A Synthetic Poly(A) Tail Targeting Extracellular CIRP Inhibits Sepsis

Sepsis is a deadly inflammatory syndrome caused by the dysregulated host response to infection (1). In the United States, there are ∼1 million sepsis cases yearly with a mortality rate of up to 40%, costing more than $24 billion annually (2, 3). Pathogen-associated molecular patterns (PAMPs), such as LPS, present in the bacterial cell wall initiate infectious inflammatory responses by recognizing pattern recognition receptors (PRRs) such as TLR4 (4). In contrast, 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 to aggravate mortality rates (5).

Cold-inducible RNA-binding protein (CIRP) is an 18-kDa RNA chaperone. CIRP binds the 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, whereas 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 could offer a promising approach to preventing the onset of ALI caused by sepsis. A recent study identified a 15-aa small peptide derived from eCIRP to target the eCIRP/TLR4 axis (12). Nonetheless, its short half-life and the possible interference with the binding of other TLR4 ligands may make it challenging to implement as an effective and specific eCIRP antagonist. Thus, identifying a new molecule that directly neutralizes eCIRP to impede its binding to TLR4 could be a promising eCIRP antagonist to prevent inflammation.

Because 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 potent inhibitor for eCIRP. With the aid of computational modeling, we revealed that a 12-base poly(A) tail optimally binds to eCIRP. Because RNA mimic is susceptible to nuclease degradation, to increase its stability, we have engineered the 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 current study, we named this oligonucleotide A₁₂ (AAAAAAAAAAAA) and sought to investigate its efficacy as an eCIRP inhibitor in sepsis. In this study, we demonstrate that A₁₂ targets eCIRP to attenuate inflammation, protect mice from ALI, and improve survival after sepsis.

Male C57BL/6 mice (8–12 wk old) were purchased from Charles River Laboratories (Wilmington, MA). Mice were housed in a temperature-controlled room with 12-h intermittent light and dark cycles and fed a standard mouse chow diet with water. Male 7- to 9-wk-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’s Guidelines for the Care and Use of Laboratory Animals and were approved by The Feinstein Institutes for Medical Research’s Animal Care and Use Committee.

The amino acid sequences of mouse CIRP (P60824), high mobility group box 1 (HMGB1) (P63158), histone H3 (P02301), and TLR4 (Q9QUK6) were retrieved from the UniProt database. The structure models of CIRP, HMGB1, histone H3, and TLR4 were generated using template-based modeling approach I-TASSER (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 a 4-fs time step after structure perturbations. The refinement process enhanced certain parameters such as increased Rama favored residues and led to a decrease in poor rotamers. The protein–protein docking was performed using the iATTRACT tool with conformational flexibility of binding partners (14). In the docking process potential energy is precalculated on a grid and then interactions are calculated by interpolations from the nearest grid point. Moreover, the docking process includes several energy minimizations steps. For protein–protein docking the 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 the HDOCK tool to generate protein–RNA complexes. The HDOCK is a fast Fourier transform–based translational search algorithm, which is optimized by an iterative knowledge-based scoring function. The interactions between A₁₂ and CIRP, HMGB1, and histone H3 were calculated using the PDBePISA tool, and the complexes were visualized using the PyMOL and Chimera tools (15). Interactions between TLR4 and CIRP with or without A₁₂ were also assessed in the same manner.

Recombinant mouse CIRP (denoted as eCIRP) was produced, and its purity and efficacy were validated 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).

OpenSPR (Nicoya, ON, Canada) was used to examine the direct interaction between eCIRP and A₁₂ (17). eCIRP was immobilized on the surface of a carboxyl sensor as a ligand, and various concentrations of A₁₂ (15.6–250 nM) were injected as an analyte and a K_D value was calculated. To determine the effect of A₁₂ on the binding of eCIRP to TLR4, eCIRP (500 nM) incubated with various concentrations of A₁₂ (50–500 nM) were injected over a high-sensitivity NTA sensor immobilized with TLR4 (catalog no. 1478-TR-050; R&D Systems, Minneapolis, MN). In addition, various concentrations of eCIRP (62.5–500 nM) were incubated with either 50, 100, or 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).

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 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 A₁₂ for an optimal time for each experiment.

Peritoneal macrophages were incubated with FITC-labeled eCIRP at 50 nM in the presence and absence of 500 nM A₁₂ for 15 min. Cells were washed with PBS and subsequently stained with allophycocyanin anti-mouse F4/80 Ab (catalog no. 123116; BioLegend, San Diego, CA) for 1 h. Confocal microscopy images were obtained using a Zeiss LSM 900 confocal microscope equipped with a ×63 objective. The samples were also subjected to flow cytometry using a FACSymphony flow cytometer (BD Biosciences) and subsequently analyzed by FlowJo software (Tree Star, Ashland, OR).

Fluorescence resonance energy transfer (FRET) analysis was performed as described previously (18). Peritoneal macrophages were cultured with 50 nM eCIRP with or without 500 nM A₁₂ for 15 min. Cells were incubated with rabbit anti-mouse CIRP Ab (catalog no. 10209-2-AP; Proteintech, Rosemont, IL) for 1 h and subsequently stained with Cy3-conjugated AffiniPure F(ab′)₂ donkey anti-rabbit IgG (catalog no. 711-166-152; Jackson ImmunoResearch Laboratories). Cells were also stained with allophycocyanin anti-mouse TLR4 Ab (catalog no. 145406; BioLegend) or allophycocyanin anti-mouse CD11b Ab (catalog no. 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), wherein a FRET unit = (E3_both − E3_none) – [(E3_{allophycocyanin} − E3_none) × (E2_both/E2_{allophycocyanin})] – [(E3_Cy3 − E3_none) × (E1_both/E1_Cy3)].

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% Nonidet P-40, 5% glycerol, 2 mM Na₃VO₄, and protease/phosphatase inhibitor cocktails (pH 7.4) (Roche Diagnostics, Basel, Switzerland). 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 Abs against p-p38 (catalog no. 9211S; Cell Signaling Technology, Danvers, MA), total p38 (catalog no. 9212S; Cell Signaling Technology), IκBα (catalog no. 10268-1-AP; Proteintech), and β-actin (MilliporeSigma, Burlington, MA) followed by reaction with fluorescence-labeled secondary Abs (anti-mouse IgG, catalog no. 926-68070; anti-rabbit IgG, catalog no. 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 (National Institutes of Health). Because of the nearly identical m.w. of the proteins of interest and the possibility that protein bands can persist even after membrane stripping and reprobing, we ran the same amount of proteins for each lane on separate gels. Subsequently, we individually probed each membrane with the specific Ab of interest.

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 Web site.

To determine the in vivo half-life of A_12, Cy3-labeled A₁₂ (0.05 nmol/g body weight [BW]) was 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. Importantly, note that performing repeated blood draws from a single mouse is not feasible. Therefore, we used rats for this experiment. There are two phases of half-life. The first sharp slope is the distribution phase (α phase). This step is followed by the elimination phase (β 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, that is, the β 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.

Polymicrobial sepsis was induced in mice by cecal ligation and puncture (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 22G needle. A vehicle (PBS) or 0.5 nmol/g BW A₁₂ was 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 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/kg 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 d for survival studies.

Serum samples or cell culture supernatants were analyzed by ELISA kits for IL-6 and TNF-α (both from BD Biosciences, Franklin Lakes, NJ). Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (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 manufacturer’s instructions.

Total RNA was extracted from homogenized lung tissues using TRIzol reagent (Invitrogen, Thermo Fisher Scientific). cDNA was synthesized using murine leukemia virus 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 StepOnePlus real-time PCR machine (Applied Biosystems). The sequences of primers used in this study are as follows: β-actin, 5′-CGTGAAAAGATGACCCAGATCA-3′ (forward), 5′-TGGTACGACCAGAGGCATACAG-3′ (reverse); IL-6, 5′-CCGGAGAGGAGACTTCACAG-3′ (forward), 5′-GGAAATTGGGGTAGGAAGGA-3′ (reverse); TNF-α, 5′-AGACCCTCACACTCAGATCATCTTC-3′ (forward), 5′-TTGCTACGACGTGGGCTACA-3′ (reverse); IL-1β, 5′-CAGGATGAGGACATGAGCACC-3′ (forward), 5′-CTCTGCAGACTCAAACTCCAC-3′ (reverse); KC, 5′-GCTGGGATTCACCTCAAGAA-3′ (forward), 5′-ACAGGTGCCATCAGAGCAGT-3′ (reverse); MIP2, 5′-CCAACCACCAGGCTACAGG-3′ (forward), 5′-GCGTCACACTCAAGCTCTG-3′ (reverse).

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 (Thermo Fisher Scientific). Absorbance was measured at 460 nm to calculate myeloperoxidase (MPO) activity, which was normalized to a protein amount measured by protein assay reagent from Bio-Rad (Hercules, CA).

Formalin-fixed, paraffin-embedded lung tissue blocks were sectioned at 5-μm thickness and placed on glass slides. Lung tissue sections were stained with 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 a TUNEL assay kit (Roche Diagnostics, Indianapolis, IN) according to the manufacturer’s protocol and examined under a Nikon Eclipse Ti-S fluorescence microscope (Nikon, Melville, NY). The microscopy assessments of lung tissue sections were performed at ×200 magnification.

Data represented in the figures are expressed as mean ± SEM. ANOVA was used for one-way comparison among multiple groups, and significance was determined by the Student–Newman–Keuls (SNK) test. A 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).

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, a 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 Fig. 1). A₁₂ was predicted to interact with the N-terminal RNA-binding domain of eCIRP by forming two hydrogen bonds (Fig. 1A). The root mean square deviation, 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 with normal unmutated eCIRP (Fig. 1B). eCIRP was shown to have a much stronger affinity than other chromatin-associated DAMPs (e.g., HMGB1, histone H3) that are not bona fide RNA-binding proteins (Fig. 1C, 1D). 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 the 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) (Fig. 1E). Taken together, A₁₂ selectively binds to eCIRP with a high affinity, suggesting its potential as an inhibitor of eCIRP.

eCIRP is known as a ligand of TLR4 (16), and thus we next investigated the effect of A₁₂ on 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 ΔiG and decreased ΔGdiss (Fig. 2A). SPR further revealed that A₁₂ dose-dependently inhibited the binding of eCIRP to TLR4 (Fig. 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 (Fig. 2G) and quantified by flow cytometry (Fig. 2H, 2I). Furthermore, FRET analysis revealed that A₁₂ significantly inhibited the binding of eCIRP to TLR4 (Fig. 2J). As a negative control, significantly less binding was observed between eCIRP and CD11b (pan marker of macrophages) compared with eCIRP and TLR4 (Fig. 2J). These data indicate that A₁₂ interferes with the binding of eCIRP to TLR4, suggesting that A₁₂ inhibits TLR4 activation mediated by eCIRP.

eCIRP has been shown to induce an 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 proinflammatory signaling in macrophages as determined by p38 phosphorylation and IκBα degradation, which reflect the activation of MAPK and NF-κB pathways, respectively (Fig. 3A, 3B, Supplemental Fig. 2). Interestingly, treatment with A₁₂ significantly inhibited p38 phosphorylation and IκBα degradation induced by eCIRP in macrophages (Fig. 3A, 3B). 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 expression levels of these transcription factors were dramatically decreased by A₁₂ treatment (Fig. 3C, 3D). These data indicate that A₁₂ significantly attenuates the activation of proinflammatory signaling pathways induced by eCIRP.

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

Our previous study revealed significantly higher levels of eCIRP in the serum of sepsis patients (19). In this study, 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 with sham mice (Fig. 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 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, TNF-α, AST, ALT, and LDH in the serum were significantly elevated in CLP mice. In contrast, those parameters were dramatically lower in A₁₂-injected mice by 45, 47, 27, 32, and 24%, respectively (Fig. 5B–F), indicating that 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, TNF-α, 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 (Fig. 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, whereas A₁₂-injected mice were protected from those histological changes (Fig. 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 Fig. 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 (Fig. 5Q). These findings demonstrate that A₁₂ attenuates inflammation, alleviates ALI, and improves survival in sepsis (Fig. 6).

In the current 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 MAPK 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 (Fig. 6). Intracellular CIRP acts as an RNA chaperone, whereas eCIRP, released from stressed or damaged cells during sepsis, serves as a potential DAMP (16). Our previous studies have demonstrated that 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 the binding of A₁₂ to eCIRP prevented the activation of the TLR4 pathway, as evidenced by the inhibition of p38 phosphorylation and IκBα 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 critical 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.

Different kinds of oligonucleotide drugs and vaccines are clinically available at present, but their predominant use 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 diverse applications of using oligonucleotides other than modulating gene expressions. Because 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 intracellular 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). Taken 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 a 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 that contributes to inflammatory disorders (18). TREM-1 and TLR4 act synergistically because 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. Similar to 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. In contrast, our approach of specifically targeting the ligand eCIRP by A₁₂ to inhibit its binding to the receptors suggests that 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 proinflammatory 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 proinflammatory activity and potential to inhibit eCIRP from binding to multiple receptors present an 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 PRR 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 complementary 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 current 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 because 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 nonimmune cells express PRRs, including TLR4, which induces MAPK 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 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 cells, or endothelial cells, which are all exaggeratedly activated during inflammatory conditions, including sepsis (4). However, a cell-specific approach is awaited to confirm this theory. In this study, we focused on the lung injury, as lungs are significantly impacted by CLP, and our previous studies provide compelling evidence for the contribution of eCIRP to ALI (9, 10). Additionally, in the clinical context, ALI is a prevalent complication that contributes to mortality in sepsis (5). Although our study primarily focused 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 those 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 noteworthy 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 with the peripheral blood. In fact, we have previously observed notably higher eCIRP levels in the peritoneal cavity compared with 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 relies on blood parameters. Unfortunately, performing repeated blood draws in mice is highly invasive, making it difficult to monitor them closely after the procedure. Although 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. Although not all nucleotide sequences exhibited this property, miRNA-130b-3p was found to attenuate the inflammatory response of eCIRP. Because our previous study involved several nucleotide mimics that served as controls, we did not use 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, A₁₂ targets eCIRP to act against systemic inflammation and ALI and improve survival in sepsis. Other diseases that 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.

The authors have no financial conflicts of interest.

We acknowledge the BioRender software service for preparing the visual abstract.