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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.


INTRODUCTION
Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative therapy for many immune and hematologic malignant and non-malignant disorders. Once transplanted, allogeneic cells support myeloid and immunological reconstitution, and require a degree of immune tolerance against allogeneic antigens. Allogeneic T cells contained in the transplant attack and destroy the recipient organs including intestine, liver, and skin contributing to graft failure, and graft-versus-host disease (GvHD).
Interrupting the CD40/CD40L signal can be achieved by silencing CD40 gene expression using small interfering RNA (siRNA). These short double-stranded RNA sequences quench target gene expression.
To overcome the challenge of rapid siRNA degradation in circulation before reaching target cells siRNA compounds were prepared whereby siRNA via its poly-dA extension at the 5′-end of the siRNA binds fungal (schizophyllan [SPG]) 1,3-β-glucan [15][16][17]. The siRNA-β-glucan complex like the native 1,3-β-glucan or galectins, and annexins on apoptotic cells can interact with the cellular-anchored DC-associated C-type lectin (Dectin1, CLEC-7A). Monocytes, or conventional and plasmacytoid DCs mainly express Dectin1 [18]. The Dectin1-β-glucan/siRNA complex interaction takes advantage of the fact that APCs express Dectin1 and that the complex via Dectin1 is internalized into lysosomal endosomes ensuring that attached cargo is incorporated into the cell. Here, we tested a siRNA targeting CD40 complexed with SPG (siCD40-SPG) that targets CD40 expression on Dectin1+ cells in a lethal murine allogeneic bone marrow transplantation (BMT) model.

Immunohistochemistry
Automated immunohistochemistry was performed using the VEN-

Dectin1+ cells coexpress CD40 in aGvHD tissues
Because CD40 expression by DCs enhances aGvHD, we reasoned that reducing the expression of CD40 on Dectin1+ DCs [19] siCD40-SPG compounds would suppress aGvHD in murine allogeneic BMT models. We established a murine major histocompatibility complex-mismatched BMT model of lethal aGvHD by injecting BM cells and splenocytes from C57/Bl6 mice i.v. into irradiated (2 × 4 Gy) CBF1 mice ( Figure 1A). The BM/splenocyte transplantation was performed on day 0. Groups of mice receiving irradiation without further cell transplantation (irradiation group) or irradiation followed by the transplantation of CBF1 mouse-derived BM cells and splenocytes (syngeneic group) were included to control for irradiation or non-GvHD, but transplantationprocedure-associated effects. Because the siRNA compounds require the presence of Dectin1 to enter cells, Dectin1 expression was examined on splenocytes of mice >80 days after irradiation or mice 34 days after allogeneic BMT ( Figure 1B). After irradiation, Dectin1 expression on splenocytes was lower compared to mice 34 days after allogeneic BMT as determined by FACS ( Figure 1B). Further characterization showed that roughly 50% of Dectin1+ cells co-stained for CD40 ( Figure 1B). Within the Dectin-1+ cell population that coexpressed CD40, around 10% of cells also co-stained with Ly6C, an antigen that can be found on macrophage/DC precursors ( Figure 1B). These data confirmed that 50% of Dectin1+ splenocytes coexpressed CD40 after allogeneic BMT and that these cells contained a subpopulation of Ly6C+ macrophage/DC precursors.
Two of the tested siRNA compounds targeted the same gene sequence but differed in their chemical modifications (NJA-312 and NJA-302), while one targeted a different CD40 sequence (NJA-515).
Two administration schedules were tested for the compound NJA-312: three injections (referred to as NJA-312low) and five injections (NJA-312high).
The frequency of Dectin1+CD40+Ly6C+ splenocytes was similar between NJA-312low and carrier-treated mice after allogenic BMT controls on day 34 ( Figure 1C). In surviving mice by day 73, a lower frequency of Dectin1+CD40+Ly6C+ cells was observed in NJA-312hightreated splenocytes of allogeneic BMT mice compared to syngeneic BMT controls ( Figure 1C). These data suggested that administration of NJA-312low did not change the frequency of Dectin1+CD40+Lyc+ splenocytes and that NJA-312high suppressed Dectin1+CD40+Lyc+ splenocytes in long-term surviving mice.

NJA-312 prolong survival in allogenic BMT mice
Survival and bodyweight loss are reliable indicators of aGvHD in murine models [20]. CBF1 mice transplanted with CBF1 cell following irradiation served as controls (syngeneic BMT). In the syngeneic group, all mice survived. Few or no mice survived long-term in this lethal aGvHD model where mice had been transplanted with allogenic hematopoietic cells of C57BL6 cell origin (allogeneic BMT).
NJA-302 and NJA-312, but not NJA-515 showed similar survival (data not shown). The analysis was continued using NJA-312. NJA-312high, but not NJA-312low injections prolonged survival compared to carrier-injected allogeneic BMT mice ( Figure 1D). Long-term surviving mice still showed a lower bodyweight when compared to syngeneic BMT or irradiation controls ( Figure 1E). NJA-312high, but not NJA-312low-injected mice showed bodyweight loss during the first 14 days

Improved thymic regeneration after NJA-312high treatment in aGvHD mice
Carrier and NJA-312-treated allogeneic BMT mice showed typical signs of aGvHD in the thymus with atrophy and hypocellularity by day 34 (Figure 2A). Surviving NJA-312high-treated mice showed a nearly complete restoration of the thymic architecture by day 72, indicative of thymic regeneration.
Earlier studies showed that blockade of CD40/CD40L interaction prevents GvHD by reducing donor CD4+ but not CD8+ T-cell expansion [22]. We assessed the intrathymic T-cell development. On day 34 after BMT, the relative distribution of thymocytes subpopulations was abnormal ( Figure 2B). The proportion of double-positive (DP, and CD8+ cells made up around 28% and 10%, respectively, of the whole thymocyte population ( Figure 2B), indicating that thymic regeneration had occurred in the surviving NJA-312high mice.

Delayed T and B-cell engraftment, but stable lymphoid chimerism of long-term survivors after NJA-312 treatment
Given that aGvHD results in a severe anti-allogeneic CTL response, we next used FACS analysis to examine the percentage of CD3+ and B220+ lymphocytes of recipient (H-2K d+ K b+ ) or donor (H-2K d− K b+ ) origin in splenocytes of allogenic BMT mice treated with or without NJA-312low (Table 1). By day 34, we observed a trend toward a delayed CD3 T and B220 B donor cell engraftment in vivo in NJA-312-treated, compared to carrier-treated, allogenic BMT mice. This prompted us to also analyze T and B cells in the surviving mice on day 73 (Table 1). Therefore, on day 73, with no carrier-treated mice available for analysis, we compared T-cell reconstitution between NJA-312high-treated allogeneic BMT mice and irradiated mice that did not receive BMT (

NJA-312-treated mice show less tissue damage in the spleen and liver
Dectin-1 mRNA is predominantly detected in the spleen and thymus DCs [23]. Smaller spleens were found in the carrier-and NJA-312low-treated allogeneic BMT compared to post-irradiation or syngeneic BMT mice ( Figure 3A) indicating aGvHD-driven tissue damage even after NJA-312low treatment. Histopathological findings revealed marked lymphoid atrophy, hypocellularity, fibrosis, and disappearance of lymphoid follicles in spleens of carrier-and NJA-312low-treated allogeneic BMT mice. NJA-312low-derived spleens retained higher splenic cellularity ( Figure 3B). Long-term surviving mice showed an average spleen size and no pathological histological findings ( Figure 3A,B). In the H&E-stained liver sections of control mice, massive infiltration of mononuclear cells and total scar were observed. In the liver of NJA-312low-treated mice, periportal infiltration of mononuclear cells and fibrosis was found ( Figure 3B).
To examine the extent of myeloid cell infiltration, especially monocytes, macrophages, granulocytes, and natural killer cells, immunohistochemistry with antibodies against CD11b (blue signal) and F4/80 (brown staining) ( Figure 3C) and only CD11b alone ( Figure 3D) were performed. The number of CD11b+F4/80+ and periportal CD11b+ cells was similar in liver tissues of both groups ( Figure 3C,D).
These data implicated that NJA-312low treatment suppressed myeloid F4/80+ cell expansion in the spleen and liver.
Because severe aGvHD and a Th1-skewed inflammatory marrow environment suppress hematopoiesis and donor HSC engraftment [24], we examined BM sections. Local hypocellularity was observed in one out of three BM sections of the carrier-treated, but not NJA-312low-treated aGvHD mice at day 34 after BMT ( Figure S1B) indicating that NJA-312 treatment was not myelosuppressive and did not cause graft failure. Immunohistochemical analysis revealed increases in immunoreactive CD11b+, F4/80+, and CD3+ cells in the dermis of NJA-312low-treated, compared to carrier-treated, allogenic BMT mice ( Figure 4B-D). These data indicate that NJA-312 was less effective in suppressing the generation of inflammatory infiltrates in the skin, perhaps due to the low Dectin1 expression reported in skin macrophages and DCs [25]. It has been reported that Dectin1 on macrophages promotes intestinal inflammation [26]. No pathological changes were observed in colon tissues of carrier and NJA-312 mice ( Figure S1A).

siCD40 compounds enhanced expansion of CD8+ cells in the spleen and thymus, but not the skin
Both host and donor DCs contribute to inducing donor T-cell tolerance against host tissues in mice undergoing allo-HSCT. CD8+ DCs have been described in both the thymus and spleen, constituting 70% of the thymus DCs and 20% of the spleen DCs that can express Dectin1.
These cells suppress donor T-cell response and impair severe GvHD [27]. We performed CD8a staining on spleen and thymus tissue sections 34 days after allogeneic BMT. CD8+ cells were increased in spleen and thymus tissues of NJA-312-treated mice. The CD8+ cells, especially in the thymus and skin, presented as large cells with round nuclei, centrally located nucleoli, bland and dispersed chromatin, and flattening of adjacent nuclear membrane, suggesting that they might be DCs. The dermis of carrier-and NJA-312-treated animals showed similar numbers of infiltrating CD8+ cells ( Figure 5C). These data indicate that CD8+ cell expansion occurred primarily in the thymus, but not the skin, of NJA-312-treated mice.

Reduced number of CCR2+ cell infiltrates in spleen and liver of NJA-312-treated mice
The interactions between CCR2 and CCL2 are involved in Tcell/monocyte/macrophage migration resulting in the accumulation of CCR2+ cells in aGvHD tissues [21]. In addition, Dectin1 is crucial for  Figure 6A,B), and a reduced number of cells strongly stained for CCR2 ( Figure 6C). In contrast, skin sections of NJA-312-treated mice showed more infiltrating CCR2+ cells in the dermis ( Figure 6D). These data indicated that NJA-312 treatment reduced the CCR2+ cell influx into the liver and spleen, but not skin.

IL17A expression is modulated in hematopoietic and non-hematopoietic cells in aGvHD tissues
Dectin1 stimulation during Candida infection induces Th17 responses [28]. Studies on the role of IL17A are controversial with some indicating IL17A as a promoter or suppressor of GvHD [29,30]. Therefore, we assessed IL17A expression in aGvHD-prone tissues. While IL17A expression under non-pathological conditions is found mainly on T cells (data not shown), hematopoietic and non-hematopoietic cells in tissue Although no lung manifestations of aGvHD had been detected for either carrier-or NJA-312low-treated mice, strong IL17A staining was found in epithelial cells especially of NJA-312low-treated mice ( Figure   S1B). These data suggest that NJA-312low treatment acted organ specifically, reducing tissue IL17A expression in liver and splenocytes but increasing it in skin and lung.
Our study demonstrated that siRNA targeting of CD40 coupled to
Recently, siCD40-SPG compounds have been shown to reduce CD40 expression on DC cells after injection in cynomolgus monkeys [19].  In conclusion, siCD40/SPG compounds improved immunological reconstitution and survival in murine aGvHD models and impaired tissue damage in extramedullar organs, but not the skin. Mechanistically siCD40-SPG compounds impaired CD11b+ and CCR2+ cell accumulation and reduced IL17A expression ( Figure 6I). The use of PAMP receptors like Dectin1 for the entry of siRNA into inflammatory cells is intriguing as it seems to result in tissue-specific modulation of the inflammatory niche. It seems to be a therapeutic strategy to block alloreactivity, but studies will be necessary to determine whether or not it compromises the graft versus tumor effects.

CONFLICT OF INTEREST
The authors declare they have no conflicts of interest.

DATA AVAILABILITY STATEMENT
Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.

ETHICS STATEMENT
Animal procedures were approved by the institutional animal care committee of The Institute of Medical Science, the University of Tokyo, Japan.

SUPPORTING INFORMATION
Additional supporting information may be found in the online version of the article at the publisher's website.