A synthetic poly(A) tail targeting eCIRP inhibits sepsis

Abstract

Sepsis is an infectious inflammatory disease that often results in acute lung injury (ALI). Cold-inducible RNA-binding protein (CIRP) is an intracellular RNA chaperon that binds to mRNA’s poly(A) tail. However, CIRP can be released in sepsis, and extracellular CIRP (eCIRP) is a damage-associated molecular pattern, exaggerating inflammation, ALI, and mortality. Here, we developed an engineered poly(A) mRNA mimic, AAAAAAAAAAAA, named A₁₂ with 2′-O-methyl ribose modification and terminal phosphorothioate linkages to protect it from RNase degradation, exhibiting an increased half-life. A₁₂ selectively and strongly interacted with the RNA-binding motif of eCIRP, thereby preventing eCIRP’s binding to its receptor, TLR4. In vitro treatment with A₁₂ significantly decreased eCIRP-induced macrophage MAP kinase and NF-κB activation and inflammatory transcription factor upregulation. A₁₂ also attenuated pro-inflammatory cytokine production induced by eCIRP in vitro and in vivo in macrophages and mice, respectively. We revealed that treating cecal ligation and puncture-induced sepsis with A₁₂ significantly reduced serum organ injury markers and cytokine levels and ALI, decreased bacterial loads in the blood and peritoneal fluid, ultimately improving their survival. Thus, A₁₂’s ability to attenuate the clinical models of sepsis sheds lights on inflammatory disease pathophysiology and prevention of the disease progress.

Introduction

Sepsis is a deadly inflammatory syndrome caused by the dysregulated host response to infection (1). In the US, there are approximately 1 million sepsis cases with a mortality rate of up to 40%, costing more than $24 billion annually (2, 3). Pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharides (LPS), present in the bacterial cell wall initiate infectious inflammatory responses by recognizing pattern recognition receptors (PRRs) such as toll-like receptor 4 (TLR4) (4). On the other hand, damage-associated molecular patterns (DAMPs) are endogenous molecules released from stressed or damaged cells that also induce aberrant immune responses to cause organ injury via PRRs in sepsis (4). The lungs are susceptible to severe inflammation and injury, given the rapid infiltration of neutrophils. Thus, sepsis is often accompanied by acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) to aggravate mortality rates (5).

Cold-inducible RNA-binding protein (CIRP) is an 18-kD RNA chaperone. CIRP binds poly(A) tail of mRNA, regulating the translation of its target mRNA (6, 7). Under stressed or inflammatory conditions, CIRP is released from cells through active or passive release mechanisms, and extracellular CIRP (eCIRP) serves as a new DAMP (6, 8). eCIRP promotes proinflammatory responses by binding to TLR4 (6). It has been shown that the injection of healthy mice with eCIRP causes inflammation and ALI, while CIRP^−/− mice are protected from systemic inflammation and ALI in sepsis (9, 10). Sepsis patients have shown elevated levels of serum eCIRP, which correlate with the severity of sepsis (11). Thus, targeting eCIRP may provide a novel avenue to prevent the development of ALI caused by sepsis. A recent study identified a 15-amino acids (aa) small peptide derived from eCIRP to target eCIRP-TLR4 axis (12). Nonetheless, due to its short half-life and the possible interference with the binding of other TLR4 ligands may make it challenging to implement it as an effective and specific eCIRP’s antagonist. Thus, identifying a new molecule that directly neutralizes eCIRP to impede its binding to TLR4 could be a promising eCIRP’s antagonist to prevent inflammation.

Since CIRP binds to the poly(A) tail of mRNA, a synthetic RNA that mimics the poly(A) tail of mRNA likely binds to eCIRP and can be a novel inhibitor for eCIRP. With the aid of computational modeling, we revealed that a 12-base poly(A) tail optimally binds to the eCIRP. Since RNA mimic is susceptible to nuclease degradation, to increase its stability, we have engineered poly(A) tail of mRNA mimic by methylating 2′-O ribose in every nucleotide and incorporating phosphorothioate linkages at the terminal adenosine residues. This synthetic poly(A) showed a strong affinity with eCIRP. In the present study, we named this oligonucleotide A₁₂ and sought to investigate its efficacy as an eCIRP inhibitor in sepsis. Here, we demonstrate that A₁₂ targets eCIRP to attenuate inflammation, protect mice from ALI, and improve survival after sepsis.

Materials and Methods

Animals

Male C57BL/6 mice, 8–12-week-old, were purchased from Charles River Laboratories (Wilmington, MA). Mice were housed in a temperature-controlled room with 12-hour (h) intermittent light and dark cycles and fed a standard mouse chow diet with water. Male 7–9-week-old Sprague–Dawley rats (Charles River Laboratories) were housed in a temperature-controlled room with 12-h intermittent light and dark cycles and fed a standard rat chow diet with water. All animal experiments were preformed following the National Institutes of Health guidelines for the care and use of laboratory animals and were approved by our Institutional Animal Care and Use Committee (IACUC).

Computational modeling

The amino acid sequences of mouse CIRP (P60824), high mobility group box 1 (HMGB1) (P63158), Histone H3 (P02301) and TLR4 (Q9QUK6) were retrieved from Uniprot database. The structure models of CIRP, HMGB1, Histone H3, and TLR4 were generated using template-based modeling approach ITasser (Iterative Threading ASSEmbly Refinement) (13). The model structures were built using templates with maximum percentage identity, sequence coverage and confidence. The structure models were refined using repetitive relaxations by short molecular dynamics simulations for mild (0.5 ps) and aggressive (0.8 ps) relaxations with 4 fs time step after structure perturbations. The refinement process enhanced certain parameters such as increased Rama favored residues and decrease in poor rotamers. The protein-protein docking was performed using ATTRACT tool with conformational flexibility of binding partners (14). In the docking process potential energy is pre-calculated on a grid and then interactions are calculated by interpolations from nearest grid point. Moreover, docking process includes several energy minimizations steps. For protein-protein docking GRAMM tool was also used which calculates intermolecular potential energy based on grid representations of molecules. A₁₂ was docked into protein structures including CIRP, HMGB1, and Histone H3 using HDock tool to generate protein-RNA complexes. The HDock is a FFT based translational search algorithm, which is optimized by iterative knowledge-based scoring function. The interactions between A₁₂ and CIRP, HMGB1, and Histone H3 were calculated using PDBePISA tool, and the complexes were visualized using PyMOL and Chimera tools (15). Interaction between TLR4 and CIRP in with or without A₁₂ was also assessed in the same manner.

Synthesis of eCIRP and A₁₂

Recombinant mouse (rm) CIRP (denoted as eCIRP) was produced and validated its purity and efficacy in our laboratory as previously described (16). A₁₂ (AAAAAAAAAAAA, with 2′-O-methyl ribose modification and terminal phosphorothioate linkages) or Cy3-labeled A₁₂ was synthesized by Integrated DNA Technologies (Coralville, IA).

Surface plasmon resonance (SPR)

OpenSPR (Nicoya, ON, Canada) was used to examine the direct interaction between eCIRP and A₁₂ (17). eCIRP was immobilized on the surface of carboxyl sensor as a ligand and various concentrations of A₁₂ (15.6 nM-250 nM) were injected as an analyte and a K_D value was calculated. To determine the effect of A₁₂ on eCIRP’s binding to TLR4, eCIRP (500 nM) incubated with various concentrations of A₁₂ (50 nM-500 nM) were injected over a high sensitivity NTA sensor immobilized with TLR4 (catalog 1478-TR-050; R&D, Minneapolis, MN). In addition, various concentrations of eCIRP (62.5 nM-500 nM) were incubated with either 50, 100, 500 nM of A₁₂ and subsequently injected over a TLR4 immobilized chip to determine K_D values. The real-time interaction data were analyzed by TraceDrawer (Nicoya). Data were globally fitted for 1:1 binding (one- to-one model).

Treatment of peritoneal macrophages with eCIRP and A₁₂

Peritoneal cells were collected using peritoneal lavage with PBS and subsequently cultured in RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, MP Biomedicals, Irvine, CA), 1% penicillin-streptomycin, and 2 mM glutamine. After 4 h, nonadherent cells were removed, and adherent cells, primarily macrophages, were used for subsequent studies. Peritoneal macrophages were treated with 50 nM eCIRP together with 0, 50 or 500 nM of A₁₂ for optimal time for each experiment.

Immunofluorescence assay

Peritoneal macrophages were incubated with FITC-labeled eCIRP at 50 nM in the presence and absence of 500 nM A₁₂ for 15 minutes. Cells were washed with PBS and subsequently stained with APC anti-mouse F4/80 Ab (catalog 123116; BioLegend, San Diego, CA) for 1 h. Confocal microscopy images were obtained using a Zeiss LSM900 confocal microscope equipped with a 63× objective. The samples were also subjected to flow cytometry using FACSymphony Flow Cytometer (BD Biosciences) and subsequently analyzed by FlowJo software (Tree Star, Ashland, OR).

Fluorescence resonance energy transfer (FRET)

FRET analysis was performed as described previously (18). Peritoneal macrophages were cultured with 50 nM eCIRP with or without 500 nM A₁₂ for 15 minutes. Cells were incubated with rabbit anti-mouse CIRP Ab (catalog 10209–2-AP; ProteinTech) for 1 h and subsequently stained with Cy3-conjugated AffiniPure F(ab’)₂ donkey anti-rabbit IgG (code 711–166-152; Jackson ImmunoResearch Laboratories). Cells were also stained with APC anti-mouse TLR4 Ab (catalog 145406; BioLegend) or APC anti-mouse CD11b Ab (catalog 101212; BioLegend). Fluorescence was measured on a Synergy Neo2 at 566 nm upon excitation at 488 nm (E1), at 681 nm after excitation at 630 nm (E2), and at 681 nm after excitation at 488 nm (E3). The transfer of fluorescence was calculated as FRET units following a previous study (18). FRET unit = (E3_both − E3_none) − ([E3_APC − E3_none] × [E2both/E2_APC]) − ([E3_Cy3 − E3none] × [E1both/E1Cy3]).

Western blotting

Proteins were extracted from peritoneal macrophages treated with eCIRP and A₁₂ using extraction buffer containing 25 mM Tris, 0.15 M NaCl, 1 mM EDTA, 1% NP-40, 5% glycerol, 2 mM Na₃VO₄, and protease/phosphatase inhibitor cocktails (Roche Diagnostics, Basel, Switzerland), pH 7.4. Lysates were run on 4%-12% gradient polyacrylamide gels for electrophoresis. Gels were transferred into nitrocellulose membranes and blocked with 3% BSA TBST. The membranes were reacted with primary antibodies (Abs) against phospho (p) p38 (catalog 9211S, Cell Signaling Technology, Danvers, MA), total p38 (catalog 9212S, Cell Signaling Technology), IκBα (catalog 10268–1-AP; ProteinTech, Rosemont, IL), and β-actin (MilliporeSigma, Burlington, MA) followed by reaction with fluorescence-labeled secondary Abs (anti-mouse IgG, catalog 926–68070; anti-rabbit IgG, catalog 926–32211; Li-Cor Biosciences, Lincoln, NE). The blots were detected using an Odyssey FC Dual-Mode Imaging system (Li-Cor Biosciences). The densitometry intensities of the bands were measured by ImageJ software (NIH).

PCR array

Peritoneal macrophages treated with 50 nM eCIRP and 500 nM A₁₂ for 4 h were subjected to RT² profiler PCR array mouse transcription factors (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol. The data were analyzed using the GeneGlobe Data Analysis Center on the QIAGEN website.

In vivo administration of eCIRP and A₁₂

To determine the in vivo half-life of A_12, Cy3-labeled A₁₂ (0.05 nmol/g BW) was intravenously (i.v.) injected into a rat. A₁₂ levels in the serum were monitored over time. The half-life studies involved the need for repeated blood draws from the same animal. It is important to note that performing repeated blood draws from a single mouse is not feasible. Therefore, we utilized rats for this experiment. There are two phases of half-life. The first sharp slope is the distribution phase (alpha phase). This step is followed by the elimination phase (beta phase). The half-life in our manuscript represents the amount of time required for the plasma concentration to decline by 50% during the elimination phase, i.e., the beta half-life (elimination half-life). To determine the effects of A₁₂ on inhibiting eCIRP-induced inflammation, mice were i.p. injected with 0.25 nmol/g BW eCIRP simultaneously with a vehicle (PBS) or 0.5 nmol/g BW A₁₂. Four hours after the injection, the blood was drawn to isolate the serum to assess inflammatory and injury markers.

Mouse model of sepsis

Polymicrobial sepsis was induced in mice by CLP (16). Mice were anesthetized with isoflurane, and a midline abdominal incision was created. The cecum was ligated with a 4–0 silk suture 1 cm proximal from its distal extremity and punctured twice for a 20 h study and once for a survival study using a 22-gauge needle. A vehicle (PBS) or 0.5 nmol/g BW A₁₂ was intraperitoneally (i.p.) delivered, and the wound was then closed in layers. Sham animals were subjected to a laparotomy without CLP. Following the surgery, 1 mL of normal saline was subcutaneously (s.c.) injected to avoid surgery-induced dehydration and 0.05 mg/kg buprenorphine was s.c. injected as an analgesic. Imipenem (0.5 mg/g BW) was also s.c. injected for survival studies. Twenty hours after the surgery, the blood and lungs were harvested. Mice were observed for 10 days for survival studies.

Assessment of cytokines and organ injury markers in serum and supernatants

Serum samples or cell-culture supernatants were analyzed by ELISA kits for IL-6 and TNFα (both from BD Biosciences, Franklin Lakes, NJ). Serum levels of AST, ALT, and LDH were determined using specific colorimetric enzymatic assays (Pointe Scientific, Canton, MI). Absorbance was measured on a Synergy Neo2 (Agilent Technologies, Santa Clara, CA) according to the manufacturers’ instructions.

Real-time quantitative reverse transcription PCR

Total RNA was extracted from homogenized lung tissues using TRIzol reagent (Invitrogen, Thermo Fisher Scientific). cDNA was synthesized using MLV reverse transcriptase (Applied Biosystems, Thermo Fisher Scientific), and PCR was performed with forward and reverse primers and SYBR Green PCR Master Mix (Applied Biosystems) using a Step One Plus real-time PCR machine (Applied Biosystems). The sequences of primers used in this study are as follows: β-Actin, 5′-CGTGAAAAGATGACCCAGATCA-3′ (forward), and 5′-TGGTACGACCAGAGGCATACAG-3′ (reverse); IL-6, 5′-CCGGAGAGGAGACTTCACAG-3′ (forward), and 5′-GGAAATTGGGGTAGGAAGGA-3′ (reverse); TNFα, 5′-AGACCCTCACACTCAGATCATCTTC-3′ (forward), and 5′-TTGCTACGACGTGGGCTACA-3′ (reverse); IL-1β, 5′-CAGGATGAGGACATGAGCACC-3′ (forward), and 5′-CTCTGCAGACTCAAACTCCAC-3′ (reverse); KC, 5′-GCTGGGATTCACCTCAAGAA-3′ (forward), and 5′-ACAGGTGCCATCAGAGCAGT-3′ (reverse); MIP2, 5′-CCAACCACCAGGCTACAGG-3′ (forward), and 5′-GCGTCACACTCAAGCTCTG-3′ (reverse).

Myeloperoxidase assay

Lung tissues were homogenized in KPO₄ buffer containing 0.5% hexadecyltrimethylammonium bromide (Sigma-Aldrich, St. Louis, MO). After centrifugation, the supernatants were diluted in reaction solution containing O-Dianisidine dihydrochloride (Sigma-Aldrich) and H₂O₂ (ThermoFisher Scientific). Absorbance was measured at 460 nm to calculate myeloperoxidase (MPO) activity, which was normalized to a protein amount measured by the Bio-Rad protein assay reagent (Hercules, CA).

Lung histology and TUNEL assay

Formalin fixed and paraffin embedded lung tissue blocks were sectioned at 5 μm thickness and placed on glass slides. Lung tissue sections were stained with hematoxylin & eosin (H&E) and observed under a light microscope. Lung injury was assessed according to a scoring system established by the American Thoracic Society. Scores ranged from 0 to 2 and were based on the presence of neutrophils in the alveolar and interstitial spaces, hyaline membranes, proteinaceous debris in the airspaces, and alveolar septal thickening. To determine the presence of apoptotic cells, lung tissue sections were stained with terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL) assay kit (Roche Diagnostics, Indianapolis, IN) according to the manufacturer’s protocol and examined under a fluorescent microscopy (Nikon Eclipse Ti-S, Melville, NY). The microscopy assessments of lung tissue sections were performed at 200× magnification.

Statistical analysis

Data represented in the figures are expressed as mean ± SEM. ANOVA was used for one-way comparison among multiple groups, and the significance was determined by the Student Newman-Keuls (SNK) test. The paired Student t-test was applied for two-group comparisons. Survival rates were analyzed by the Kaplan-Meier estimator and compared using a log-rank test. Significance was considered for p ≤ 0.05 between study groups. Data analyses were carried out using GraphPad Prism graphing and statistical software (GraphPad Software, San Diego, CA).

Results

A₁₂ selectively binds to eCIRP with a high affinity

We first determined the interaction between A₁₂ and eCIRP. We screened different lengths of poly(A) tails (A₈, A₁₀, A₁₄, A₁₆), which are implemented with the same chemical modifications (2′-O-methylation and phosphorothioate linkages), in terms of their affinity with eCIRP using computational modeling. Among all, 12-base poly(A), A₁₂, was predicted to have the strongest affinity with eCIRP as indicated by lower binding energy (ΔiG) and higher free energy of dissociation (ΔGdiss) (Supplemental Figure 1A–D). A₁₂ was predicted to interact with the N-terminal RNA-binding domain of eCIRP by forming two hydrogen bonds (Figure 1A). The root mean square deviation (RMSD), the difference in atomic distance induced by the incorporation of another molecule, was 0.60 Å between eCIRP only and eCIRP with A₁₂, indicating that A₁₂ caused the conformational change of eCIRP. Mutations in the RNA-binding domain of eCIRP (Asn68 and Gln81 to Ala68 and Ala81, respectively) markedly decreased its affinity to A₁₂, as demonstrated by the higher ΔiG and lower ΔGdiss values compared to normal unmutated eCIRP (Figure 1B). eCIRP was shown to have a much stronger affinity than other chromatin-associated DAMPs (e.g., HMGB1, Histone H3) which are not bona fide RNA-binding proteins (Figure 1C, D). Thus, A₁₂ is predicted to selectively bind to the N-terminal RNA-binding domain of eCIRP. To rigorously confirm the binding between A₁₂ and eCIRP, we implemented surface plasmon resonance (SPR) or Biacore assay tool. Consistent with the computational analysis, SPR revealed that A₁₂ binds to eCIRP with an extremely high affinity (K_D = 2.05 × 10⁻⁹ M) (Figure 1E). Taken together, A₁₂ selectively binds to eCIRP with a high affinity, suggesting its potential as an inhibitor of eCIRP.

Figure 1. A₁₂ binds to eCIRP with a high affinity.

Three-dimensional (3D) computational prediction of molecular binding between A₁₂ and (A) CIRP, including the distance (Ȧ) of hydrogen bonds, (B) CIRP with mutations in an RNA-binding domain (Asn68 and Gln81 to Aln68 and Aln81, respectively), (C) HMGB1, and (D) Histone H3. Lower binding energy (ΔiG) and higher free energy of dissociation (ΔGdiss) indicate higher-affinity interaction. (E) SPR between eCIRP and A₁₂. A calculated K_D value is shown. SPR experiments were performed at least 2 times, generating similar findings.

Presence of A₁₂ inhibits the interaction between eCIRP and TLR4

eCIRP is known as a ligand of TLR4 (16), thus we next investigated the effect of A₁₂ on the eCIRP-TLR4 interaction. A computational modeling predicted that the interaction between eCIRP and TLR4 was impaired in the presence of A₁₂ as indicated by increased binding energy (ΔiG) and decreased free energy of dissociation (ΔGdiss) (Figure 2A). SPR further revealed that A₁₂ dose-dependently inhibited the binding of eCIRP to TLR4 (Figure 2B–F). We then assessed A₁₂-mediated inhibition of eCIRP-TLR4 interaction in live peritoneal macrophages. A₁₂ inhibited the binding of eCIRP to macrophage cell membrane as observed by microscopy (Figure 2G) and quantified by flow cytometry (Figure 2H, I). Furthermore, FRET analysis revealed that A₁₂ significantly inhibited the binding of eCIRP to TLR4 (Figure 2J). As a negative control, significantly less binding was observed between eCIRP and CD11b (pan marker of macrophages) compared to eCIRP and TLR4 (Figure 2J). These data indicate that A₁₂ interferes with the binding of eCIRP to TLR4, suggesting A₁₂ inhibits TLR4 activation mediated by eCIRP.

Figure 2. A₁₂ interferes with the interaction between eCIRP and TLR4.

(A) 3D computational prediction of molecular binding between CIRP and TLR4 in the presence and absence of A₁₂. (B) SPR between TLR4 and 500 nM eCIRP with various doses (0–500 nM) of A₁₂. SPR between TLR4 and different doses (62.5–500 nM) of eCIRP in the presence of (C) 0, (D) 50, (E) 100, and (F) 500 nM A₁₂. Calculated K_D values for each dose of A₁₂ are shown. SPR experiments were performed at least 2 times, generating similar findings. (G) Microscopy images of peritoneal macrophages incubated with fluorescent-labeled CIRP (green fluorescence) in the presence and absence of A₁₂. F4/80 (red fluorescence) serves as a cell surface marker. Experiments were performed 3 times. (H) Representative histograms and (I) median fluorescent intensity (MFI) of FITC-labeled eCIRP’s binding to peritoneal macrophages. Experiments were performed 3 times, and representative data was used for analysis. Data are expressed as mean ± SEM (n=3 samples/group) and compared by one-way ANOVA and SNK test. *p<0.05 vs. PBS, ^#p<0.05 vs. eCIRP alone. (J) FRET analysis of interaction between eCIRP and TLR4 with or without A₁₂ in peritoneal macrophages. CD11b serves as a negative control. Experiments were performed 2 times, and all data were used for analysis. Data are expressed as mean ± SEM (n=6 samples/group) and compared by one-way ANOVA and SNK test. *p<0.05 vs. eCIRP-CD11b, ^#p<0.05 vs. eCIRP-TLR4.

Treatment with A₁₂ inhibits eCIRP-induced pro-inflammatory signaling in macrophages

eCIRP has been shown to induce inflammatory response in macrophages (6). We first assessed the effects of A₁₂ on eCIRP-induced inflammatory signaling in vitro using primary mouse peritoneal macrophages. We found that eCIRP activated pro-inflammatory signaling in macrophages as determined by p38 phosphorylation and IκBα degradation, which reflect the activation of MAP kinase and NF-κB pathways, respectively (Figure 3A, B, Supplemental Figure 2). Interestingly, treatment with A₁₂ significantly inhibited p38 phosphorylation and IκBα degradation induced by eCIRP in macrophages (Figure 3A, B). Furthermore, PCR array data revealed that eCIRP upregulated a cluster of transcription factors implicated in inflammatory signal transduction such as Nfkb1, Ets2, Rela, and Stat2 in macrophages. However, the expressions of these transcription factors were dramatically decreased by A₁₂ treatment (Figure 3C, D). These data indicate A₁₂ significantly attenuates the activation of pro-inflammatory signaling pathways induced by eCIRP.

Figure 3. A₁₂ inhibits eCIRP-induced pro-inflammatory signaling in macrophages.

Peritoneal macrophages were treated with eCIRP with 50 and 500 nM of A₁₂. After 30 minutes, cells were lysed to assess (A) p38 phosphorylation and (B) IκBα degradation by Western blotting. Representative blots and corresponding bar diagram are shown. The Western blot experiments were performed 2–3 times, and all data were used for analysis. Data are expressed as mean ± SEM (n = 4–6 samples/group) and compared by one-way ANOVA and SNK test. *p<0.05 vs. PBS, ^#p<0.05 vs. eCIRP alone. PBS control was normalized as 1. (C) A clustergram of PCR array for mouse transcription factors of peritoneal macrophages treated with eCIRP and A₁₂ for 4 h. Experiments were performed 3 times, and representative data is shown. (D) Most upregulated genes by eCIRP stimulation from the PCR array are shown. Data are expressed as mean ± SEM (n = 3 samples/group).

Treatment with A₁₂ attenuates eCIRP-induced cytokine production in macrophages and mice

Next, we investigated whether A₁₂ inhibits cytokine production induced by eCIRP. Our in vitro study shouwed that eCIRP increased the release of pro-inflammatory cytokines, IL-6 and TNFα, from macrophages (Figure 4A, B). However, A₁₂ significantly inhibited IL-6 and TNFα production induced by eCIRP in a dose-dependent manner (Figure 4A, B). Interestingly, A₁₂ did not significantly alter the production of IL-6 and TNFα by the macrophages induced by LPS or HMGB1 (Supplemental Figure 3), supporting A₁₂’s functional specificity to eCIRP. We then assessed the effects of A₁₂ on eCIRP-induced inflammation in vivo by injecting eCIRP with or without A₁₂. In vivo half-life of A₁₂ in serum was calculated to be over 2 h (Figure 4C, D), indicating that A₁₂ is stable under the in vivo condition to exhibit its inhibitor effects. eCIRP injection induced systemic inflammation as determined by the elevated levels of IL-6 and TNFα in the serum. A₁₂ treatment significantly attenuated the levels of IL-6 and TNFα increased by eCIRP by 42% and 39%, respectively (Figure 4E, F). Thus, our data reveal A₁₂ as an effective inhibitor of eCIRP to reduce cytokine production in vitro and in vivo.

Figure 4. A₁₂ attenuates eCIRP-induced cytokine production in macrophages and mice.

Peritoneal macrophages were treated with eCIRP with 50 and 500 nM of A₁₂. After 2 h, (A) IL-6 and (B) TNFα levels in the supernatants were measured by ELISA. Experiments were performed 2 times, and all data were used for analysis. Data are expressed as mean ± SEM (n=4 samples/group) and compared by one-way ANOVA and SNK test. *p<0.05 vs. PBS, ^#p<0.05 vs. eCIRP alone. (C, D) In vivo half-life of A₁₂ was determined in rat serum by i.v. injecting Cy3-labed A₁₂. Experiments were performed 3 times, and all data were used for analysis. Mice were i.p. injected with 250 nmol/kg eCIRP simultaneously with a vehicle (PBS) or 500 nmol/kg A₁₂. 4 h after the injection, the blood was drawn to assess the serum levels of (E) IL-6 and (F) TNFα. Experiments were performed 3 times, and all data were used for analysis. Data are expressed as mean ± SEM (n=8 samples/group) and compared by one-way ANOVA and SNK test. *p<0.05 vs. PBS, ^#p<0.05 vs. vehicle.

A₁₂ alleviates systemic inflammation and ALI to improve survival in sepsis

Our previous study revealed significantly higher levels of eCIRP in the serum of sepsis patients (19). Here, we induced sepsis in mice by CLP and harvested the blood 20 h after the operation. The levels of eCIRP in the serum were significantly elevated in CLP mice compared to sham mice (Figure 5A). To investigate the potential of A₁₂ as an eCIRP inhibitor to improve outcomes in sepsis, mice were induced sepsis by CLP and i.p. administered with a vehicle or A₁₂ at the time of abdominal closure. After 20 h of sepsis, blood and lungs were collected. We found that IL-6, TNFα, AST, ALT, and LDH in the serum were significantly elevated in CLP mice. By contrast, those parameters were dramatically lower in A₁₂-injected mice by 45%, 47%, 27%, 32%, and 24%, respectively (Figure 5B–F), indicating A₁₂ attenuates systemic inflammation and organ injuries in sepsis. We also evaluated inflammation and tissue injury in the lungs of septic mice. CLP significantly increased mRNA levels of cytokines and chemokines, including IL-6, TNFα, IL-1β, KC, and MIP2, as well as MPO activity in the lungs, whereas A₁₂ significantly decreased those parameters by 65%, 53%, 45%, 74%, 64%, and 24%, respectively (Figure 5G–L), indicating that A₁₂ prevented lung inflammation and neutrophil influx in sepsis. Histological analysis showed a severe tissue injury and an increase in apoptotic cells in the lungs of septic mice, while A₁₂-injected mice were protected from those histological changes (Figure 5M–P). We have recently reported that eCIRP causes dysfunction in bacterial phagocytosis of macrophages during sepsis (20). Thus, we also assessed the bacterial loads in the blood and peritoneal lavage of CLP mice with or without A₁₂ administration. We found that treatment of septic mice with A₁₂ decreased the bacterial contents in both compartments (Supplemental Figure 4). We then investigated the effect of A₁₂ on the survival of septic mice induced by CLP. A₁₂ administration at the end of the surgery significantly improved survival in septic mice (Figure 5Q). These findings demonstrate that A₁₂ attenuates inflammation, alleviates ALI, and improves survival in sepsis.

Figure 5. A₁₂ alleviates systemic inflammation and ALI to improve survival in sepsis.

Mice were induced sepsis by CLP with i.p. instillation of a vehicle (PBS) or 500 nmol/kg A₁₂ at the end of the procedure. 20 h after the surgery, the blood and lungs were harvested. (A) Serum levels of eCIRP in sham and CLP mice. Experiments were performed 3 times, and all data were used for analysis. Data are expressed as mean ± SEM (n=4–6 samples/group) and compared by paired student’s t-test. *p < 0.05 vs. Sham. Serum levels of (B) IL-6, (C) TNFα, (D) AST, (E) ALT, and (F) LDH. mRNA levels of (G) IL-6, (H) TNFα, (I) IL-1β, (J) KC, and (K) MIP2, and (L) MPO activity in the lungs. (M) Representative images of H&E-stained lung tissues and (O) lung injury score. (N) Representative images of TUNEL staining (green fluorescence) and nuclear counterstaining (blue fluorescence) and (P) numbers of TUNEL positive cells/HPF in lung tissues. Magnification 200×. Scale bar: 100 μm. Experiments were performed 3 times, and all data were used for analysis. Data are expressed as mean ± SEM (n=6–10 samples/group) and compared by one-way ANOVA and SNK test. *p<0.05 vs. Sham, ^#p<0.05 vs. vehicle. (Q) 10-day survival study of septic mice induced by CLP with i.p. instillation of a vehicle or 500 nmol/kg A₁₂ at the end of the procedure. n = 25 mice/group. Survival rates were analyzed by the Kaplan-Meier estimator using a log-rank test. *p<0.05 vs. vehicle.

Discussion

In the present study, we have demonstrated that the poly(A) mRNA mimic, A₁₂, has demonstrated its remarkable inhibitory effects on eCIRP-induced inflammation by targeting TLR4-mediated activation of p38 MAP kinase and NF-κB in macrophages. This critical mechanism of action effectively suppresses the inflammatory response triggered by eCIRP. Furthermore, our in vivo experiments have yielded promising results. Upon administration of A₁₂ to mice, we observed significantly improved outcomes in ALI and increased survival rates in sepsis. These findings underscore the potential preventative value of A₁₂ in treating inflammatory conditions and provide a strong rationale for further exploration of its clinical applications. (Figure 6). Intracellular CIRP acts as an RNA chaperone, while extracellular CIRP, released from stressed or damaged cells during sepsis, serves as a potential DAMP (16). Our previous studies have demonstrated that extracellular CIRP or eCIRP acts as a ligand for TLR4 to execute distinct functions from intracellular CIRP (16). In this study, we have shown that A₁₂ could directly bind eCIRP and inhibited the binding of eCIRP to TLR4. Consequently, we have concluded that A₁₂’s binding to eCIRP prevented the activation of the TLR4 pathway, as evidenced by the inhibition of p38 phosphorylation and IkBa degradation in our present study. Besides supportive care, no definitive disorder-specific drugs are available for ALI or sepsis. Several clinical trials, including the ones targeting PAMPs (i.e., LPS), cytokines, or neutrophil elastase, have failed after decades of research for those disorders (21). DAMPs are the potential targets to overcome this failure, considering the studies showing their significant contribution to ALI and sepsis (4). eCIRP is a novel DAMP involved in the pathogenesis of these disorders (6). Together with the present findings, A₁₂ can potentially overcome the challenges of developing an effective drug for preventing the progress of ALI and sepsis by targeting eCIRP.

Figure 6. Summary of findings.

During sepsis, eCIRP stimulates TLR4 on macrophages to activate p38 MAPK and NF-kB, leading to the production of inflammatory mediators to cause ALI and death. A₁₂ neutralizes eCIRP to attenuate ALI and improve survival in sepsis.

Different kinds of oligonucleotide drugs and vaccines are clinically available these days, but their predominant usage is to modulate gene expressions intracellularly. Because of their hydrophilic properties, oligonucleotides do not readily pass through the plasma membrane and often require a delivery system to enhance membrane permeability (22). For example, COVID-19 mRNA vaccines are carried in liposomes to increase their uptake by cells (23). Despite the rapid expansion of their clinical application, the utilization of oligonucleotides in the extracellular space has yet to be studied. Our present study gives insight into the novel ways of using oligonucleotides other than modulating gene expressions. Because of their presence without liposome can be mainly outside the cells, the oligonucleotide-dependent cell activation would be limited given that the receptors for oligonucleotides, such as TLR7 and TLR9, are located inside the cells (24). In fact, macrophages treated with only A₁₂ did not show an increase in inflammatory parameters, and no apparent adverse effects were observed in rodents injected with only A₁₂ (data not shown). Moreover, one can adjust the rationale of oligonucleotide-based treatment, whether to target intra or extracellular proteins by using with or without liposomal molecules, which help them to penetrate inside the cells. To date, most of the oligonucleotide drugs have been designed to be delivered locally or to the liver (22). For instance, a recent study has shown that a poly(A) mRNA mimic, which targets intracellular RNA-binding proteins to inhibit the protection of COX2 mRNA by the RNA-binding proteins, reduces COX-2-dependent pains when it is delivered locally at the site of injury (25). Together, our study focusing on the extracellular role of the RNA mimic to target a DAMP, eCIRP, by systemic delivery reflects its unique strategy to control inflammatory disorders mediated by DAMPs.

We have demonstrated that A₁₂ inhibits the binding of eCIRP to TLR4, which is arguably the most important and well-studied PRR. However, recent phase 2 and phase 3 clinical trials pharmacologically blocking TLR4 have failed to yield any survival benefit in septic patients (26, 27). Given that TLR4 plays a crucial role as an innate immune receptor, responsible for initiating immune responses upon recognition of various PAMPs to eliminate pathogens, it is essential to consider the implications of targeting TLR4 directly. Previous studies in TLR4-deficient mice have revealed that these animals exhibit compromised innate immune defense, which could potentially hinder their ability to fight off pathogens effectively (28, 29). As a result, pharmacologically blocking TLR4 alone might not be beneficial, especially when it comes to improving overall survival, due to its immunosuppressive nature. Instead of directly targeting TLR4, an alternative and potentially more effective approach to controlling hyperinflammation and tissue injury in sepsis could involve blocking or neutralizing the putative ligand(s) of TLR4. This strategy aims to mitigate the downstream inflammatory cascade without suppressing the overall immune response. This is where the unique properties of A₁₂ come into play. Triggering receptor expressed on myeloid cells-1 (TREM-1) is another PRR which contributes to inflammatory disorders (18). TREM-1 and TLR4 act synergistically since TREM-1 is an amplifier of the TLR4 pathway and TLR4 can increase TREM-1 expression (30, 31). Therefore, blocking the eCIRP-TLR4 axis will presumably interfere with the TREM-1 pathway. Like other DAMPs, eCIRP recognizes not only TLR4, but also several other receptors, such as TREM-1 and IL-6R (18, 32). Given those multiple receptors and the possible existence of other unknown receptors for eCIRP, competitive inhibition of eCIRP by blocking its receptors using antagonists for specific receptors may not provide sufficient effects to improve the disease outcomes. On the other hand, our novel approach of specifically targeting the ligand eCIRP by A₁₂ to inhibit its binding to the receptors suggests A₁₂ has the potential to prevent the binding of eCIRP to TREM-1 and IL-6R as well as other unknown receptors in addition to TLR4. The binding site of A₁₂ on the eCIRP motif did not overlap with the site where eCIRP binds to TLR4. Thus, a possible explanation for decreased interaction between eCIRP and TLR4 caused by A₁₂ could be the conformational change of eCIRP following interaction with A₁₂. Consequently, it is possible that A₁₂ might also inhibit the binding of eCIRP to other receptors in addition to TLR4 and attenuate the pro-inflammatory effects of eCIRP. Collectively, A₁₂ represents a promising candidate for overcoming the challenges faced by direct pharmacological blockade of TLR4. Its ability to specifically neutralize eCIRP’s pro-inflammatory activity and potential to inhibit eCIRP from binding to multiple receptors present a novel and potentially more effective approach to combat hyperinflammation in sepsis.

Although we have demonstrated the specificity of A₁₂ to eCIRP by showing its decreased binding capacity and inhibitory effects with other PAMPs and DAMPs, we acknowledge that there remains a possibility of A₁₂ exerting off-target effects by interacting with other PRRs’ ligands. It is important to recognize that, in addition to the ligands we have evaluated in this study, there are numerous molecules capable of activating TLR4. Furthermore, various other PAMPs and DAMPs exist for different PRRs, which play critical roles in immune responses, such as Pam3Cys for TLR2 and extracellular RNAs for TLR3 and TLR7 (33, 34). Theoretically, A₁₂ may have the potential to bind extracellular RNAs enriched in uracil through complimentary pairing. Considering the myriad of possibilities, it is indeed practically challenging to cover all the ligands for all PRRs and their potential interactions with A₁₂ in the scope of the present study. A more comprehensive approach would be required to fully delineate the interaction of A₁₂ with different PRR ligands, providing a broader understanding of the pharmacological properties of A₁₂.

In our in vitro studies, we used macrophages to assess the effects of A₁₂ on inflammation since macrophages are regarded as one of the primary sources of proinflammatory cytokines and chemokines especially in sepsis. Nevertheless, many cell types concomitantly play a role in the pathophysiology of inflammatory disorders including sepsis (4). Different kinds of immune and non-immune cells express PRRs including TLR4, which induces MAP kinase and NF-κB activation, and eCIRP has been shown to affect the status of different cell types. For example, eCIRP directly activated neutrophils to induce the formation of neutrophil extracellular traps (NETs) via TLR4 and TREM-1, leading to ALI in sepsis (35, 36). It has also been shown that eCIRP induces apoptosis and pyroptosis in lung endothelial cells to cause tissue injury in sepsis (9, 10). Thus, it is presumable that A₁₂ may also exhibit its inhibitory effects in neutrophils, lymphocytes, epithelial or endothelial cells, which are all exaggeratedly activated during inflammatory conditions including sepsis (4). However, cell-specific approach is awaited to confirm this theory. In this paper, we focused on the lung injury, as lungs are significantly impacted by cecal ligation and puncture (CLP) and our previous studies provide compelling evidence for the contribution of extracellular CIRP (eCIRP) to ALI (9, 10). Additionally, in the clinical context, ALI is a prevalent complication that contributes to mortality in sepsis (5). While our study primarily emphasized on lung injuries, we acknowledge that other organs may have been affected more severely by CLP-induced sepsis. Even though we did not specifically evaluate injuries in other organs in this study, we assessed serum AST, ALT, and LDH levels, which not only reflect the injury of lungs but also any organs/tissues during sepsis.

eCIRP levels in the serum of our CLP mice were similar to that of septic patients (19), supporting the clinical relevance of our sepsis model. For in vitro experiments, the dose of eCIRP was referred to our previous studies where we screened the optimal concentration of eCIRP (16). It is to be noted that the in vitro concentration of eCIRP is significantly higher than the measured levels of eCIRP in the blood of septic patients and mice. Several factors need to be considered to explain this disparity in eCIRP concentrations. During sepsis, the concentration of eCIRP could be much higher in the local area where it directly acts on resident cells, such as peritoneal macrophages, compared to the peripheral blood. In fact, we have previously observed notably higher eCIRP levels in the peritoneal cavity compared to the blood, even though the peritoneal samples were diluted during lavage (20). Moreover, it is possible that a significant proportion of eCIRP in the body of septic patients or mice forms complexes with its cell-surface receptors, considering the high affinity between those molecules.

In this study, the timing of A₁₂ administration was at the end of the surgical procedure. It would be more clinically relevant to administer A₁₂ after the diagnosis of sepsis. However, it is important to acknowledge the challenges associated with accurately diagnosing sepsis in mice without invasive procedures. Currently, sepsis diagnosis in clinical settings primarily rely on blood parameters. Unfortunately, performing repeated blood draws in mice is highly invasive, making it difficult to monitor them closely after the procedure. While administering A₁₂ at a later stage would enhance the clinical relevance of our study, it still presents limitations such as variability in sepsis development, even following the same procedure of CLP. Together, it becomes challenging to determine whether mice have developed sepsis by the time of A₁₂ administration. Thus, the use of higher animals would be necessary to carry out more clinically relevant therapeutic interventions.

We have previously discovered miR-130b-3b as an endogenous eCIRP inhibitor (19). In that study, we employed various nucleotide sequences to investigate their ability to bind to eCIRP and attenuate its inflammatory effects. While not all nucleotide sequences exhibited this property, miRNA-130b-3p was found to attenuate eCIRP’s inflammatory response. Since our previous study involved several nucleotide mimics that served as controls, we did not utilize another nucleotide as a control in the preset study. miR-130b-3p also forms hydrogen bonds with eCIRP through adenosines in the same way as A₁₂. This finding suggests that adenosines might play a significant role in the hydrogen bond formation. It is plausible that eCIRP recognizes diverse nucleotide motifs, considering the sequence dissimilarity between miR-130b-3p and A_12. This observation opens an intriguing avenue for further exploration in future studies. We believe that investigating the specific nucleotide motifs recognized by eCIRP will provide valuable insights into its interaction mechanisms and may uncover additional functional implications.

In conclusion, a novel mRNA mimic A₁₂ targets eCIRP to act against systemic inflammation and ALI and improve survival in sepsis. Other diseases which are exacerbated by eCIRP, such as rheumatoid arthritis, ulcerative colitis, and chronic obstructive pulmonary disease, could also benefit from A₁₂ treatment (6). Pharmacokinetics, toxicity, and higher animal testing should be done to move to the next level of implementing this drug in patient-oriented clinical conditions.

Key points:

• A₁₂ (AAAAAAAAAAAA) interacts with eCIRP, preventing eCIRP’s binding to TLR4.

• A₁₂ significantly decreases eCIRP-induced sterile inflammation.

• Treating septic mice with A₁₂ ameliorates ALI and improves their survival.

Abbreviations

CLP

cecal ligation and puncture

DAMP

damage-associated molecular pattern

eCIRP

extracellular cold-inducible RNA-binding protein

HMGB1

high mobility group box 1

LPS

lipopolysaccharides

PAMPs

pathogen-associated molecular patterns

TLR

Toll-like receptor

NETs

neutrophil extracellular traps