Recombinant IL-7/HGFβ Hybrid Cytokine Enhances T Cell Recovery in Mice Following Allogeneic Bone Marrow Transplantation

<div><p>T cell immunodeficiency is a major complication of bone marrow (BM) transplantation (BMT). Therefore, approaches to enhance T cell reconstitution after BMT are required. We have purified a hybrid cytokine, consisting of IL-7 and the β-chain of hepatocyte growth factor (HGFβ) (IL-7/HGFβ), from a unique long-term BM culture system. We have cloned and expressed the IL-7/HGFβ gene in which the IL-7 and HGFβ genes are connected by a flexible linker to generate rIL-7/HGFβ protein. Here, we show that rIL-7/HGFβ treatment enhances thymopoiesis after allogeneic BMT. Although rIL-7 treatment also enhances the number of thymocytes, rIL-7/HGFβ hybrid cytokine was more effective than was rIL-7 and the mechanisms by which rIL-7 and rIL-7/HGFβ increase the numbers of thymocytes are different. rIL-7 enhances the survival of double negative (DN), CD4 and CD8 single positive (SP) thymocytes. In contrast, rIL-7/HGFβ enhances the proliferation of the DN, SP thymocytes, as well as the survival of CD4 and CD8 double positive (DP) thymocytes. rIL-7/HGFβ treatment also increases the numbers of early thymocyte progenitors (ETPs) and thymic epithelial cells (TECs). The enhanced thymic reconstitution in the rIL-7/HGFβ-treated allogeneic BMT recipients results in increased number and functional activities of peripheral T cells. Graft-versus-host-disease (GVHD) is not induced in the rIL-7/HGFβ-treated BMT mice. Therefore, rIL-7/HGFβ may offer a new tool for the prevention and/or treatment of T cell immunodeficiency following BMT.</p> </div

The survival of pre-selection DP and the proliferation of DN and SP thymocytes were enhanced by rIL-7/HGFβ.

<p>(A-F, I) Lethally irradiated BALB/c mice were injected with TCD-BM from B6 mice and treated with cytokines as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082998#pone-0082998-g001" target="_blank">Figure 1</a>. On day 30 after BMT, (A) The percentages of Annexin V⁺ cells in each thymocyte subset were analyzed by flow cytometry. (B) Representative histograms of intracellular staining for Bcl-2 by DN thymocytes. (C) Relative mean fluorescence intensity (MFI) <u>+</u> SD of Bcl-2 by DN thymocytes from the cytokine-treated BMT mice. (D) Representative histograms of intracellular staining for Bcl-xL by DP thymocytes. (E) Relative MFI of Bcl-xL by DP thymocytes. (F) rIL-7/HGFβ treatment enhances the survival of CD69^- pre-selection DP thymocytes. (G, H) rIL-7/HGFβ maintains the survival and the expression of Bcl-xL of DP thymocytes <i>in </i><i>vitro</i>. Purified DP thymocytes were cultured in the presence of equimolar amounts of rIL-7/HGFβ (30 ng/ml), rIL-7 (10 ng/ml) and/or rHGFβ (20 ng/ml), or PBS for 24 and 48 hours. Cells were analyzed for (G) annexin V⁺ cells by flow cytometry and (H) the expression of Bcl-xL by Western blot. (I) the cytokine-treated allo-BMT mice were injected i.p. with BrdU. The percentages of BrdU⁺ cells by thymocyte subsets were then analyzed by flow cytometry 4 hour later. The data are representative of 2 independent experiments with 4-6 mice per group. * P<0.05 compared with PBS-treated mice.</p

The number of ETPs and TECs was significantly increased in allo-BMT recipients after rIL-7/HGFβ treatment.

<p>Lethally irradiated BALB/c mice were injected with TCD-BM from B6 mice and treated with cytokines as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082998#pone-0082998-g001" target="_blank">Figure 1</a>. On day 30 after BMT, ETPs and TECs were analyzed. (A) Representative flow cytometric profiles showing the percentage of donor-origin lineage^- c-kit⁺ IL-7Rα^- CD44⁺CD25^- ETPs in total thymocytes. (B) Number of donor-origin ETPs in the cytokine-treated BMT mice. (C) Representative flow cytometric profiles showing the percentage of CD45^-EpCAM⁺MHC II⁺Ly51⁺ cTECs and CD45^-EpCAM⁺MHC II⁺Ly51^- mTECs in total TECs of the rIL-7/HGFβ-treated BMT mice. (D) Number of total TECs, cTEC and mTECs in the cytokine-treated BMT mice. The data are representative of 2 independent experiments with 4-6 mice per group. * P<0.05 compared with PBS-treated mice. ** P<0.05 compared with rIL-7 and/or rHGFβ-treated mice. </p

The number of thymocyte subsets was significantly increased in allo-BMT recipients after rIL-7/HGFβ treatment.

<p>Lethally irradiated mice (BALB/c, 4-10 week old) were injected i.v. with 2 X10⁶ TCD-BM from B6 mice. Groups of mice were then injected i.p. with the equimolar amounts of rIL-7/HGFβ (15 μg), rIL-7 (5 μg) and/or rHGFβ (10 μg), or PBS at 2-day intervals from days 1 to 26 after BMT. The number of (A) total thymocytes, donor-origin (B) CD4 and CD8 DN, (C) CD4 and CD8 DP, (D) CD4 SP, and (E) CD8 SP thymocytes was analyzed on day 30 after BMT. Means <u>+</u> S.D. are presented. The data are representative of 2 independent experiments with 4-6 mice per group. * P<0.05 compared with PBS-treated mice. ** P<0.05 compared with rIL-7 and/or rHGFβ-treated mice. </p

The number of total and naïve T cells in the spleen of allo-BMT recipients was significantly increased after rIL-7/HGFβ treatment.

<p>Lethally irradiated BALB/c mice were injected with TCD-BM from B6 mice and treated with cytokines as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082998#pone-0082998-g001" target="_blank">Figure 1</a>. On day 30 after BMT, the number of total (A) CD4 and (C) CD8 splenic T cells; and donor-origin naive (CD62L^hi CD44^lo) (B) CD4 and (D) CD8⁺ splenic T cells was evaluated by flow cytometry. The data are representative of 2 independent experiments with 4-6 mice per group. * P<0.05 compared with PBS-treated mice. ** P<0.05 compared with rIL-7 and/or rHGFβ-treated mice.</p

rIL-7/HGFβ-treated allo-BMT recipients do not develop GVHD.

<p>Lethally irradiated BALB/c mice were injected with TCD-BM from B6 mice. Syngeneic BMT (both BM and recipients from B6 mice) were used as controls. Both syngeneic and allo-BMT recipients were treated with rIL-7/HGFβ (15 μg) or PBS at 2-day intervals from days 1 to 26 after BMT. (A) weights and (B) histopathological analysis of signs of GVHD in liver, small bowel (SB) and large bowel (LB) were conducted on day 75 after BMT. (C) Splenocytes harvested from the allo-BMT recipients were used as effector cells for MLRs. Splenocytes from normal non-BMT B6 mice were used as controls. The effector cells were cultured with irradiated splenocytes (as stimulators) from normal non-BMT BALB/c, B6, and CBA mice, respectively. Cell proliferation was determined by BrdU incorporation. Mean OD in MLRs/OD in spontaneous proliferation with splenocytes from all-BMT recipients or normal B6 mice as effectors and splenocytes from BALB/c, B6, and CBA mice as stimulators were 0.21/0.20, 0.20/0.20, 2.97/0.21, as well as 2.95/0.21, 0.21/0.20, and 3.15/0.22, respectively. Data are shown as stimulation index. (A-C) The data are representative of 2 independent experiments of 5 mice each group. </p

Peripheral T cells in the rIL-7/HGFβ-treated allo-BMT recipients are functional.

<p>Lethally irradiated BALB/c mice were injected with TCD-BM from B6 mice and treated with cytokines as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082998#pone-0082998-g001" target="_blank">Figure 1</a>. On day 30 after BMT, (A) splenic CD3⁺ T cells were stimulated with anti-CD3 and anti-CD28 antibodies (5 μg/ml), and cell proliferation was determined by BrdU incorporation 4 days later. (B) splenic CD3⁺ T cells were stimulated with Con A (4 μg/ml) and cell proliferation was determined by BrdU incorporation 4 days later. (A and B) Data are shown as stimulation index. (C and D) Splenocytes were stimulated with phorbol myristate acetate and ionomycin, and stained with antibodies for cell surface markers and intercellular cytokines (H-2^<b>b</b>, CD4, CD8, IL-2, IFN-γ, and TNFα). The percentage of IL-2, IFN-γ and TNFα positive cells in donor-origin CD4⁺ and CD8⁺ T cells was determined by flow cytometry. The data are representative of 2 independent experiments with 4-6 mice per group. * P<0.05 compared with PBS-treated mice. ** P<0.05 compared with rIL-7 and/or rHGFβ-treated mice.</p

Peptide-anchored neutrophil membrane-coated biomimetic nanodrug for targeted treatment of rheumatoid arthritis

Abstract Macrophage polarization determines the production of cytokines that fuel the initiation and evolution of rheumatoid arthritis (RA). Thus, modulation of macrophage polarization might represent a potential therapeutic strategy for RA. However, coordinated modulation of macrophages in the synovium and synovial fluid has not been achieved thus far. Herein, we develop a biomimetic ApoA-I mimetic peptide-modified neutrophil membrane-wrapped F127 polymer (R4F-NM@F127) for targeted drug delivery during RA treatment. Due to the high expression of adhesion molecules and chemokine receptors on neutrophils, the neutrophil membrane coating can endow the nanocarrier with synovitis-targeting ability, with subsequent recruitment to the synovial fluid under the chemotactic effects of IL-8. Moreover, R4F peptide modification further endows the nanocarrier with the ability to target the SR-B1 receptor, which is highly expressed on macrophages in the synovium and synovial fluid. Long-term in vivo imaging shows that R4F-NM@F127 preferentially accumulates in inflamed joints and is engulfed by macrophages. After loading of the anti-inflammatory drug celastrol (Cel), R4F-NM@F127-Cel shows a significant reduction in hepatotoxicity, and effectively inhibits synovial inflammation and alleviates joint damage by reprogramming macrophage polarization. Thus, our results highlight the potential of the coordinated targeted modulation of macrophages as a promising therapeutic option for the treatment of RA