Research ArticleSepsis

Targeting Siglecs with a sialic acid–decorated nanoparticle abrogates inflammation

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Science Translational Medicine  02 Sep 2015:
Vol. 7, Issue 303, pp. 303ra140
DOI: 10.1126/scitranslmed.aab3459

Stopping sepsis

Sepsis is a dreaded diagnosis; clinicians have few tools to fight this generalized inflammatory response to infection that too often results in death. A new nanoparticle described by Spence et al. may prove to be a welcome weapon in the antisepsis arsenal. The nanoparticles are coated with di(α2→8) N-acetylneuraminic acid (NANA), which mimics sialic acid, the natural ligand for a critical anti-inflammatory receptor found on macrophages. This so-called Siglec receptor (sialic acid–binding immunoglobulin-like lectin-E) down-regulates macrophage activation by inflammatory signals released during infection and tissue damage, thereby interrupting the chain of events leading to sepsis. The authors demonstrate that the nanoparticle boosts this anti-inflammatory response in culture, and also show that it improves survival in two mouse models of generalized sepsis and one of pulmonary injury. Most encouraging for the ultimate utility of this nanoparticle in human patients, the nanoparticle is effective in human macrophages and in a sophisticated ex vivo model of human lung edema.

Abstract

Sepsis is the most frequent cause of death in hospitalized patients, and severe sepsis is a leading contributory factor to acute respiratory distress syndrome (ARDS). At present, there is no effective treatment for these conditions, and care is primarily supportive. Murine sialic acid–binding immunoglobulin-like lectin-E (Siglec-E) and its human orthologs Siglec-7 and Siglec-9 are immunomodulatory receptors found predominantly on hematopoietic cells. These receptors are important negative regulators of acute inflammatory responses and are potential targets for the treatment of sepsis and ARDS. We describe a Siglec-targeting platform consisting of poly(lactic-co-glycolic acid) nanoparticles decorated with a natural Siglec ligand, di(α2→8) N-acetylneuraminic acid (α2,8 NANA-NP). This nanoparticle induced enhanced oligomerization of the murine Siglec-E receptor on the surface of macrophages, unlike the free α2,8 NANA ligand. Furthermore, treatment of murine macrophages with these nanoparticles blocked the production of lipopolysaccharide-induced inflammatory cytokines in a Siglec-E–dependent manner. The nanoparticles were also therapeutically beneficial in vivo in both systemic and pulmonary murine models replicating inflammatory features of sepsis and ARDS. Moreover, we confirmed the anti-inflammatory effect of these nanoparticles on human monocytes and macrophages in vitro and in a human ex vivo lung perfusion (EVLP) model of lung injury. We also established that interleukin-10 (IL-10) induced Siglec-E expression and α2,8 NANA-NP further augmented the expression of IL-10. Indeed, the effectiveness of the nanoparticle depended on IL-10. Collectively, these results demonstrated a therapeutic effect of targeting Siglec receptors with a nanoparticle-based platform under inflammatory conditions.

INTRODUCTION

Sepsis is the single most frequent cause of death in hospitalized patients. Recent statistics have estimated occurrence worldwide as higher than 19 million cases per year. Despite 8 million sepsis-caused deaths annually, there is no effective treatment. Current treatments are supportive and often ineffective, with survivors tending to show persistent critical illness, which presents as long-term organ dysfunction (1, 2). In addition, about 25% of patients with severe sepsis progress to develop acute respiratory distress syndrome (ARDS) (3), and mortality from ARDS is about 25 to 30% (4). The excessive proinflammatory responses that contribute to organ dysfunction in sepsis are typically initiated and driven by the Toll-like receptors (TLRs), which recognize pathogen-derived constituents such as lipopolysaccharide (LPS), bacterial lipoproteins, and nonmethylated CpG DNA (5). Additionally, TLRs trigger sterile inflammatory responses, driven by damage-associated molecular patterns.

Sialic acid–binding immunoglobulin-like lectins (Siglecs) were originally identified as cell surface transmembrane receptors found primarily on hematopoietic cells and are capable of inhibiting TLR signaling (6). Hence, Siglecs have attracted attention as diagnostic and therapeutic biomarkers in various diseases, including acute myeloid leukemia, lymphoma, rheumatological disease, and allergy (79). One member of this family, Siglec-E (and its human orthologs Siglec-7 and Siglec-9), is predominantly found on macrophages and neutrophils. Siglec-E is an important regulator of neutrophil infiltration during pulmonary inflammation (10) and can abrogate TLR-mediated responses through tyrosine-specific Src homology-2 domain–containing phosphatase-2 (SHP-2) recruitment (11). Likewise, the human ortholog Siglec-7 is present on various immune cell subsets and, upon activation, recruits SHP-1 to down-regulate inflammatory mediators (12, 13).

The natural ligands for Siglecs are sialic acids, which are derivatives of N-acetylneuraminic acid (NANA), a monosaccharide with a nine-carbon backbone that can be linked to other sugars, thereby generating considerable diversity (14). Sialic acids act as signaling molecules, activating Siglecs expressed on the surface of immune cells both in cis and in trans to initiate inhibitory signal transduction. Recognition of sialic acids by the Siglecs is particularly dependent on the point-to-point saccharide linkage (α2→3, α2→6, and α2→8), with different Siglec receptors having distinct binding preferences (15). We therefore have examined the therapeutic potential of targeting Siglecs in models of acute inflammation. Previous studies by us (16) and others (17) have used complexed antibody strategies to engage Siglec receptors. However, the ability of bivalent antibodies to oligomerize receptors in vivo translates poorly to patients (18). As an alternative approach, here we specifically target and activate Siglec receptors using a sialylated nanoparticle.

RESULTS

α2,8 NANA-NP target Siglec-E on macrophages to limit LPS-driven proinflammatory cytokine production in vitro

Biodegradable and biocompatible 150-nm poly(lactic-co-glycolic acid) (PLGA) nanoparticles were prepared with a salting-out formulation method (19) and decorated with α2,8 NANA (α2,8 NANA-NP) before physicochemical characterization (fig. S1). We then evaluated the capacity of α2,8 NANA-NP to elicit anti-inflammatory effects in peritoneal macrophages stimulated for 12 hours with LPS. Exposure of macrophages to α2,8 NANA-NP significantly attenuated LPS-induced production of the proinflammatory cytokines tumor necrosis factor–α (TNF-α) and interleukin-6 (IL-6) (Fig. 1, A and B). These effects could be specifically attributed to the surface display of α2,8 NANA on the nanoparticles, because a series of controls did not produce similar anti-inflammatory effects: These included free α2,8 NANA alone, nonfunctionalized nanoparticles (Nude-NP), or nanoparticles decorated with glucosamine (Glucosamine-NP). Furthermore, the anti-inflammatory effects depended on both the dose and surface density of α2,8 NANA, with increasing sialic acid concentrations (1.4 to 15 μg) eliciting greater inhibitory effects. Moreover, the inhibitory effects of a single treatment of α2,8 NANA-NP persisted for 24 hours, with minimal batch-to-batch variation (fig. S1).

Fig. 1. α2,8 NANA-NP target Siglec-E on macrophages to limit LPS-driven proinflammatory cytokine production in vitro.

(A and B) C57BL/6 peritoneal macrophages were stimulated with LPS (100 ng/ml) ± α2,8 NANA-NP or appropriate controls (125 μg/ml) for 12 hours. Controls included α2,8 NANA alone (α2,8 NANA; equivalent amount as attached to α2,8 NANA-NP surface), nonfunctionalized nanoparticles (Nude-NP; 125 μg/ml), and nanoparticles displaying a control polysaccharide (Glucosamine-NP; 125 μg/ml). Supernatants were assayed for TNF-α (Α) and IL-6 (B) by enzyme-linked immunosorbent assay (ELISA). Statistical significance was assessed by one-way analysis of variance (ANOVA) with post hoc Tukey test (***P < 0.001, in comparison to LPS only). Data are expressed as means + SEM (n = 3 independent experiments in triplicate). (C) C57BL/6 peritoneal macrophages were stimulated with LPS (100 ng/ml) or medium alone for 24 hours. mRNA was extracted, converted to complementary DNA (cDNA), and analyzed by real-time polymerase chain reaction (PCR) for Siglece gene expression. Data are expressed as fold change relative to untreated samples and normalized to β-actin gene expression. Data are expressed as means + SEM (n = 3 independent experiments in triplicate). (D and E) C57BL/6 peritoneal macrophages (D) or BMDMs (E) were treated with the stated dose of LPS for 0, 12, and 24 hours. Macrophages were lysed in radioimmunoprecipitation assay (RIPA) buffer before analysis of Siglec-E and γ-tubulin protein by Western blot (representative images of n = 4 independent experiments). (F) C57BL/6 BMDMs were stimulated with LPS (1 ng/ml) overnight to up-regulate Siglec-E expression. After 2-hour preblocking with anti–Siglec-E antibody (Ab) (10 μg/ml), BMDMs were stimulated with LPS (100 ng/ml) ± α2,8 NANA-NP or Nude-NP (125 μg/ml) for 12 hours. Supernatants were assayed for TNF-α by ELISA. Statistical significance was assessed by one-way ANOVA with post hoc Tukey test (***P < 0.001, in comparison to LPS only). Data are expressed as means + SEM (n = 3 independent experiments in triplicate). (G) C57BL/6 peritoneal macrophages were incubated with Siglec-E–specific short hairpin RNA (shRNA) or scrambled shRNA encapsulated GeneCellin nanoporters for 48 hours. Macrophages were then stimulated with LPS (100 ng/ml) ± α2,8 NANA-NP or Nude-NP (125 μg/ml) for 24 hours. Supernatants were assayed for TNF-α by ELISA. Statistical significance was assessed by one-way ANOVA with post hoc Tukey test (**P < 0.01, in comparison to scrambled shRNA LPS only). Data are expressed as means + SEM (n = 3 independent experiments in triplicate). (H) Confocal fluorescence microscopy images of unstimulated RAW 264.7 cells (left panels) or RAW 264.7 cells incubated with coumarin 6–loaded Nude-NP (125 μg/ml) (green; middle panels) or coumarin 6–loaded α2,8 NANA-NP (green; right panels) for 3 hours. Nuclei were distinguished by TO-PRO-3 staining (blue) (representative images of n = 3 independent experiments). (I) Confocal fluorescence microscopy images of RAW 264.7 cells incubated with coumarin 6–loaded α2,8 NANA-NP (125 μg/ml) (green; left panels). Alternatively, RAW 264.7 cells were preincubated with control [immunoglobulin G1 (IgG1); middle panels] or free anti–Siglec-E (right panels) antibodies for 3 hours and then incubated with coumarin 6–loaded α2,8 NANA-NP (green). Nuclei were distinguished by TO-PRO-3 staining (blue) (representative images of n = 3 independent experiments).

Subsequently, we examined the specificity of the nanoparticle toward Siglec-E. Treatment of peritoneal macrophages with LPS induced Siglec-E mRNA and protein (Fig. 1, C and D). LPS also induced Siglec-E on bone marrow–derived macrophages (BMDMs) (Fig. 1E). We then sought to confirm that receptor engagement was required for the anti-inflammatory effects of these nanoparticles. In an initial experiment, we showed that α2,8 NANA-NP showed enhanced binding to recombinant Siglec-E Fc protein in vitro compared to nonfunctionalized control nanoparticles (fig. S2). Next, the effects of the nanoparticles were tested in cells in which surface Siglec-E had been preblocked with a nonagonistic antibody. This receptor blocking significantly impeded the anti-inflammatory capacity of α2,8 NANA-NP compared to controls (Fig. 1F). To conclusively rule out that the effect was a result of antibody cross-linking, we depleted Siglec-E in cells with RNA interference. Depletion of Siglec-E mRNA eliminated the anti-inflammatory effects of α2,8 NANA-NP (Fig. 1 G). The specificity of α2,8 NANA-NP for Siglec-E was further demonstrated by microscopy studies, in which we observed increased adherence of nanoparticles decorated with α2,8 NANA to macrophages (Fig. 1H). This preferential binding of α2,8 NANA-NP was markedly reduced by preblocking with Siglec-E antibody (Fig. 1I). Finally, we investigated the clustering of Siglec-E on the plasma membrane of macrophages with fluorescent α2,8 NANA-NP (fig. S2). Siglec-E localization was enhanced at areas of high α2,8 NANA-NP density compared with Nude-NP or Siglec-E nonagonistic antibody controls. This clustering was indicative of increased receptor activation (20, 21). Collectively, these data demonstrate that the anti-inflammatory effects of α2,8 NANA-NP toward murine macrophages occur via enhanced interactions with Siglec-E molecules and are Siglec-E–dependent.

α2,8 NANA-NP attenuate inflammatory effects in murine models of systemic inflammation

The therapeutic potential of α2,8 NANA-NP in vivo was next examined in a mouse model of LPS-induced systemic inflammation. Mice were challenged with an intraperitoneal injection of a lethal dose of LPS (6 mg/kg) and treated with either α2,8 NANA-NP or Nude-NP intraperitoneally at 0 hours. In this lethal model, seven of eight animals treated with Nude-NP reached experimental endpoint within 32 hours, whereas animals treated with α2,8 NANA-NP remained healthy (Fig. 2A). The survival benefits conferred by α2,8 NANA-NP corresponded with a significant reduction in serum TNF-α (Fig. 2B) and increased levels of IL-10 (Fig. 2C). Having demonstrated that α2,8 NANA-NP could prophylactically prevent death in LPS-induced systemic inflammation, we next examined efficacy after the onset of inflammation (defined as a clinical score of 2: piloerection, huddling, and diarrhea). α2,8 NANA-NP (2 mg) or controls were administered at the peak of inflammation (2 hours after LPS), in accordance with previous reports (22). Similar to prophylactic treatment, α2,8 NANA-NP significantly enhanced survival (Fig. 2D), and analogous attenuation of serum TNF-α (Fig. 2E) and elevated IL-10 (Fig. 2F) were observed.

Fig. 2. α2,8 NANA-NP attenuate systemic inflammation in murine models.

(A) C57BL/6 mice (n = 8 per group) were treated with intraperitoneal injections of LPS (6 mg/kg) in conjunction with 2 mg of α2,8 NANA-NP or Nude-NP, or treated with 2 mg of α2,8 NANA-NP only. Survival was monitored for 50 hours. Statistical significance was assessed by Kaplan-Meier log-rank χ2 test (***P < 0.001). (B and C) At 24 hours after injection, blood samples were taken from mice by tail vein puncture, and serum was assayed for TNF-α (B) and IL-10 (C) by ELISA. Statistical significance was assessed by one-way ANOVA with post hoc Tukey test (***P < 0.001). Data are expressed as means + SEM. (D) C57BL/6 mice (n = 4 per group) were treated with intraperitoneal (i.p.) injections of LPS (6 mg/kg) [or phosphate-buffered saline (PBS) for α2,8 NANA-NP–only control]. After 2 hours, 2 mg of α2,8 NANA-NP or Nude-NP was injected intraperitoneally. Survival was monitored for 50 hours. Statistical significance was assessed by Kaplan-Meier log-rank χ2 test (**P < 0.01). (E and F) At 24 hours after LPS injection, blood samples were taken from mice by tail vein puncture, and serum was assayed for TNF-α (E) and IL-10 (F) by ELISA. Data are expressed as means + SEM. (G) C57BL/6 mice (n = 8 per group) were treated with intraperitoneal injections of clodronate liposomes or PBS control liposomes at −48 and −24 hours. All groups were then treated with intraperitoneal injections of LPS (6 mg/kg) in conjunction with 1 mg of α2,8 NANA-NP or Nude-NP. Survival was monitored for 50 hours. Statistical significance was assessed by Kaplan-Meier log-rank χ2 test (***P < 0.001, between control liposome + α2,8 NANA-NP and control liposome + Nude-NP groups). (H) Cecal ligation and puncture (CLP) was performed on C57BL/6 mice (n = 8 to 10 per group), followed by intraperitoneal injection of 2 mg of α2,8 NANA-NP, 2 mg of Nude-NP, equivalent dosage of α2,8 NANA alone, or 100 μg of dexamethasone 2 hours after surgery and every 24 hours subsequently. Survival was monitored for 9 days. Statistical significance was assessed by Kaplan-Meier log-rank χ2 test (***P < 0.001, between α2,8 NANA-NP and Nude-NP groups). (I) Clinical scoring after CLP used the following criteria: score 0, no symptoms; score 1, piloerection and huddling; score 2, piloerection, huddling, and diarrhea; score 3, lack of interest in surroundings and severe diarrhea; score 4, decreased movement and listless appearance; score 5, loss of self-righting reflex. Data are expressed as means ± SEM. (J) Body temperature after CLP. Data are expressed as means ± SEM.

As recent reports had indicated that Siglec-E was present on other innate immune cells, particularly neutrophils (10), we questioned whether the therapeutic effects of α2,8 NANA-NP were solely a result of effects on macrophages in vivo. Therefore, mice were treated intraperitoneally with clodronate liposomes to deplete macrophages (fig. S3) (23) before administration of LPS and nanoparticles. No abnormal clinical signs were observed subsequent to depletion. However, the therapeutic benefit of α2,8 NANA-NP was lost in macrophage-depleted mice, with no significant difference in survival observed between the Nude-NP and α2,8 NANA-NP groups (Fig. 2G). We also examined the binding of α2,8 NANA-NP to cell populations in peritoneal exudate. We observed that both macrophages (CD11b+F4/80+) and neutrophils (CD11b+Ly6G+) were Siglec-E–positive, although Siglec-E expression was greater on macrophages. This was reflected in greater binding of α2,8 NANA-NP by macrophages, whereas neutrophil binding was negligible (fig. S4). Together, these results indicated that α2,8 NANA-NP–mediated effects in vivo were a consequence of binding to Siglec-E expressed on macrophages.

Thus far, the validation of α2,8 NANA-NP as a viable therapy was solely based on experimental models with LPS-driven inflammation. Therefore, we next evaluated the nanoparticles in the CLP model of polymicrobial sepsis. Inflammation in this model is driven by a multitude of TLR ligands and is much more representative of clinical sepsis and other acute inflammatory disorders (24, 25). Mice were anesthetized, and CLP was performed as described (25). At 2 hours after surgery and every 24 hours subsequently, mice were treated with α2,8 NANA-NP, Nude-NP, or associated controls. α2,8 NANA-NP markedly prolonged survival; all control animals failed to survive beyond 8 days (Fig. 2H). Although all groups initially developed clinical signs of sepsis, α2,8 NANA-NP attenuated further progression of illness from day 2 onward (Fig. 2I). In addition, core body temperature stabilized in the α2,8 NANA-NP animals compared to controls (Fig. 2J). Collectively, these data indicate that α2,8 NANA-NP can produce therapeutic effects in a clinically relevant model of inflammation.

α2,8 NANA-NP attenuate inflammatory effects in murine models of lung injury

Having established the efficacy of α2,8 NANA-NP in systemic inflammation and the CLP model of sepsis, the application of the nanoparticles in murine lung injury was next investigated. Intratracheal instillation of LPS in mice induces neutrophilic alveolitis and enhanced proinflammatory cytokine responses, features representative of the clinical characteristics of ARDS (2628). After intratracheal instillation of LPS, mice were treated with 2 mg of α2,8 NANA-NP or Nude-NP intraperitoneally 2 hours after the endotoxin. Bronchoalveolar lavage (BAL) fluid neutrophilia was attenuated upon treatment with α2,8 NANA-NP in comparison to mice that received Nude-NP (Fig. 3A). Conversely, macrophage numbers within total BAL cell populations were enhanced by α2,8 NANA-NP (Fig. 3B). Cytokine analyses revealed that lung homogenate IL-10 levels were significantly elevated after α2,8 NANA-NP administration (Fig. 3C).

Fig. 3. α2,8 NANA-NP attenuate pulmonary inflammation in murine models.

(A and B) C57BL/6 mice (n = 7 to 8 per group) were treated with 20 μg of LPS intratracheally. After 2 hours, 2 mg of α2,8 NANA-NP or Nude-NP was injected intraperitoneally. Mice were sacrificed at 24 hours after LPS instillation, BAL fluid was collected, and the lungs were then excised for further analyses. Neutrophil (A) and macrophage (B) counts in BAL fluid, as quantified from cytospin images. Statistical significance was assessed by Mann-Whitney U test (**P < 0.01). Data are expressed as means + SEM. (C) Lung homogenate supernatants were assayed for IL-10 by ELISA. Statistical significance was assessed by Mann-Whitney U test (*P < 0.05). Data are expressed as means + SEM. (D) CLP was performed on C57BL/6 mice (n = 12 to 13 per group at outset of experiment), followed by intratracheal instillation of 20 μg of α2,8 NANA-NP or Nude-NP 6 to 8 hours after surgery. Clinical scoring after CLP used the following criteria: score 0, no symptoms; score 1, piloerection and huddling; score 2, piloerection, huddling, and diarrhea; score 3, lack of interest in surroundings and severe diarrhea; score 4, decreased movement and listless appearance; score 5, loss of self-righting reflex. Statistical significance was assessed by Student’s t test (*P < 0.05). Data are expressed as means ± SEM. (E) Survival was monitored for 96 hours. (F and G) CLP was performed on C57BL/6 mice (n = 7 to 11 per group), followed by treatment as described in (D). Mice were sacrificed at 30 hours after treatment, and BAL fluid was collected. Total cell (F) and neutrophil (G) counts in BAL fluid. Statistical significance was assessed by Student’s t test (*P < 0.05). Data are expressed as means + SEM.

CLP has been used to model pulmonary inflammation resulting from a systemic insult (29, 30). We therefore investigated the therapeutic efficacy of α2,8 NANA-NP in this indirect model of lung injury. At 6 to 8 hours after CLP, mice were treated intratracheally with 20 μg of α2,8 NANA-NP or Nude-NP. Clinical scores were significantly lower after treatment with α2,8 NANA-NP transiently at 24 and 36 h time points (Fig. 3D), reflected in reduced mortality, albeit non-significantly, up to 72 h mortality rate (Fig. 3E). These findings were consistent with the observed reductions in BAL fluid total cell counts (Fig. 3F) and neutrophil numbers (Fig. 3G). Collectively, these data suggest that α2,8 NANA-NP represent a viable therapeutic in murine models of lung injury with both intraperitoneal and intratracheal administration routes.

IL-10–induced Siglec-E up-regulation is potentiated by α2,8 NANA-NP

We next examined the mode of action of these potent anti-inflammatory nanoparticles. In previous murine studies, SHP-2 was recruited to the cytoplasmic domains of Siglec-E after receptor activation (11). We confirmed the presence of increased amounts of immunoprecipitated SHP-2 bound to Siglec-E in cells treated with α2,8 NANA-NP in comparison to Nude-NP–treated controls (lanes 4 to 6 versus lanes 7 to 9) (Fig. 4A). Indicative of an anti-inflammatory response, α2,8 NANA-NP elevated the nuclear factor κB (NFκB) p50/p65 ratio within the nucleus (Fig. 4B). A key observation from our earlier data was that serum IL-10 was elevated in mice treated with α2,8 NANA-NP (Fig. 2, C and F). However, the ability of α2,8 NANA-NP to enhance production of IL-10 was ablated in the absence of myeloid differentiation primary response gene 88 (MyD88) (Fig. 4C), confirming previous reports that MyD88 was required for LPS-stimulated IL-10 production (Fig. 4D) (31).

Fig. 4. α2,8 NANA-NP induce SHP-2 recruitment to Siglec-E to promote downstream anti-inflammatory responses.

(A) C57BL/6 peritoneal macrophages were pretreated with α2,8 NANA-NP or Nude-NP (125 μg/ml) for 24 hours before stimulation with LPS (10 ng/ml) for 0, 12, or 24 hours. Macrophages were lysed in RIPA buffer and immunoprecipitated (IP) using anti–Siglec-E antibody. The immunoprecipitate was blotted (IB) for SHP-2, whereas whole-cell lysate was blotted for Siglec-E and SHP-2 protein expression (representative image of n = 3 independent experiments). (B) C57BL/6 peritoneal macrophages were treated with α2,8 NANA-NP or Nude-NP (125 μg/ml) in conjunction with LPS (100 ng/ml). Nuclear localization of p50 was determined using the p50/p65 EZ-TFA colorimetric assay. Data presented as fold ratio of p50/p65 compared to control untreated cells. Statistical significance was assessed by one-way ANOVA with post hoc Tukey test (**P < 0.01). Data are expressed as means + SEM (n = 3 independent experiments in duplicate). (C) Immortalized wild-type (WT) or MyD88-deficient (MyD88−/−) BMDMs were stimulated with LPS (100 ng/ml) ± α2,8 NANA-NP (125 μg/ml) for 24 hours. Supernatants were assayed for IL-10 by ELISA. Statistical significance was assessed by two-way ANOVA with post hoc Bonferroni test (*P < 0.05). Data are expressed as means + SEM (n = 3 independent experiments in duplicate). (D) Immortalized WT or MyD88−/− BMDMs were stimulated with LPS (100 ng/ml) or medium alone for 24 hours. Supernatants were assayed for IL-10 by ELISA. Statistical significance was assessed by two-way ANOVA with post hoc Bonferroni test (***P < 0.001). Data are expressed as means + SEM (n = 3 independent experiments in duplicate).

Previous studies had indicated that Siglec-E expression is dependent upon MyD88 function (11). Therefore, we examined expression of Siglec-E induced by both LPS and IL-10 in more detail. Similar up-regulation of both Siglec-E mRNA and protein was confirmed in peritoneal macrophages treated with either stimulus (Fig. 5, A and B). In parallel studies, preblocking the IL-10 receptor resulted in marked ablation of Siglec-E levels on treatment with either LPS or IL-10 (Fig. 5C). Together, these data demonstrated that IL-10 regulated the expression of Siglec-E. We hypothesized that the effect of the nanoparticles would therefore be dependent on IL-10. This was confirmed using IL-10−/− mice, in which administration of α2,8 NANA-NP was unable to rescue these animals from LPS-induced mortality (Fig. 5D) and did not reduce serum TNF-α in comparison to wild-type controls (Fig. 5E).

Fig. 5. Siglec-E is induced by IL-10 and the anti-inflammatory effects of α2,8 NANA-NP are IL-10–dependent.

(A) C57BL/6 peritoneal macrophages were stimulated with LPS (100 ng/ml) or IL-10 (10 ng/ml) for 24 hours. mRNA was extracted, converted to cDNA, and analyzed by real-time PCR for Siglece gene expression. Data are expressed as fold change relative to untreated samples and normalized to β-actin gene expression. Data are expressed as means + SEM (n = 3 independent experiments in triplicate). (B) C57BL/6 peritoneal macrophages were stimulated as in (A). Macrophages were lysed in RIPA buffer before analysis of Siglec-E and γ-tubulin protein expression by Western blot (representative image of n = 3 independent experiments). (C) C57BL/6 peritoneal macrophages were pretreated with IL-10 receptor blocking antibody or IgG1 isotype control (10 μg/ml) overnight. Macrophages were then stimulated with LPS (100 ng/ml) or IL-10 (10 ng/ml) for 24 hours. Macrophages were lysed in RIPA before analysis of Siglec-E and γ-tubulin protein expression by Western blot (representative image of n = 3 independent experiments). (D) C57BL/6 WT or IL-10−/− mice (n = 8 per group) were treated with intraperitoneal injections of LPS (6 mg/kg) in conjunction with 1 mg of α2,8 NANA-NP or Nude-NP. Survival was monitored for 50 hours. Statistical significance was assessed by Kaplan-Meier log-rank χ2 test (**P < 0.01 between WT + α2,8 NANA-NP and WT + Nude-NP groups). (E) At 12 hours after injection, blood samples were taken from mice by tail vein puncture, and serum was assayed for TNF-α by ELISA. Statistical significance was assessed by two-way ANOVA with post hoc Bonferroni test (***P < 0.001 between WT + α2,8 NANA-NP and WT + Nude-NP groups). Data are expressed as means + SEM.

α2,8 NANA-NP exhibit negligible toxicity in preclinical models

To analyze possible side effects of α2,8 NANA-NP, we also assessed indicators of toxicity. To mimic the treatment regime of Fig. 2, mice were treated with a single intraperitoneal dose of α2,8 NANA-NP or PBS. We saw no alterations in lung, liver, or kidney histology among groups. Furthermore, α2,8 NANA-NP did not alter serum lactate dehydrogenase concentrations or animal weights. Similarly, no toxic effects were observed after weekly intraperitoneal administration of α2,8 NANA-NP over 28 days or in a bolus-localized pulmonary delivery model (fig. S5).

α2,8 NANA-NP elicit anti-inflammatory effects in human cell and whole-lung ex vivo perfusion models

The translational potential of the nanoparticle was next explored in human models. Siglec-7 and Siglec-9 are the human orthologs of murine Siglec-E and represent translational targets for α2,8 NANA-NP. Flow cytometry analyses revealed that both receptors were present on primary human monocyte-derived macrophages (MDMs) (Fig. 6A) and monocytes (Fig. 6F). Treatment of MDMs with α2,8 NANA-NP significantly attenuated LPS-induced secretion of proinflammatory TNF-α (Fig. 6B), IL-6 (Fig. 6C), and IL-8 (Fig. 6D), and further enhanced concentrations of the anti-inflammatory cytokine IL-10 (Fig. 6E). Furthermore, α2,8 NANA-NP limited the activation of NFκΒ by preventing the degradation of IκB (inhibitor of nuclear factor κB) (fig. S6). We observed comparable results with primary human monocytes, and although the degree of proinflammatory cytokine inhibition was less pronounced in comparison to MDMs, TNF-α was nevertheless significantly attenuated (Fig. 6, G to I). To assess possible toxicity, we examined IL-1β secretion in human MDMs. Analogous to PBS controls, α2,8 NANA-NP did not induce IL-1β production (fig. S5).

Fig. 6. α2,8 NANA-NP promote anti-inflammatory effects in human cell and tissue assays.

(A) Primary human MDMs (n = 4 donors) were stimulated with LPS (500 ng/ml) for 18 hours. MDMs were stained with Siglec-7–phycoerythrin (PE) and Siglec-9–fluorescein isothiocyanate (FITC) or appropriate isotype control antibodies and analyzed by flow cytometry. Representative plots are shown. (B to D) Primary human MDMs (n = 6 to 13 donors) were pretreated with LPS (10 ng/ml) for 1 hour before addition of α2,8 NANA-NP or Nude-NP (25 μg/ml) for a further 12 hours. Supernatants were assayed for TNF-α (B), IL-6 (C), and IL-8 (D) by ELISA. Data are presented as % change in cytokine levels relative to MDMs stimulated with LPS only. Statistical significance was assessed by Mann-Whitney U test (*P < 0.05, ***P < 0.001). Data are expressed as means ± SEM. (E) Primary human MDMs (n = 3 donors) were stimulated with LPS (10 ng/ml) ± α2,8 NANA-NP (50 μg/ml). Supernatants were assayed for IL-10 by ELISA. Data are expressed as means + SEM. (F) Primary human monocytes (n = 2 donors) were stimulated with LPS (10 ng/ml) for 24 hours. Monocytes were stained with Siglec-7–PE and Siglec-9–FITC or appropriate isotype control antibodies and analyzed by flow cytometry. Representative plots shown. (G to I) Primary human monocytes (n = 7 donors) were stimulated with LPS (10 ng/ml) ± α2,8 NANA-NP or Nude-NP (50 μg/ml) for 18 hours. Supernatants were assayed for TNF-α (G), IL-6 (H), and IL-8 (I) by ELISA. Data are presented as % change in cytokine levels relative to monocytes stimulated with LPS only. Statistical significance was assessed by Mann-Whitney U test (*P < 0.05). Data are expressed as means ± SEM. (J) Human lungs (n = 3 per group) were injured ex vivo by intrabronchial instillation of 6 mg of LPS. Lungs were treated simultaneously with saline or 5 mg of α2,8 NANA-NP, which were introduced into the perfusate to model systemic administration. At 4 hours after treatment, lung tissue sections were excised for assessment of the wet/dry ratio (*P < 0.05). Data are expressed as means ± SEM.

We next investigated the feasibility of this treatment in attenuating inflammation in a human ex vivo lung perfusion (EVLP) model (fig. S7) (3234). Injury was induced by intrabronchial instillation of LPS, and α2,8 NANA-NP or saline control treatment was added to the perfusate to model systemic administration in a randomized, blinded manner. Pulmonary edema, the hallmark of ARDS, was measured by determining tissue wet/dry ratios (Fig. 6J), and BAL fluid IL-10 was also analyzed (fig. S6). Compared to controls, treatment with α2,8 NANA-NP induced a significant reduction in wet/dry ratios, indicating the attenuation of pulmonary edema. BAL IL-10 concentrations were increased by α2,8 NANA-NP treatment; these results are consistent with IL-10 mediation of the therapeutic effects of α2,8 NANA-NP on pulmonary edema in the EVLP model. Taken together, these data demonstrate that the nanoparticle elicits anti-inflammatory effects on human cells and tissues, highlighting its translational potential.

DISCUSSION

Previously, we have demonstrated that multivalent display of antibodies on the surface of nanoparticles can facilitate targeting to a given cell type and also elicit receptor clustering and activation, triggering downstream responses that are not achievable with free antibody (16, 3538). Here, we devised an alternative strategy to induce specific Siglec activation and produce therapeutic effects. In lieu of antibodies, we used a physiological ligand of mouse Siglec-E and its human equivalents (39). We demonstrated that α2,8-linked disialic acid elicited anti-inflammatory effects only when coated onto PLGA nanoparticles. We then showed that these nanoparticles produced therapeutically useful anti-inflammatory effects in a range of murine and human inflammatory models.

Boyd et al. (11) showed that activated Siglec-E recruited SHP phosphatases and ameliorated LPS-induced signaling by reduction of IL-6 and TNF-α secretions. The downstream effects of this nanoparticle engagement in both murine and human model systems agreed with the previously reported findings. We showed that α2,8 NANA-NP can specifically target Siglec-E and reduce inflammatory cytokine production in models of systemic inflammation and localized lung injury. McMillan et al. (10) reported that lung neutrophilia was exacerbated in a murine model of ARDS in a Siglec-E–deficient background. Similarly, we found that nanoparticle-mediated activation of Siglec-E attenuated pulmonary neutrophil counts.

In response to reports verifying the expression of Siglec-E on other cell types such as neutrophils (10, 39), we examined the cellular targets of α2,8 NANA-NP. Upon intraperitoneal administration of fluorescent α2,8 NANA-NP, macrophages appeared to be the primary target, expressing higher levels of Siglec-E compared to neutrophils. The function of the nanoparticles appeared to be neutrophil-independent because the ability of these nanoparticles to rescue animals from lethal inflammation was attenuated after macrophage depletion. Hypothetically, in contrast to macrophages, α2,8 NANA-NP may be unable to cross-link Siglec-E on neutrophils to the extent needed for sufficient activation. Recently, a newly described innate response activator B cell population was shown to play an important role in bacterial clearance and sepsis-associated cytokine responses (40). Our data suggest that macrophage populations are the predominant targeted cell population by α2,8 NANA-NP and are largely responsible for our observed anti-inflammatory effect. However, our experiments cannot completely exclude that the functionalized nanoparticles could directly or indirectly influence these newly described innate effector B cells.

Our previous studies suggested that Siglec-E, and potentially other human equivalents, was up-regulated in a negative feedback process to return cells to homeostasis. Although LPS was able to potently up-regulate the expression of Siglec-E in mouse macrophages, increases in expression of human orthologs was not significant in human MDMs, confirming other reports (41). While investigating this dichotomy, we noted that LPS could enhance IL-10 production in both settings and that this was further potentiated on cotreatment with α2,8 NANA-NP. Others have reported that LPS-induced IL-10 is important for attenuating proinflammatory responses and maintaining tolerance, and thus, this highlights an important role for Siglecs in homeostatic and anti-inflammatory responses (42).

Our alternative approach to targeting Siglec receptors extends the findings of Boyd et al. (11). We report that IL-10 is fundamental for the downstream expression of Siglec-E after LPS stimulation. Moreover, we confirm our previous finding that this Siglec-E induction pathway is also MyD88-dependent. We propose that this negative feedback pathway is exploited by α2,8 NANA-NP to block proinflammatory responses and enhance resolution (fig. S7). In an inflammatory setting, α2,8 NANA-NP engage Siglec-E and potentiate inhibitory pathways, including recruitment of SHP-2 and NFκB p50 dimer translocation to the nucleus. Elevated production of IL-10 then leads to further Siglec-E expression, enhancing the available receptors for nanoparticle binding. Activation of Siglec-E by cross-linking nanoparticles potently induces further secretion of IL-10. An important feature of this approach is that the restoration of normal immune cell function is possible because of the biocompatible and biodegradable nature of this PLGA platform.

The consequences of Siglec-E activation observed here with our nanoparticle are consistent with results from an indirect approach to Siglec modulation in which sialidase inhibitors preserve Siglec-G signal transduction, inhibiting sepsis progression (43). Läubli et al. (44) have recently described the inhibitory effects of Siglec-9 engagement on human neutrophils and macrophages with tumor-expressed sialic acid. Other data show the strategies evolved by bacteria and viruses to evade immunosurveillance mechanisms (45, 46) and enhance dissemination and survival. A variety of sialic acid–bearing “nanocarriers” has been described before. For instance, different sialic acid variants have been exploited to selectively deliver chemotherapeutics to B cells or to enhance nanosurface hydrophilicity, thus improving bioavailability (4750). In another report, liposomes decorated with high-affinity Siglec-7 ligands enabled efficient delivery of mycobacterial ligands to dendritic cells, resulting in a robust T cell immune response (51). Similarly, a liposomal formulation was designed to target macrophage-expressed sialoadhesin, which also elicited T cell activation (52).

In conclusion, we have developed a Siglec-targeting approach that is distinct from other reported strategies. Here, we have shown that nanoparticle surface display of sialic acid promotes oligomerization and activation of Siglecs on macrophages. We have exploited this effect to elicit therapeutically useful effects in murine models of systemic and pulmonary inflammation. We have also demonstrated the translational potential of α2,8 NANA-NP in human tissue, including an EVLP model, an advanced preclinical model for lung-targeting therapeutics. Together, our findings validate this nanotherapeutic approach and confirm Siglecs as druggable anti-inflammatory targets.

MATERIALS AND METHODS

Mice

C57BL/6 specific pathogen–free wild-type mice were purchased from Harlan Laboratories and used at 6 to 8 weeks. IL-10−/− (C57BL/6 background) mice were a gift of the Mouse Genetics Cologne Foundation (53). All experiments were sanctioned by the UK Home Office and approved by Queen’s University Belfast Ethical Review Committee or Irish Department of Health and Children and Trinity College Dublin BioResources Ethical Review Board.

Cell preparation and culture

Peritoneal macrophages were collected through injection of sterile PBS into the peritoneal cavity of wild-type mice. Cells were lavaged and washed once with PBS before counting and adherence to culture plates for 2 hours. Nonadherent cells were discarded, whereas adherent cells were collected by vigorous washing and characterized as macrophages by fluorescence-activated cell sorting (FACS) as CD11b+F4/80+ cells. The murine macrophage RAW 264.7 cell line was obtained from the American Type Culture Collection, and immortalized wild-type or MyD88−/− murine BMDMs were a gift from A. Bowie (Trinity College, Dublin). The above cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% low-endotoxin fetal calf serum, 1% penicillin/streptomycin, and 1% l-glutamine (subsequently referred to as complete DMEM) (PAA Laboratories) during stimulations at 37°C, 5% CO2. Primary murine BMDMs were prepared from C57BL/6 wild-type mice and cultured for 1 week in complete DMEM. Differentiation was driven by granulocyte-macrophage colony-stimulating factor (GM-CSF) derived from L929 supernatant. Human monocytes were isolated from healthy donor buffy coats by centrifugation across a Ficoll-Paque gradient (GE Healthcare), followed by adherence purification. Cells were matured in RPMI supplemented with 10% low-endotoxin fetal calf serum and 1% penicillin/streptomycin. Differentiation of monocytes to macrophages was achieved through the addition of GM-CSF (10 ng/ml) (PeproTech) to the culture medium for 6 days.

Study design

The hypothesis of this investigation was that PLGA nanoparticles coated with a sialic acid ligand would inhibit inflammatory responses of macrophages and monocytes by binding to their cognate Siglec receptor on the cell surface. Investigations were designed to evaluate the impact of sialic acid–coated nanoparticles compared to uncoated nanoparticle and ligand controls. This hypothesis was tested in various human and murine models of inflammation, and experimental replication is defined within the appropriate figure legends. In vivo studies were carried out on littermate animals to minimize variances between groups. The number of animals within each study arm is denoted within the appropriate figure legends. Ethical approval for the use of buffy coat residue from anonymized healthy blood donors for monocyte isolation was obtained from the Northern Ireland Blood Transfusion Service. Experiments were repeated from multiple donors to minimize bias. Human lungs for the EVLP studies were obtained and used with approval from the International Institute for the Advancement of Medicine. Ethical approval for the use of lungs and human donor blood in the EVLP model was obtained from Queen’s University Belfast School of Medicine, Dentistry and Biomedical Sciences School Research Ethics Committee. Both EVLP and CLP studies were conducted in a randomized and blinded manner.

Evaluation of the anti-inflammatory effects of α2,8 NANA-NP

Peritoneal macrophages were stimulated with Escherichia coli (100 ng/ml) R515 LPS (InvivoGen) ± nanoparticles as specified in the appropriate figure legends. Murine BMDMs were stimulated with LPS (1 ng/ml) overnight to up-regulate Siglec-E. BMDMs were washed twice in serum-free DMEM before resting for 2 hours. BMDMs were restimulated with LPS (100 ng/ml) ± nanoparticles.

Fluorescence microscopy imaging

RAW 264.7 cells were seeded on glass coverslips in six-well plates at 2.5 × 105 cells per well and cultured overnight. Cells were stimulated for 3 hours with coumarin 6–loaded nanoparticles (0.25 μg/ml) in serum-free DMEM. Cells were washed three times with ice-cold PBS, fixed in 4% paraformaldehyde for 1 hour, and washed a further three times as above. Cells were incubated for 30 min in Hepes buffer (0.1 M, pH 8) and washed again as above. For nuclear staining, cells were incubated with a 1:200 solution of TO-PRO-3 iodide (Invitrogen) and subsequently washed three times with ice-cold PBS. Cells were visualized by mounting coverslips onto microscope slides using SlowFade Gold reagent (Invitrogen). Sample analyses were performed using confocal scanning laser microscopy (Leica Confocal TCS Sp2) with laser illumination at 488 nm for green fluorescence, 543 nm for red fluorescence, and 633 nm for far-red fluorescence.

Attenuation of Siglec-E activity

Blocking of Siglec-E–dependent binding was achieved by 2-hour pretreatment of cells with anti–Siglec-E (10 μg/ml) (Antibodies-online) or IgG isotype (Fusion Antibodies) before stimulation with LPS, nanoparticles, or controls. Wild-type peritoneal macrophages were plated at 5 × 105 cells per well in 24-well plates. Siglec-E gene silencing was accomplished by transfecting macrophages with GeneCellin nanoporters encapsulating 2 μg of scrambled shRNA or Siglec-E–specific shRNA (OriGene) (11) for 48 hours as per the manufacturer’s instructions. After transfection, macrophages were stimulated for another 24 hours with LPS (100 ng/ml) ± nanoparticles (125 μg/ml).

Immunoblotting and evaluation of NFκB

Immunoprecipitation of Siglec-E (R&D Systems) and analyses of SHP-2 (Santa Cruz Biotechnology) by Western blot were performed as described previously (11) using lysates from LPS ± nanoparticle–treated C57BL/6 macrophages. γ-Tubulin was probed as a loading control (Sigma-Aldrich). Nuclear translocation of NFκB p50 and p65 subunits was assessed using the colorimetric NFκB p50/p65 EZ-TFA Transcription Factor Assay (Millipore) according to the manufacturer’s instructions.

Model of LPS-induced systemic inflammation

C57BL/6 wild-type or IL10−/− mice were treated with intraperitoneal injections of LPS (6 mg/kg) ± nanoparticles or controls dispersed in PBS. Mice were monitored over 50 hours as per UK Home Office guidelines. Mice were culled immediately at a humane endpoint noted by loss of self-righting and insensitivity to touch. Serum was sampled from tail vein bleeds at specified time points and analyzed by ELISA. Serum was obtained by allowing blood to clot at room temperature for 30 min before centrifugation at 14,000g for 10 min.

Model of LPS-induced acute lung inflammation

C57BL/6 wild-type mice were anesthetized and suspended on an endotracheal intubation platform (Penn-Century). A LS-2-M laryngoscope (Penn-Century) was used to illuminate the trachea, and a blunt 24-gauge intravenous catheter (BD) was positioned between the vocal cords. A total volume of 50 μl was instilled into the lungs via the catheter, containing 20 μg of LPS in PBS. At 2 hours after LPS instillation, 2 mg of α2,8 NANA-NP or Nude-NP was injected intraperitoneally. Mice were then sacrificed 24 hours after LPS instillation. To facilitate collection of BAL fluid, a blunt 23-gauge needle was placed within a small opening in the upper trachea and secured in position with Mersilk suture (Ethicon). The lungs were lavaged with a total volume of 700 μl of ice-cold PBS, which was instilled in 350-μl aliquots via the tracheal cannula, followed by gentle aspiration. BAL fluid was centrifuged at 425g for 10 min at 4°C, and the cell pellets were resuspended in 100 μl of ice-cold PBS. Total viable cell counts were conducted using a hemocytometer under trypan blue exclusion. After collection of BAL fluid, lung lobes were homogenized in a TissueLyser LT (Qiagen) for 4 min. Samples were centrifuged at 18,000g for 15 min at 4°C, and supernatant cytokine levels were quantified by ELISA.

Cytocentrifugation, cell staining, and differential counts

Approximately 1 × 105 cells from each BAL fluid sample were cytocentrifuged onto coated cytoslides (Shandon, Thermo Scientific) for 5 min. After fixation in methanol for 20 min, the slides were dried at room temperature and then stained in May-Grünwald solution (VWR) for 8 min. The cytospins were washed quickly in distilled water and submerged in Giemsa stain (VWR) for 8 min. After two brief washes in PBS, the slides were dried at room temperature for 5 min, and a single drop of VectaMount AQ (Vector Laboratories) was added to each cytospin. Coverslips were then positioned, and the slides were allowed to dry overnight. Representative images were captured at ×40 magnification, using a DM5500B light microscope (Leica Microsystems) with Leica AL software (version 3.7). Differential counts were performed on a minimum of 400 white cells per cytospin.

Flow cytometric analyses

Human monocytes and macrophages were stimulated as described in Fig. 6 legend. Cells were stained with antibodies against Siglec-7–PE, Siglec-9–FITC, or isotype controls (BD). In brief, cells were stained as per the manufacturer’s instructions for 30 min on ice. Cells were washed with 3 ml of FACS buffer, centrifuged at 500g for 5 min at 4°C, and resuspended in 200 μl of FACS buffer. Cells were analyzed using a FACSCantoII flow cytometer and FACSDiva (BD) and FlowJo software (Tree Star). Representative plots shown.

Depletion of macrophages using clodronate liposomes

Clodronate liposomes or control PBS liposomes were purchased from www.clodronateliposomes.org. In brief, 300 μl of liposomes was injected intraperitoneally into appropriate mice groups 48 and 24 hours before LPS and nanoparticle treatment. Mice were monitored for survival as above, and serum was collected 24 hours after treatment for analysis of TNF-α by ELISA. Depletion of F4/80+ macrophages was assessed by flow cytometry as described previously to determine percentage of depletion (representative data to ~90% depletion as shown in fig. S3).

Induction of CLP-mediated sepsis

Polymicrobial sepsis was induced in male C57BL/6 wild-type mice using the CLP method as described (25). Briefly, after anesthesia, the cecum was ligated below the ileocecal valve after midline laparotomy. Perforation of the cecum resulted in translocation of bacteria into the peritoneum and subsequent peritonitis. A defined severity of sepsis among animals was ensured with the uniform positioning of the ligation. Postoperative fluid resuscitation was provided with 1 ml of sterile sodium chloride (37°C) administered subcutaneously. Groups of mice were given α2,8 NANA-NP or Nude-NP (2 mg per mouse), equivalent free α2,8 NANA (30 μg per mouse), or dexamethasone (0.1 mg per mouse) intraperitoneally 2 hours after surgery and every subsequent 24 hours. Temperature transponders (Plexx BV) were implanted subcutaneously before surgery, and temperature was recorded electronically (DAS-6007, Bio Medic Data Systems) before and every 12 hours after surgery. Mice were scored every 12 hours using the following criteria: score 0, no symptoms; score 1, piloerection and huddling; score 2, piloerection, huddling, and diarrhea; score 3, lack of interest in surroundings and severe diarrhea; score 4, decreased movement and listless appearance; and score 5, loss of self-righting reflex. When mice reached score 5, they were humanely killed. Analgesia was provided throughout to all mice using buprenorphine.

Detection of secreted cytokines

DuoSet ELISA kits (R&D Systems) were used to detect concentrations of TNF-α, IL-6, IL-8, IL-10, and IL-1β within samples according to the manufacturer’s instructions.

Real-time PCR

After appropriate stimulation, RNA was extracted using the RNeasy Mini Kit (Qiagen). After conversion, 50 ng of first-strand cDNA was used in SYBR Green reactions (Qiagen) using primers previously described (11). Siglec-E mRNA expression was analyzed and normalized to β-actin. Comparison of ΔΔCT values versus unstimulated levels was carried out using MxPro QPCR Software (version 4.10d) (Agilent Technologies).

EVLP model

EVLP setup and operation were as previously described (34). Briefly, lung injury was induced by intrabronchial instillation of 6 mg of LPS. Saline or 5 mg of α2,8 NANA-NP was added simultaneously to the perfusate, thereby modeling systemic delivery. At 4 hours after injury, BAL was performed, and tissue sections were excised for determination of wet/dry ratios to evaluate accumulation of alveolar fluid, as a marker of global lung injury. Tissue sections were weighed immediately to obtain the wet weight, then dried in an oven at 56°C for 48 hours, and weighed again to determine the dry weight. Wet/dry ratios were calculated by dividing the wet weight by the dry weight.

Statistical analysis

Student’s t test was used to determine significance of parametric data between two groups. Mann-Whitney U test was used to determine significance of nonparametric data between two groups. One-way ANOVA and Tukey post hoc test was used to determine significance of parametric data between multiple groups. Two-way ANOVA and Bonferroni post hoc test was used to determine significance of parametric data between wild-type and IL-10−/− mice strains. Survival significance was assessed with Kaplan-Meier plot and log-rank χ2 test. Statistical significance was denoted by asterisks in the appropriate figures (defined as *P < 0.05, **P < 0.01, ***P < 0.001) in comparison to LPS-only control unless indicated otherwise. Error bars represent ± 1 SEM.

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/7/303/303ra140/DC1

Materials and Methods

Fig. S1. Nanoparticle characterization.

Fig. S2. α2,8 NANA-NP bind to Siglec-E and induce receptor clustering.

Fig. S3. Clodronate depletion of macrophages.

Fig. S4. Differential expression of Siglec-E and uptake of fluorescent α2,8 NANA-NP by macrophages and neutrophils.

Fig. S5. α2,8 NANA-NP exhibit no toxicity in preclinical models.

Fig. S6. α2,8 NANA-NP prevent IκB degradation in THP-1 cells and enhance IL-10 production in the EVLP model.

Fig. S7. Schematic of EVLP model and proposed mechanism of action of α2,8 NANA-NP.

REFERENCES AND NOTES

  1. Acknowledgments: We thank D. Jones (Queen’s University Belfast) for his assistance with statistical analyses and L. Hanna, S. Lloyd, and M. Hanna (Queen’s University Belfast) for aid with in vivo experiments. Funding: This work was funded in part through a Medical Research Council Developmental Pathway Funding Scheme project grant MR-J014680 awarded to C.J.S., A.K., D.F.M., C.M.O., and P.G.F. and the Health and Social Care Northern Ireland R&D Division Translational Research Group for Critical Care awarded to C.J.S., C.M.O., and D.F.M. Author contributions: S.S., M.K.G., and F.F. conducted all experiments and drafted the manuscript. E.H. and S.P.S. conducted CLP experiments. U.H. and M.F. conducted EVLP experiments. J.B. performed experiments relating to IL-10–dependent responses. B.K.B. formulated nanoparticle preparations for animal studies. P.S. and E.T. characterized nanoparticle structure. D.M.S. and D.S. helped design and undertake in vitro and in vivo experiments and aided manuscript preparation. C.M.O. and D.F.M. aided in experimental design for the human in vitro and ex vivo experiments, secured ethical approval for the use of human lungs, blood, and buffy coats, and aided in data interpretation and manuscript preparation. D.C.F. provided IL-10−/− animals and aided in experimental design. S.M.A. characterized nanoparticle anti-inflammatory responses in vitro. J.A.J., P.G.F., J.F.B., and A.K. aided in experimental design and manuscript preparation. C.J.S. conceptualized the project and aided in experimental design and manuscript preparation. Competing interests: C.J.S., J.A.J., S.S., F.F., and D.F.M. hold patent US8962032 covering the use of sialic acid–bearing particles as anti-inflammatory agents. D.F.M. has been an advisor to GlaxoSmithKline, Peptinnovate, and Bayer. The other authors declare that they have no competing interests. Data and materials availability: Data and materials are available upon request from the authors. Nanoparticles are available subject to a material transfer agreement.
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