Nucleic acid-binding polymers as anti-inflammatory agents

Abstract

Dead and dying cells release nucleic acids. These extracellular RNAs and DNAs can be taken up by inflammatory cells and activate multiple nucleic acid-sensing toll-like receptors (TLR3, 7, 8, and 9). The inappropriate activation of these TLRs can engender a variety of inflammatory and autoimmune diseases. The redundancy of the TLR family encouraged us to seek materials that can neutralize the proinflammatory effects of any nucleic acid regardless of its sequence, structure or chemistry. Herein we demonstrate that certain nucleic acid-binding polymers can inhibit activation of all nucleic acid-sensing TLRs irrespective of whether they recognize ssRNA, dsRNA or hypomethylated DNA. Furthermore, systemic administration of such polymers can prevent fatal liver injury engendered by proinflammatory nucleic acids in an acute toxic shock model in mice. Therefore these polymers represent a novel class of anti-inflammatory agent that can act as molecular scavengers to neutralize the proinflammatory effects of various nucleic acids.

Pattern-recognition receptors (PRRs) allow immune cells to recognize and protect tissues from various harmful stimuli, such as pathogens and damaged cells (1, 2). They recognize a diverse set of molecular signatures termed pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) (2), and act as innate and adaptive immune sensors by recognizing such PAMPs and DAMPs (3). Toll-like receptors (TLRs) are the best characterized PRRs (4). At least 10 human and 12 mouse TLRs have been identified. Each TLR is able to recognize a particular molecular pattern. For instance, TLR2, 4, 5, 6, and 11 bind to bacterial membrane-associated molecules such as lipoprotein, lipopolysaccharide (LPS), and peptidoglycan whereas TLR3, 7, 8 and 9 recognize bacterial, viral, or endogenous nucleic acids including ssRNA, dsRNA, and unmethylated CpG-containing DNA (5). Moreover, TLRs can be classified based on their cellular localization: TLR1, 2, 4, 5, and 6 are expressed on the cell surface whereas TLR3, 7, 8, and 9 are localized mostly in the endosomal compartment (5). Activation of TLRs upon ligand binding results in signaling events that lead to the expression of immune response genes including inflammatory cytokines, immune stimulatory cytokines, chemokines, and costimulatory molecules, which augment the killing of pathogens and initiates the process of developing adaptive immunity (6, 7). Inappropriate activation of TLRs, on the other hand, contributes to development of a variety of human diseases including systemic lupus erythematosus (SLE), bacterial sepsis, inflammatory bowel disease, psoriasis, multiple sclerosis, rheumatoid arthritis, and atherosclerosis (8–10). Upon infection or injury of tissue, the infected or damaged tissue releases various intracellular factors that can recruit and activate innate immune cells. Nucleic acids that originate from host cells or intracellular microorganisms can be recognized by nucleic acid-sensing TLRs and can result in the induction of pathological inflammatory responses (Fig. S1) (2). In patients with bacterial sepsis, toxic shock, SLE or rheumatoid arthritis, hypomethylated CpG DNAs, or RNAs have been detected in extracellular compartments and these extracellular nucleic acids have been correlated to the pathogenesis of these diseases (11–13). Although inhibition of one or two nucleic acid-sensing TLRs using receptor antagonists has been demonstrated to attenuate disease progression in polymicrobial sepsis and autoimmune disease models to some extent (12, 14–16), the redundancy of the TLR family of proteins suggests that concurrent inhibition of all nucleic acid-sensing TLRs would be the most effective means to control nucleic acid-induced inflammation in humans. Because all the nucleic acid-sensing TLRs bind to RNA or DNA, albeit each TLR ligand contains a distinguishable molecular pattern, we hypothesized that agents that bind to DNAs and RNAs regardless of their sequence, structure or chemistry might be able to inhibit nucleic acid-mediated activation of all RNA- and DNA-sensing TLRs. Herein we demonstrate that certain cationic polymers can act as molecular scavengers and block the immune stimulatory effects of extracellular ssRNA, dsRNA, and unmethylated DNA.

Results

Nucleic Acid-Binding Polymers Inhibit Nucleic Acid-Mediated Activation of TLRs.

We initially evaluated six agents known to bind nucleic acids for their ability to attenuate nucleic acid-mediated activation of TLRs on macrophages: polyphosphoramidate polymer (PPA-DPA), polyamidoamine dendrimer, 1,4-diaminobutane core-PAMAM-G3 (PAMAM-G3), poly-L-lysine, β-cyclodextrin-containing polycation (CDP), hexadimethrine bromide (HDMBr), and protamine sulfate (Table S1). All of the compounds except protamine sulfate inhibit TLR3 activation by synthetic dsRNA, polyinosinic-polycytidylic acid (poly I∶C) as measured by TNFα and IL-6 production and CD80 expression (Fig. 1A and Fig. S2). Moreover three of the cationic polymers, CDP, HDMBr, and PAMAM-G3 inhibit the ability of synthetic CpG DNAs (CpG 1668) to activate TLR9 (Fig. 1A). Similarly these three polymers inhibit the ability of ssRNA-lipid complexes (ssRNA40) to activate TLR7 (Fig. 1A). Such cationic polymers are specific for nucleic acid-mediated activation of the TLRs. They do not inhibit activation of TLRs that recognize nonnucleic acid-based TLR agonists such as bacterial LPS, which activate TLR4, and Pam3CSK4, a synthetic bacterial lipoprotein, which activates the TLR2/1 complex (Fig. 1A and Fig. S2). Furthermore CDP, HDMBr, and PAMAM-G3 neutralize immune stimulatory activity of all types of nucleic acid-based TLR agonists in a variety of primary cells including B cells, fibroblasts and dendritic cells (DCs) (Fig. S3).

Fig. 1.

Nucleic acid-binding polymers neutralize the ability of nucleic acids to activate inflammatory cells. Nucleic acid-binding polymers, CDP, HDMBr and PAMAM-G3, (20 μg/mL) inhibited the activation of the murine macrophage cell line, RAW264.7, by (A) synthetic nucleic acid-based TLR agonists, ssRNA40 (TLR7), poly I∶C (TLR3), CpG1668 (TLR9) or (B) genomic DNAs from bacteria (E. coli) and calf thymus (CT DNA) (TLR9) but did not inhibit cell activation by (A) the synthetic nonnucleic acid TLR agonists, Pam3CSK4 (TLR2) and LPS (TLR4) and (B) endogenous DAMP, heparan sulfate (TLR4). To transform immunological inert mammalian CT DNA into immune stimulatory DNA CT, a DNA/DOTAP complex was prepared by incubation of 5 μg of CT-DNAs with 15 μg of DOTAP in 30 μL Hepes-buffered saline buffer (20 mM Hepes, 150 mM NaCl, pH 7.4). (C, D) To study spatiotemporal regulation of TLR9 signaling (C) bone marrow-derived plasmacytoid DCs and (D) RAW cells were incubated with CpG 1585 (1 μM) in the presence or absence of cationic polymers, CDP, HDMBr or PAMAM-G3 (20 μg/mL). (E) To study activation of cytosolic dsRNA sensors, PKR and MDA5, 1 μg of poly I∶C was complexed with 1 μL of DharmaFECT (Thermo Scientific) in 50 μL of Opti-MEM I media (Invitrogen). RAW264.7 cells and MEFs were stimulated with the poly I∶C/DharmaFECT complex, poly I∶C alone, DharmaFECT alone or PBS in the presence or absence of HDMBr or PAMAM-G3. TNFα, IL-6 and IFNα production were measured by ELISA to monitor the activation of the cells. Data represents the mean of three individual experiments. Error bar is SD; ∗ P < 0.05. ‡ Amount of cytokine = [cytokine]_{poly I∶C or poly I∶C/DharmaFECT} - [cytokine]_{PBS or DharmaFECT}

Next we asked whether the cationic polymers could neutralize the immune stimulatory activity of endogenous nucleic acids. As expected, the three polymers, CDP, HDMBr, and PAMAM-G3 inhibited bacterial DNA-induced cellular activation (Fig. 1B). Unlike bacterial DNAs, mammalian genomic DNAs are highly methylated and immunologically inert. In various pathological conditions, autoantibody, nuclear protein or endogenous cationic protein form a complex with mammalian DNAs, and these DNA-containing complexes are able to activate innate and adaptive immune cells through TLR9 signaling (reviewed in ref. 17). Consistent with previous findings, mammalian DNA (CT DNA) only weakly activated mouse macrophages to produce TNFα, but CT DNA/DOTAP (N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate) complexes induced much higher levels of TNFα production from these cells (Fig. 1B). This inflammatory cytokine production was inhibited upon treatment with cationic polymers, CDP, HDMBr, or PAMAM-G3. Moreover, the inhibitory cationic polymers did not inhibit nonnucleic acid-based endogenous DAMPs. Heparan sulfate is a strongly negatively charged polysaccharide found in cell surface or extracellular matrix. When harmful stimuli insults tissues the heparan sulfates are enzymatically cleaved from cellular or extracellular matrix macromolecules and recognized by TLR4 of innate immune cells (18). The CDP, HDMBr, and PAMAM-G3 did not inhibit heparan sulfate-mediated TNFα production by cells (Fig. 1B). This data suggest that the nucleic acid-binding polymers specifically neutralize immune stimulatory activity of ncleic acids, and this target specificity is not just governed by the electrostatic interaction between oppositely charged molecules.

It has been shown that binding of CpG oligodeoxynucleotides (ODNs) to TLR9 in the early endosomal compartment leads predominantly to a type I IFN response whereas activation in the late endosomal compartment induces inflammatory cytokines and immune stimulatory receptors but not type I IFN (19, 20). Multiple types of CpG ODNs have been identified that preferentially activate the early, late or both endosomal responses. Type A CpG ODNs are characterized by poly-G tails at both ends and a palindromic sequence in the middle, and they activate plasmacytoid DCs (pDCs) to produce type I IFNs through MyD88-IRF7 signaling pathway, but only weakly activate other immune cells to produce inflammatory cytokines (21, 22). Type B CpG ODNs contain linearized sequences and activate B cells, conventional DCs and other inflammatory cells to produce inflammatory cytokines (e.g., TNFα and IL-6) through MyD88-NF-κB signaling pathway, but not pDCs to produce type I IFN (23, 24). Interestingly, lipoplex can regulate endosomal location of CpG ODNs and consequently alter the downstream signaling of TLR9 (19). To determine if the inhibitory nucleic acid-binding polymers, CDP, HDMBr, and PAMAM-G3, form complexes with CpG ODNs that are retained in the early endosome, we evaluated whether these polymers would skew TLR9 signaling toward type I IFN expression and away from TNFα and IL-6 production. CDP inhibited TNFα production by macrophage cells after stimulation with either type A CpG ODN (CpG 1585) or type B CpG ODN (CpG 1668) but did not inhibit IFNα production from pDCs after stimulation with either type A or type B CpG ODN. By contrast, HDMBr and PAMAM-G3 inhibit both TNFα and IFNα production by pDCs and macrophage cell lines after stimulation with all types of CpG ODNs (Fig. 1 C and D).

In addition to endosomal nucleic acid-sensing TLRs microbial dsRNAs can be recognized by cytosolic dsRNA-sensing PRRs, including protein kinase R (PKR) and RIG-I-like receptor family members such as melanoma differentiation-associated gene 5 (MDA-5) and retinoic acid inducible gene-I (RIG-I). Although both endosomal TLRs and cytosolic dsRNA sensors are able to recognize exogenous dsRNAs, their activation leads to distinctive downstream signaling pathways resulting in expression of different types of genes. For example, cytosolic dsRNA-sensing PRRs induce type I IFN production through IFN regulatory factor 3 (IRF-3)-dependent pathways whereas endosomal dsRNA-sensing TLR3 induce inflammatory cytokine production through IRF-3-independent but NF-kB-dependent pathways (25). It is well established that treatment of cells with poly I∶C dsRNA complexed with a transfection reagent activates cytosolic PKR and MDA5 signaling pathways that leads to type I IFN production; whereas cells treated with poly I∶C alone predominantly induce endosomal TLR3 signaling pathways (26, 27). As shown in Fig. 1E, HDMBr and PAMAM-G3 can block poly I∶C/DharmaFECT-mediated activation of type I IFN production from treated cells. Thus HDMBr and PAMAM-G3 are able to effectively and specifically inhibit the activation of multiple nucleic acid-sensing PRRs on a variety of inflammatory cell types. Therefore, these two polymers were chosen for further study.

Nucleic Acid-Binding Polymers Neutralize Extracellular Inflammatory Nucleic Acids, Rather than Directly Inhibiting Nucleic Acid-Sensing TLRs.

The two polymers, HDMBr and PAMAM-G3, were able to neutralize the immune stimulatory activity of nucleic acid-based TLR agonists whose receptors are located in endosomal compartments. This inhibitory effect can be similarly achieved by treatment of cells with chloroquine that is an inhibitor of endosomal acidification (28–30). Nucleic acid-binding polymers containing protonable amines (-NH₂) have been shown to induce a proton sponge effect inside endosomes, which promotes osmotic swelling and disruption of the endosomal membrane (31). To determine if HDMBR and PAMAM-G3 were working by this mechanism, we studied the inhibitory effect of polymers on TLR7 activated by either a nucleic acid-based agonist (ssRNA40) or nonnucleic acid-based agonist (imidazoquinoline compound, R848). Although the polymers inhibited the activation of macrophages treated with ssRNA40, they did not inhibit stimulation of cells treated with R848. If macrophages were coincubated with ssRNA40, R848, and either nucleic acid-binding polymer, these cells generated reduced levels of inflammatory cytokines production comparable to the level from cells treated with R848 alone. By contrast, treatment of macrophages with chloroquine inhibited inflammatory cytokine production from cells treated with ssRNA40 and R848 (Fig. 2A). Furthermore, pretreatment of cells with chloroquine prior to stimulation prevented the activation of the cells with CpG ODNs. By contrast, preincubation of macrophages with the polymer-based inhibitors and subsequent removal of the polymers did not prevent inflammatory cytokine production from the cells subsequently stimulated with CpG ODNs even though pretreatment and continuous treatment of macrophages with the cationic polymers prevents CpG ODN-induced TNFα production (Fig. 2B). Thus HDMBr and PAMAM-G3 do not directly inhibit the nucleic acid-sensing TLRs but rather neutralize the nucleic acid-based receptor ligands.

Fig. 2.

Nucleic acid-binding polymers selectively inhibit nucleic acid-based TLR ligands. (A) RAW264.7 cells were coincubated with nucleic acid-binding polymers (HDMBr or PAMAM-G3), nucleic acid TLR7 agonist (ssRNA40) or nonnucleic acid TLR7 agonist (R848). Endosomal acidification inhibitor, chloroquine, was used as an inhibitor of TLR7. Cationic polymers inhibited TLR7 activation by the nucleic acid-based agonist but not by the nonnucleic acid agonist. (B) Cells were preincubated for 2 h with nucleic acid-binding polymer, chloroquine or PBS. CpG 1668 (1 μM), then, were added into cell culture after 3× washing with complete medium or nonwashing. Cells were incubated for additional 6 h and TNFα production was analyzed by ELISA. Data represents the mean of two individual experiments. Error bar is SD.

Nucleic Acid-Binding Polymers can Alter the Uptake and Intracellular Distribution of Immune Stimulatory Nucleic Acids.

Next we wanted to explore the mechanism(s) that nucleic acid-binding polymers utilize to neutralize the immune stimulatory activity of nucleic acids. We speculated that the HDMBr and PAMAM-G3 may act by inhibiting the cellular uptake of immune stimulatory nucleic acids. To evaluate this possibility we performed cellular uptake studies using fluorescently labeled CpG ODNs. PAMAM-G3 and HDMBr slightly reduced cellular uptake of CpG ODNs whereas noninhibitory cationic molecules, PPA-DPA, poly L-lysine and protamine, significantly increased intracellular uptakes of CpG ODNs (Fig. 3A). Nevertheless the mean fluorescence intensity of cells coincubated at 37 °C with combination of CpG ODNs and HDMBr or PAMAM-G3 was significantly greater than that of cells at 4 °C (CpG 1668 alone vs. CpG 1668 + HDMBr: P = 0.0493; CpG 1668 + PAMAM-G3: P = 0.0165), which suggests that cells internalized significant amounts of CpG ODNs even in the presence of HDMBr and PAMAM-G3. Thus, the polymers do not appear to exclusively act by limiting the ability of nucleic acids to associate with cells. Next, because different nucleic acid-binding polymers are known to deliver genes into cells through diverse internalization and trafficking pathways, which is influenced by molecular composition, surface charge and size of complexes (32), we investigated whether the subcellular localization of CpG ODNs was altered by the polymers. As shown in Fig. 3B, when no polymer is added, a CpG ODN is internalized into macrophages and is found in the cytosol as well as the nucleus. In the cytosol, the DNA appears to form a punctate pattern that at least in part colocalizes with TLR9. Addition of HDMBr or PAMAM-G3 to the CpG ODNs appears to not only reduce internalization of the DNA into cells but the polymers also alter CpG distribution largely toward the nucleus where it does not colocalize with TLR9. By contrast, addition of noninhibitory polymer, PPA-DPA, to CpG ODN enhances this punctate appearance in the cytosol and appears to further colocalize the DNA with TLR9 (Fig. 3B). Thus HDMBr and PAMAM-G3 appear to alter the uptake and distribution of CpG ODNs in cells that likely accounts at least in part for their ability to limit nucleic acid-mediated activation of TLR9.

Fig. 3.

Modification of cellular internalization of CpG DNAs by nucleic acid-binding polymers. (A) Cellular uptake of nucleic acids was studied by quantification of Alexa488-CpG ODNs in cells. RAW264.7 cells were incubated at 37 °C for 1 h with Alexa488-conjugated CpG 1668 (1 μM) in the presence or absence of HDMBr, PAMAM-G3, PPA-DPA, poly-L-lysine or protamine sulfate (10 μg/mL). CpG DNA internalization was analyzed by flow cytometry. Filled gray histogram: unlabeled CpG 1668. Open red histogram: Alexa488-CpG 1668 + cationic polymer. Open black histogram: Allexa488-CpG 1668 alone. To assess nonspecific uptakes or surface binding of CpG ODNs, the cells were incubated at 4 °C for 1 h with Alexa488-CpG 1668 and nucleic acid-binding polymers. (B) To study intracellular trafficking of CpG ODNs, RAW cells were incubated at 37 °C for 1 h with Alexa488-CpG 1668 and HDMBr, PAMAM-G3 or PPA-DPA. After extensive washing, the cells were stained with an anti-TLR9 Antibody (red) and DAPI (blue). Intracellular localization of CpG DNAs was analyzed by a confocal microscopy. Colocalization of CpG 1668 and TLR9 in endosomal compartments formed fluorescent puncta in yellow. Cells coincubated with CpG 1668 and noninhibitory polymer, PPA-DPA increased size and intensity of puncta comparing CpG 1668 alone whereas coincubation with CpG 1668 and HDMBr or PAMAM-G3 did not form puncta (X63).

Cationic Polymers can Protect Mice from Nucleic Acid-Induced Toxic Shock and Fatal Inflammation.

Finally, we evaluated the ability of the nucleic acid-binding polymers to control pathogenic activation of TLRs by freely circulating nucleic acids in an in vivo toxic shock model. When mice that have been sensitized with D-galactosamine (D-GalN) are challenged with TLR agonists these mice will succumb to TNFα-dependent lethal liver damage and die shortly (33, 34). Consistent with previous reports, we observed that greater than 90% of mice die following administration of D-GalN and CpG DNA or poly I∶C RNA within 48 h. Strikingly, coadministration of the nucleic acid-binding polymers, HDMBr or PAMAM-G3, following D-GalN and CpG DNA or poly I∶C injection resulted in significant protection of animals, in a dose-dependent manner, and reduced mortality by 100% in several cases (Fig. 4). By contrast such polymers are unable to protect mice from nonnucleic acid TLR activators such as LPS (Fig. S4). Thus certain nucleic acid-binding polymers are also able to bind freely circulating nucleic acids in vivo and thereby inhibit the potent nucleic acid-induced toxic shock response that such free nucleic acids engender in mice.

Fig. 4.

Nucleic acid-binding polymers limit nucleic acid-induced acute liver injury. (A) Prevention of CpG DNA- and (B) poly I∶C RNA-mediated liver injury. Mice were injected i.p. with D-galactosamine (D-GalN) + CpG 1668 or D-GalN + poly I∶C. Subsequently, PBS (black circle), HDMBr (red triangle) or PAMAM-G3 (dark green square) at the indicated amounts was administered i.p. Mice injected with D-GalN, CpG 1668, poly I∶C, HDMBr or PAMAM-G3 alone or D-GalN + GpC 1720 were used as controls (gray asterisk). Mice were monitored daily for survival.

Discussion

Our studies demonstrate that certain nucleic acid-binding polymers can act as molecular scavengers and inhibit the ability of circulating immune stimulatory nucleic acids, regardless of their sequence, chemistry, or structure, to activate a variety of nucleic acid-sensing TLRs and cytoplasmic PRRs in vitro and in vivo. Increasing evidence indicates that inappropriate activation of nucleic acid-sensing TLRs and cytoplasmic PRRs can result in a variety of human inflammatory and autoimmune diseases including SLE, sepsis, inflammatory bowel disease, psoriasis, multiple sclerosis, and rheumatoid arthritis (8–10). Thus, unique strategies to control such pathogenic activation of these receptors are needed. Unfortunately, the interconnectedness and redundancy of nucleic acid-induced TLR and PRR signaling pathways will likely limit the therapeutic efficacy of single or dual TLR/PRR inhibitors (Fig. S1). TLR signaling, irrespective of which TLR is activated, culminates in activation of MAP kinases, NF-κB, and IFN regulatory factors and engenders the production of inflammatory cytokines and/or type I IFN (5). Our proof-of-concept studies describe a unique strategy that employs nucleic acid-binding polymers to simultaneously inhibit the ability of freely circulating nucleic acids to activate all nucleic acid-sensing TLRs and cytoplasmic PRR.

We have previously shown that cationic polymers can serve as antidotes for nucleic acid aptamers and rapidly reverse the effects of such agents in vivo (35). Results in this study complement our previous findings and demonstrate that these polymers can also interact with endogenous nucleic acids to inhibit activation of nucleic acid-sensing TLRs and cytplasmic PRRs. One of the most exciting applications of nanomedicine has been to engineer nanocomplexes that can deliver exogenous drugs and genes to target tissues in a spatially and temporally controlled manner. This study shows that nucleic acid-binding polymers used for such nanotherapeutic applications can instead be applied in a unique manner to form complexes with various extracellular nucleic acids in situ. This unique concept may have several therapeutic applications. Extracellular nucleic acids released by dead and dying cells play a role not only for the aberrant inflammatory responses by overactivation of endosomal TLRs, but also for other pathological responses; e.g., microvascular permeability, blood coagulation, and thrombosis. Extracellular RNAs has been shown to directly bind to vascular endothelial-cell growth factor (VEGF) and stabilize the interaction of VEGF and cognate receptor, VEGF-R2, which lead to significantly increase the permeability of microvascular endothelial cells that is one of common pathologic conditions associated with brain tumor, head injury and ischemic stoke (36). Moreover, extracellular RNAs were found to mediate blood coagulation by activation of Factor XII and XI, proteases of the contact factor pathway of blood coagulation (37), and treatment of mice with RNase significantly delays occlusive thrombus formation in a murine model of arterial thrombosis (37). Though not directly evaluated herein, we speculate that this same strategy may also be useful for inhibiting the ability of extracellular nucleic acids from stimulating endothelial cells and blood coagulation pathways as well.

HDMBr is broadly used to facilitate viral transduction, and it can bind to and neutralize negatively charged viral membrane, resulting in decreasing of virus-cell repulsion and increasing of virus absorption and transduction (38). HDMBr is also known to neutralize highly negatively charged anticoagulation agent, heparin (39). The PAMAM dendrimer is a macromolecular platform for carrying drug, gene, siRNA and imaging agents in vivo (40). Depending on their surface chemistry PAMAM dendrimers can also act as active therapeutic agents. For example, PAMAM-G5 with alkylation at surface primary amines neutralizes LPS. Treatment with the PAMAM-G5 derivative could protect D-GalN-sensitized mice from toxic shock upon LPS challenge (41). Furthermore, Shaunak et al. has shown that anionic polymer, PAMAM-G3.5, covalently conjugated with glucosamine inhibited a variety of cellular activity including LPS-mediated TLR4 activation, fibroblast growth factor-2 (FGF-2)-mediated endothelial-cell proliferation and allogeneic mixed leukocyte reaction (42). In the current study, we found that cationic polymers, PAMAM-G3 and -G5 and HDMBr inhibited nucleic acid-sensing TLRs by neutralization of immune stimulatory nucleic acids. Although we do not clearly understand the inhibitory mechanism of polymers the HDMBr and PAMAM seems to alter intracellular distribution of immune stimulatory nucleic acids and sequester the nucleic acids from their corresponding TLRs. In the thermodynamic and stoichiometric studies, we have observed correlation between the polymer-mediated neutralization of CpG ODNs and the strength of binding between the polymer and CpG ODN (Table S2 and Fig. S5). Additional studies will be required to develop a thorough understanding of the mechanism(s) employed by various nucleic acid-binding polymers to inhibit activation of nucleic acid-sensing TLR.

Though the development of any unique therapeutic strategy requires extensive testing and development, the current study suggests that certain cationic polymers that are being utilized in a variety of nanomedicine applications may be useful for a totally unique application, to act as molecular scavengers and thereby limit nucleic acid-induced inflammation in patients with a broad spectrum of inflammatory and autoimmune diseases. It is worth noting that the nucleic acid-binding materials we evaluated herein were not originally designed with the intent of using them to control nucleic acid-induced inflammation. Therefore we anticipate that a significant translational research opportunity now exists to develop optimized nucleic acid-binding polymers for the treatment of patients with nucleic acid-induced inflammatory and autoimmune diseases.

Materials and Methods

In Vitro TLR Activation and Inhibition.

Mouse embryonic fibroblasts (MEFs), 5 × 10⁵ of RAW264.7 cells, immature DCs, or B cells were incubated for 18 h in complete medium in a 48-well culture plate. In a 96-well flat-bottom culture plate, 8 × 10⁴ of pDCs were incubated for 48 h. To activate TLR9, the complete medium was supplemented with phosphorothioate type B CpG oligodeoxynucleotide (ODN) 1668 (CpG 1668) (5′-TCCATGACGTTCCTGATGCT-3′) (IDT), type A CpG ODN 1585 (CpG 1585) (5′-GGGGTCAACGTTGAGGGGGG-3′) (1 μM) (InvivoGen) or DNAs isolated from calf thymus or Escherichia coli (5 μg/mL) (Sigma). Phosphorothioate GpC DNA 1720 (GpC 1720) (5′-TCCATGAGCTTCCTGATGCT-3′) (IDT) and CpG 1585 control (5′-GGGGTCAAGCTTGAGGGGGG-3′) (InvivoGen) were used as control DNAs. For activation of TLR2, 3 and 4, Pam3CSK4 (1 μg/mL) (InvivoGen), poly I∶C (10 μg/mL) (Amersham/GE Healthcare), LPS serotype 026:B6 (100 ng/mL) (Sigma) and heparan sulfate (kindly provided by Todd Brennan, Duke University) were added. Finally, phosphorothioate ssRNA40/LyoVec (5 μg/mL) (5′-GCCCGUCUGUUGUGUGACUC-3′), a control ssRNA41/LyoVec (5 μg/mL) (5′-GCCCGACAGAAGAGAGACAC-3′) or a synthetic Imidazoquinoline compound, R848 (5 μg/mL) (all from InvivoGen) were used to activate mouse TLR7. To inhibit TLR activation PAMAM-G1, -G3 and -G5, poly-L-lysine, HDMBr (all from Sigma), CDP (kindly provided by Mark Davis, California Institute of Technology), protamine sulfate (APP), PPA-DPA [MW 30 kDa, synthesized as previously described (43)] at various concentrations were added and cells were treated with TLR agonists. After incubation, cells were analyzed for CD80 expression by flow cytometry after staining with either phycoerythrin (PE)-labeled anti-mouse CD80 or PE-hamster IgG isotype control (all from eBioscience). Culture supernatants were collected and analyzed. The production of TNFα and IL-6 were analyzed with BD OptEIA™ ELISA sets (BD Biosciences); IFNα production was determined with a Mouse IFNα ELISA kit (PBL Biomedical Laboratories) following the manufacturer’s instructions.

Cellular Uptake of CpG DNAs.

In a 24-well culture plate, 2 × 10⁵ of RAW264.7 cells were cultured overnight. After washing three times with fresh culture medium 300 μL of cold complete medium were added onto each well and cells were incubated for 15 min at 4 °C. After cold incubation, CpG 1668 conjugated at 3′ end with Alexa-488 (1 μM) (IDT) in the presence or absence of cationic polymers (20 μg/mL) was supplemented into culture media. Fluorescently unlabeled CpG 1668 was used to assess autofluorescence. Cells were incubated at either 37 °C or 4 °C. After a 1-h incubation, cells were washed five times with cold PBS and harvested by treatment with 0.25% typsin-EDTA. After washing once with cold PBS, fluorescence intensity of cells was measured by flow cytometry.

Intracellular Localization of CpG DNA.

On a 1 mm glass cover slip in a 24-well culture plate, 2 × 10⁵ RAW264.7 cells were cultured overnight. The cells were incubated with CpG 1668-Alexa 488 (1 μM) at 37 °C. After a 1-h incubation, cells were washed five times with cold PBS and fixed with a 4% paraformaldehyde solution followed by permeablization with 0.25% Saponin and 1% BSA in PBS. The cells were blocked by incubation for 30 min with Image-iT FX signal enhancer (Invitrogen) and stained with mouse anti-human TLR9 (1/100 dilution) (Imgenex) and Cy5-conjugated goat anti-mouse IgG secondary antibody (1/1000 dilution) (Abcam). Localization of CpG DNAs and TLR9 was observed under a Zeiss LSM 510 inverted confocal microscope and images was analyzed using a Zeiss LSM image software.

Mouse TNFα-Mediated Acute Liver Injury.

TNFα-mediated acute liver injury in a D- galactosamine-sensitized mice was performed as previously described (33, 34). Briefly, C57BL/6 mice were injected i.p. with PBS (100 μL), CpG 1668 (51 μg), GpC DNA (51 μg), poly I∶C (200 μg), or LPS (2 μg) with or without D(+)Galactosamine, Hydrochloride (D-GalN) (EMD Biosciences) (20 mg). HDMBr or PAMAM-G3 (200 to 400 μg) or PBS were injected i.p. 5–10 min after toxin challenge. Viability of mice was monitored daily for a week.