siRNA against CD40 delivered via a fungal recognition receptor ameliorates murine acute graft-versus-host disease

Abstract

Acute graft-versus-host disease (aGvHD) remains a major threat to a successful outcome after allogeneic hematopoietic stem cell transplantation (HSCT). Although antibody-based targeting of the CD40/CD40 ligand costimulatory pathway can prevent aGvHD, side effects hampered their clinical application, prompting a need for other ways to interfere with this important dendritic T-cell costimulatory pathway. Here, we used small interfering RNA (siRNA) complexed with β-glucan allowing the binding and uptake of the siRNA/β-glucan complex (siCD40/schizophyllan [SPG]; chemical modifications called NJA-312, NJA-302, and NJA-515) into Dectin1+ cells, which recognize this pathogen-associated molecular pattern receptor. aGvHD was induced by the transplantation of splenocytes and bone marrow cells from C57BL/6J into CBF1 mice. Splenic dendritic cells retained Dectin1 expression after HSCT but showed lower expression after irradiation. The administration of siCD40/SPG, NJA-312, and NJA-302 ameliorated aGvHD-mediated lethality and tissue damage of spleen and liver, but not skin. Multiple NJA-312high injections prevented aGvHD but resulted in early weight loss in allogeneic HSCT mice. In addition, NJA-312 treatment caused delayed initial donor T and B-cell recovery but resulted in stable chimerism in surviving mice. Mechanistically, NJA-312 reduced organ damage by suppressing CCR2+, F4/80+, and IL17A-expressing cell accumulation in spleen, liver, and thymus but not the skin of mice with aGvHD. Our work demonstrates that siRNA targeting of CD40 delivered via the PAMP-recognizing lectin Dectin1 changes the immunological niche, suppresses organ-specific murine aGvHD, and induces immune tolerance after organ transplantation. Our work charts future directions for therapeutic interventions to modulate tissue-specific immune reactions using Pathogen-associated molecular pattern (PAMP) molecules like 1,3-β-glucan for cell delivery of siRNA.

Keywords: CCR2; CD40; Dectin1; IL17A; PAMP; acute GvHD; bone marrow transplantation; cytokine; siRNA.

FIGURE 1

Reduced graft‐versus‐host disease (GvHD) lethality in mice treated with small interfering RNA (siRNA) against CD40. (A) Experimental protocol of allogenic bone marrow transplantation (BMT): all mice received 8 Gy total body irradiation. In the allogeneic BMT group, F1 mice were given B6 donor splenocytes and BM cells. F1 mice received F1 splenocytes and BM cells to establish the syngeneic BMT group. Allogenic BMT mice were treated either with/without siRNA targeting mouse CD40 (siCD40/schizophyllan [SPG]) called NJA‐312, NJA‐302, or NJA‐515. (B) Splenocytes retrieved at day 34 after BMT were co‐stained with antibodies against Dectin1, CD40, and Ly6c. Percentages of Dectin+ and Dectin+CD11c‐ cells were determined by three‐color flourescence‐activated cell sorting (FACS) in splenocytes from mice receiving 8 Gy (irradiation, n = 2) without BMT (noBMT, 8 Gy, n = 3). (C) Splenocytes retrieved at indicated time points and treatment groups (after NJA‐312, NJA‐302, and NJA‐515 treatment) were analyzed by FACS using the following antibodies: anti‐Dectin1, anti‐Ly6c, and anti‐CD40 (n = 1–2/group). (D) Survival of mice undergoing indicated treatments was monitored daily. Data were plotted as a Kaplan–Meier curve. Statistics using SPSS software using log‐rank (Mantel‐Cox). Left panel: indicated groups are shown (same data as left panel). The number of mice per group were as follows: *NJA‐312low (n = 11); >80 d irradiation (n = 12); >80 syngeneic BMT (n = 10); d73 NJA‐302 (n = 14); d73 NJA‐515 (n = 15); d73 NJA‐312high (n = 13); d34 allog BMT (n = 25). (E) Bodyweight was determined weekly (n = 7 per treatment group). Left panel: all groups are shown. Right panel: only indicated groups are shown (same data as left panel). *p < 0.05; **p < 0.01, ***p < 0.0001

FIGURE 2

NJA‐312 treatment does not prevent acute thymus atrophy but results in normal T‐cell development in surviving mice. (A) Hematoxylin and eosin (H&E)‐stained thymus sections of indicated treatment groups and time points (magnification bar = 200 μm). (B) Percentage of cells expressing CD4‐CD8+, CD4+CD8+, and CD4+CD8‐ on thymocytes as analyzed by FACS. Only live thymocytes, as defined by propidium iodide exclusion were analyzed (n = 2–3 per group). (C) Immunoreactive CD11b+ c (blue signal) and F4/80+ cells (brown signal) on thymus sections 34 days after allogeneic bone marrow transplantation (BMT) in the carrier‐ and NJA‐312low‐treated mice (magnification bar = 50 μm). Inserts in (D) show isotype controls (bar = 100 μm)

FIGURE 3

Treatment with NJA‐312 impairs liver and spleen organ damage in graft‐versus‐host disease (GvHD) mice. (A) Macroscopic images of spleens of different treatment groups at indicated time points. (B) Hematoxylin and eosin (H&E)‐stained spleen and liver tissue sections of different treatment groups at indicated time points. Magnification of main image (bar = 100 μm), inserts (bar = 100 μm). Yellow dotted lines encircle periportal areas with fibrosis and leukocyte infiltrates. (C) On day 34 immunoreactive CD11b (blue signal) and F4/80 (brown signal) were determined on liver sections of allogenic bone marrow transplantation (BMT) mice treated with carrier or NJA‐312 and representative images are shown (bar = 100 μm). Insert image shows isotype staining (bar = 200 μm). (D) Representative images of immunoreactive CD11b cells in liver tissue sections of allogenic BMT mice treated with carrier or NJA‐312 (magnification bar = 200 μm)

FIGURE 4

Targeting CD40 using the small interfering RNA (siRNA) compounds did not prevent skin acute graft‐versus‐host disease (aGvHD). (A) Hematoxylin and eosin (H&E)‐stained liver and spleen tissue sections of different treatment groups at indicated time points. Magnification of main image (bar = 100 μm). (B) Representative images of immunoreactive CD11b cells in liver tissue sections of allogenic bone marrow transplantation (BMT) mice treated with carrier or NJA‐312 (magnification bar = 100 μm). (C) Representative images of skin sections from animals 34 days after BMT stained with CD11b (blue signal) and co‐stained with F4/80 (brown signal) (bar = 100 μm). Arrows indicate examples of positive cells in both sections. (D) Representative images of skin sections from animals 34 days after BMT stained with CD3 (red signal) in the upper panels (bar = 100 μm). Arrows indicate examples of CD3+ cells in all images. Isotype controls are given

FIGURE 5

Focal expansion of large CD8+‐expressing cells in thymi of NJA‐312low‐treated mice, but not the skin. (A–C) Representative images of immunoreactive CD8a in the spleen (A), thymus (B), and skin (C) in tissues were retrieved on day 34 after bone marrow transplantation (BMT). Red dotted lines encircle areas of CD8+ cell expansion (upper panels magnification bar = 1 mm; lower panels magnification bar = 100 μm). Arrows indicate examples of positive cells in sections

FIGURE 6

NJA‐312low treatment altered IL17A and CCR2 expression in an organ‐specific manner in acute graft‐versus‐host disease (aGvHD) tissues. (A–D) Representative images of CCR2‐stained tissues section of the liver (A), spleen (B), thymus (C), and skin (D) of the carrier‐ or NJA‐312‐treated allogeneic bone marrow transplantation (BMT) mice. Immunoreactivity for CCR2 (E–G) in sections of liver (E), spleen (F), thymus (G), and skin (H) is depicted as brown staining. Strongly IL17A‐expressing cells were indicated using arrows. Magnification of spleen and liver (bar = 50 μm) and skin (bar = 20 μm) images. Inserts show isotype controls in (E), (F), and (H) (bar = 1 mm). The (G) insert gives a low magnification of IL17A‐stained thymus sections. (I) siCD40/schizophyllan (SPG) can incorporate into Dectin1+ cells where it reduces CD40 expression on CD40+ cells like myeloid cells (macrophages or dendritic cells [DCs]) thereby preventing further costimulation of T cells via CD40/CD40L interaction