells were incubated with anti-NS1 antibody, followed by anti-mouse IgG conjugated to AlexaFluor488, and DAPI. The expression of DENV NS1 was analyzed by fluorescence microscopy. E-F) Purified B cells were cultured as in <i>D</i> and stained with anti-3H5 or anti-NS1 antibodies. A representative dot blot is shown in E and the average of the percentage of NS1⁺ cells obtained from 3 individual donors is shown in F.</p

DENV infection of PBMC promotes IgG secretion.

<p><b>A)</b> PBMCs or purified B cells were mock-treated or cultured with DENV2. After 12 days p.i., the supernatants were harvested and IgG secretion was evaluated by ELISA. Squares (mock) and triangles (DENV) indicate individual donors. <b>B)</b> PBMCs obtained from DENV immune (serum IgG+) and DENV naïve (serum IgG-) donors were mock-treated or cultured with DENV2, and IgM secretion was evaluated after 12 days. Stimulation index was calculated as the ratio between IgM concentration obtained from DENV and mock treated cultures. Dots indicate individual donors. <b>C-D)</b> PBMCs, CD3-CD27- cells (naïve Bcells), or CD3-CD27- and CD19+ cells (coculture between naïve B cells and non B cells), from the same donor were mock treated or DENV-infected. After 12 days p.i., the supernatants were harvested and IgM <b>(C)</b> or IgG <b>(D)</b> secretion were evaluated by ELISA. Bars indicate the average and SD of, at least, two independent experiments.</p

B cell infection by DENV promotes MAPK phosphorylation.

<p>B lymphocytes were mock-treated or cultured with DENV2 (MOI = 1). The cells were harvested after 2h, 24h or 48h p.i., and the expression of ERK, p38 and JNK MAPK were analyzed in the cell lysates by western blotting, using the indicated antibodies. The bars indicate the ratio between the analyzed phosphorylated protein and the corresponding non phosphorylated one; β actin staining were performed as a loading control and is shown in the bottom of the figure. Data are representative of three independent experiments.</p

Bone marrow mesenchymal cells improve muscle function in a skeletal muscle re-injury model

Skeletal muscle injury is the most common problem in orthopedic and sports medicine, and severe injury leads to fibrosis and muscle dysfunction. Conventional treatment for successive muscle injury is currently controversial, although new therapies, like cell therapy, seem to be promise. We developed a model of successive injuries in rat to evaluate the therapeutic potential of bone marrow mesenchymal cells (BMMC) injected directly into the injured muscle. Functional and histological assays were performed 14 and 28 days after the injury protocol by isometric tension recording and picrosirius/Hematoxilin & Eosin staining, respectively. We also evaluated the presence and the fate of BMMC on treated muscles; and muscle fiber regeneration. BMMC treatment increased maximal skeletal muscle contraction 14 and 28 days after muscle injury compared to non-treated group (4.5 ± 1.7 vs 2.5 ± 0.98 N/cm2, p<0.05 and 8.4 ± 2.3 vs. 5.7 ± 1.3 N/cm2, p<0.05 respectively). Furthermore, BMMC treatment increased muscle fiber cross-sectional area and the presence of mature muscle fiber 28 days after muscle injury. However, there was no difference in collagen deposition between groups. Immunoassays for cytoskeleton markers of skeletal and smooth muscle cells revealed an apparent integration of the BMMC within the muscle. These data suggest that BMMC transplantation accelerates and improves muscle function recovery in our extensive muscle re-injury model

Bone marrow mesenchymal cells improve muscle function in a skeletal muscle re-injury model.

Skeletal muscle injury is the most common problem in orthopedic and sports medicine, and severe injury leads to fibrosis and muscle dysfunction. Conventional treatment for successive muscle injury is currently controversial, although new therapies, like cell therapy, seem to be promise. We developed a model of successive injuries in rat to evaluate the therapeutic potential of bone marrow mesenchymal cells (BMMC) injected directly into the injured muscle. Functional and histological assays were performed 14 and 28 days after the injury protocol by isometric tension recording and picrosirius/Hematoxilin & Eosin staining, respectively. We also evaluated the presence and the fate of BMMC on treated muscles; and muscle fiber regeneration. BMMC treatment increased maximal skeletal muscle contraction 14 and 28 days after muscle injury compared to non-treated group (4.5 ± 1.7 vs 2.5 ± 0.98 N/cm2, p<0.05 and 8.4 ± 2.3 vs. 5.7 ± 1.3 N/cm2, p<0.05 respectively). Furthermore, BMMC treatment increased muscle fiber cross-sectional area and the presence of mature muscle fiber 28 days after muscle injury. However, there was no difference in collagen deposition between groups. Immunoassays for cytoskeleton markers of skeletal and smooth muscle cells revealed an apparent integration of the BMMC within the muscle. These data suggest that BMMC transplantation accelerates and improves muscle function recovery in our extensive muscle re-injury model