Cold-inducible RNA-binding protein (CIRP) were first discovered in mouse testis more than two decades ago (1). It came to the attention of researchers as a result of its expression during moderate cold stress. The RG/RGG region of CIRP is critical for mediating CIRP phase separation in vitro and stress granules (SGs) association in cells (2). As a RNA-binding protein (RBP), CIRP plays important roles in transcription, pre-mRNA processing/transport, mRNA degradation, translation, and non-coding RNA processing. Studies have shown that the 3`-UTR binding sites of CIRP are enriched within 100 nucleotides upstream of the polyadenylation sites, and UU and UUU are most likely the core recognition sequences of CIRP (3). CIRP consists of an N-terminal RNA-recognition motif (RRM) and a C-terminal arginine-rich region. The X-ray quaternary structure of the CIRP RRM has been resolved recently and four important residues with possible involvement in protein-nucleic acid binding have been identified (4). Expression of CIRP during cold stress modulates RNA stability and translation in hibernating mammals that reduce their body temperature from 37°C to as low as 0~5°C during bouts of dullness (5). CIRP is translocated from the nucleus to the cytoplasm, whereby they stabilize mRNAs. They can regulate mRNA transcripts and protect them from degradation for future protein synthesis. Studies have also shown that the expression of CIRP can also be regulated by several kinds of stress conditions suggesting that CIRP is generally expressed as stress-response proteins. Besides, CIRP is widely expressed in a large variety of tissues and cells.

According to the literature, iCIRP has been implicated in multiple cellular processes such as cell proliferation (6), cell survival (7, 8), telomere maintenance (9), circadian modulation (10), DNA repair (11) and tumor formation and progression (12, 13). Intracellular CIRP (iCIRP) can migrate from the nucleus to the cytoplasm via methylation-dependent mechanism in response to stress and regulate the stability of mRNA through their binding sites on the 3′-UTR of their target mRNAs (3). The translocation of CIRP is dependent on GSK3β and CK2. Both GSK3β and CK2 cause phosphorylation of CIRP and affect its cellular localization and pretreatment of cells with either CK2 inhibitors or GSK3β inhibitors prior to UV treatment significantly reduce CIRP localization to the cytosol (14). These results suggest that both methylation and phosphorylation of CIRP is required for CIRP translocation to the cytosol upon stress stimulus. Some studies have demonstrated that both CK2 sites and GS3Kβ sites on CIRP have overlapping functional role in regulating CIRP translocation to the cytosol, but only GSK3β sites are involved in the RNA-binding activity of CIRP, in response to UV radiation (14, 15).

In contrast to its functions in the intracellular space, extracellular CIRP (eCIRP) has been discovered recently as a damage-associated molecular pattern (DAMP) capable of triggering inflammation in various inflammatory conditions (16), including sepsis (17), neuroinflammation (18)and ischemia-reperfusion injury (19–24). For example, administering exogenous CIRP to healthy mice was shown to induce lung injury through vascular leakage, neutrophil infiltration, local production of pro-inflammatory cytokines, and activating the NLRP3 inflammasome in the vascular endothelial cells of the lung (25). Altogether, a fair number of studies have investigated the potential role of eCIRP in different models of inflammatory diseases, such as sepsis (16, 26–29), acute pancreatitis (30), and Alzheimer’s disease (31). The important pro-inflammatory role of eCIRP suggests that targeting eCIRP may be of potential therapeutic importance in controlling inflammatory diseases. Nevertheless, the translation of such findings into clinical practice still remains nascent field to be explored.

Here, we summarize how eCIRP, especially exosome derived CIRP, amplify inflammation in different inflammatory conditions with the aim of providing new insights for the development of novel targeted therapies.

CIRP release mechanisms include necrosis, lysosome-mediated release, extracellular traps, and exosomes (32).CIRP can be released either via inflammasome activation or passively following cell death (33). Necroptosis, apoptosis, pyroptosis, and ferroptosis might contribute to the passive release of the CIRP (32). Studies have shown that CIRP is among the main factors that induces mitochondrial DNA (mtDNA) fragmentation in damaged tissues (34). However, according to an in-vitro study on macrophages, it has been shown that cell necrosis does not trigger the passive release of CIRP (16).

Active release of CIRP is mediated mainly by lysosomes and exosomes. When cells are stimulated by oxidative stress, hypothermia, ultraviolet (UV) radiation, etc. CIRP are transferred to the extracellular space through the lysosomal pathway (12, 33). CIRP migrates from the nucleus to cytoplasmic SGs via a methylation-dependent mechanism and act as translational repressors. Stressors such as oxidative stress, endoplasmic reticulum (ER) stress, osmotic shock, and heat shock may lead to the methylation of iCIRP (35). Oxidative stress leads to CIRP migration to SGs without altering their expression (36). SGs are dynamic cytoplasmic foci in which stalled translation initiation complexes accumulate in cells that are subjected to environmental stress. The relocation of CIRP to the SG also occurs in osmotic stress, heat shock and in response to endoplasmic reticulum stress (36). CIRP/hnRNP A18 is an RNA-binding factor consisting of an RNA recognition motif (RRM) at the N-terminus and a region at the c-terminus containing multiple repeats of the RGG motif. Methylation is important for the recruitment of CIRP from the nucleus to the SGs. As mentioned earlier, TIA-1 has been shown to be the main mediator of SGs formation (37). In the absence of TIA-1 or TIAR proteins, CIRP is still recruited to SGs. Since CIRP does not contain any signaling peptides, their secretion cannot be mediated by the classical Endoplasmic Reticulum-Golgi-dependent pathway. In one study, biochemical fractionation revealed that CIRP is enriched in the lysosomal compartment of macrophages undergoing hypoxia, suggesting that CIRP is released through lysosomal secretions (16, 35). As a class of DAMPs, eCIRP conforms to the general mechanism of the extracellular release of DAMPs (32). The well-studied carriers of DAMPs during active release are the secreted lysosomes and exosomes, both of which are normally secreted by exocytosis (16, 38).

Lysosomal secretion is a typical feature of stressed cells and has been also demonstrated to be a release pathway for eCIRP (16, 32). The synaptotagmin (Syt) assay found that lysosomal secretion can be initiated by stimulating cell surface receptors as well as through increasing intracellular Ca²⁺. Syt mobilizes lysosomes to the microtubule organizing center, where lysosomes are associated with kinesin motility (39). The motor proteins further transport the lysosome near the secretion site, where the lysosome travels to the docking site using actin-based movements. The docking and fusion of lysosomes with the plasma membrane are mediated by Rabs and SNARE (Soluble NSF Attachment Protein Receptors) complexes, respectively (39, 40).

The overexpression of CIRP has been consistently witnessed in different organs and species (from amphibians to humans) upon mild hypothermia or cold stress (10), which supports the plausible protective role of CIRP in adaptation of these species to cold stress. However, CIRP homologs in different species responded differently to different environmental stresses. Besides this, CIRP is also overexpressed upon UV radiation, mild hypoxia and low glucose conditions, but is under expressed in response to heat stress stimulus and upon treatment with inflammatory cytokines such as TNF-α and TGF-β (12), underscoring that CIRP is general stress responsive protein. While, CIRP is overexpressed upon mild hypoxia, the chronic hypoxia results in completely opposite genetic outcome, that is low CIRP expression (13). Glycogen synthase kinase 3β (GSK3β) is an important serine/threonine kinase, which is involved in various biochemical processes in cells, for instance cell metabolism, cell cycle, transcription, vesicular transport and others (41). Activation of GSK3β has shown to increase the transcription of CIRP (16). CIRP is also upregulated by IGF1 (17). As expected, the transcription of the CIRP homolog in Salmon can be upregulated by osmotic stress, but not by cold stress (18) as expected to be respond differently in a species-specific manner. Similar to this, there exist different homologs of CIRP in Xenopus cells including xCIRP, xCIRP-1 and xCIRP-2. All homologs express and respond differently to different environmental stimulus. For instance, low temperature (cold stress) induces overexpression of xCIRP and xCIRP 2, but not xCIRP-1 (9, 19, 20). This implies that the CIRP is a general stress protein and its expression and response to a particular stress differs significantly between species.

An essential mode of intercellular communication is the transport of information components such as proteins, nucleic acids, and lipids via exosomes (42, 43), extracellular vesicles measuring between 40 and 100 nm in diameter and found in various bodily fluids (44). Exosomes support the interior environment’s balance under normal physiological settings (45). However, these exosomes may have radically altered contents in a state of excessive inflammation. Many studies have shown that miRNAs such as miR-155 (46) and miR-21 (47) transported by exosomes play a crucial role in inflammation. It has been shown that CIRs can be mainly released outside the cell in two ways: passively through necrosis and actively through lysosomes (32). However, Murao et al. (48), discovered that CIRP might stimulate inflammatory cell aggregation and activation through the exosome secretion.

Murao and colleagues found that the serum exosomes of LPS-injected and CLP-operated mice contained significantly higher amounts of eCIRP than those in control animals. Similarly, activation of macrophages with LPS resulted in a significant increase in the release of exosomal CIRP. Based on these findings, it appears that exosome-carried CIRP are a substantial source of eCIRP. Accordingly, the presence of eCIRP in exosomes could be justified by the presence of the lipid bilayer on the surface of exosomes, which can protect proteins or microRNAs from being degraded during transport process. CD63 is a well-known marker used to identify exosomes (49). Studies have revealed that CIRP can stably bind to CD63. Furthermore, they discovered that exosome biogenesis and release inhibitors (50) could not only block exosome release but also simultaneously decrease the expression of eCIRP. With the exposure of inflammatory cells to exosomes, eCIRP present on the surface of the exosomes stimulates the release of pro-inflammatory cytokines and migration of neutrophils by binding to cell surface receptors (48) ( Figure 1 ). However, there is no direct evidence of eCIRP release from the interior of exosome/luminal vesicles to the surface. Through literature review, it was found that there were transmembrane proteins on the exosome surface, such as lysosome-associated membrane protein (LAMP) and transferrin receptor (TfR) (51).Therefore, we hypothesized that proteins carried in exosomes cross the phospholipid bimolecular wind of exosomes through the stimulation of specific signaling molecules to reach the surface, and CIRP is no exception. Taken together, these findings support the notion that exosomes are mediators of CIRP release that targeting exosomal CIRP may represent a novel treatment strategy.

In inflammatory diseases, eCIRP acts as an inflammatory amplifier by binding to its receptors. TLR4, TREM-1, and IL-6R are three receptors to which CIRP binding occurs, as mentioned in the Table 1 . TLR4 is a member of an important class of molecules involved in adaptive immunity and acts as a bridge between adaptive and innate immunity. CIRP binds to TLR4-MD2, a complex formed in systemic inflammation induced by hemorrhagic shock and sepsis (16). Hemorrhagic shock increases eCIRP levels and activates STING through the TLR4/MyD88/TRIF pathway to exacerbate inflammation (55). In lung fibroblasts, eCIRP amplifies pro-inflammatory cytokines in a TLR4-dependent manner, triggering pulmonary fibrosis (54). As a receptor for eCIRP, TREM-1 plays a vital role in ischemia-reperfusion in the liver, intestine, and other organs (60), and induces inflammation in macrophages and neutrophils (35), forming NETs (61). In hemorrhagic shock and sepsis, TREM-1 can also be a key target of eCIRP (16, 56, 62). In the alveoli, TREM-1 can act as a target to trigger inflammation in alveolar type II cells (57). One study showed a strong binding affinity between eCIRP and IL-6R (58). Supporting this finding, it is well known that CIRP can skew the macrophages towards the M2 phenotype and that using IL-6R antibodies CIRP can inhibit M2 polarization (58). In neurological diseases, eCIRP activates the IL-6Rα/STAT3/Cdk5 pathway in neurons, inducing neuroinflammation (59). Combining the above two studies, we can find that IL-6R plays a double-edged sword role in inflammation. It can improve the tolerance of macrophages to endotoxin in sepsis, and can also promote microglial inflammation in the nervous system.

Delivery systems use specialized carriers to facilitate the aggregation of drugs in their target tissues or cells in order to increase their efficacy and usage (91). Combining targeted medications with nanocarriers is one of the most important means of increasing the effectiveness of targeted therapeutics (92–94). Exosomes generated from intracellular membranes (95) have many benefits over conventional nanocarriers, including good biocompatibility and targeting (96), easy modification of the membrane surface (97), and the capacity to traverse the blood-brain barrier and placental barrier (98). As a new means of drug delivery, exosomes are gaining attention (99).

Exosome-based targeted drug delivery systems have been summarized in several studies (100). Naturally occurring exosomes can be used as carriers for a wide range of therapeutic agents such as natural products (101–104), synthetic pharmaceuticals (105), nucleic acid drugs (106, 107), and protein-based peptides or proteins (108). Exosomes can penetrate biological barriers that regular nanomaterials cannot, and they exhibit good efficacy. The exosome-carried CIRP brings a more severe inflammatory response to the disease while providing us with a potential therapeutic strategy (48, 109). Using exosomes that have been artificially modified may be a way to reduce the eCIRP-induced inflammatory response. On one hand, adding peptides or proteins to the surface of exosomes inhibits the activation of downstream signaling pathways by targeting inflammatory cell surface receptors (110, 111) and on the other hand, exosomes directly loaded with natural products through incubation (108), electroporation (112), sonication (113), and transfection exert anti-inflammatory effects. In conclusion, these methods may address the low water solubility and bioavailability of natural compounds (114). Moreover, the drug loading and modification (115) of EVs may enhance the targeting ability of the drug and prevent its rapid oxidation.

Since the first description of CIRP two decades ago, the physiopathological and biological roles of CIRP have been extensively studied. Although we have gained significant achievements in unraveling the role of CIRP in diseased etiology and pathogenesis; however, there are still many unanswered questions, for instance, what is the role of CIRP in chronic inflammatory diseases such as obesity, diabetes and other kinds of diseases. It has been proved that iCIRP participates in cell proliferation, cell survival, apoptosis, and circadian rhythm, telomere maintenance, and carcinoma progression through regulating mRNA stability as an RNA chaperone (28).

Whereas little is known about the function of exosome-carried CIRP and its role in inflammation. Here we summarized former and recent studies on the roles of eCIRP, especially exosome-derived CIRP, in inflammatory disease. One of the most notable recent developments is that eCIRP has been shown to play an important part in various types of ALI through participating in different inflammatory pathways. The discovery of eCIRP’s role as an important DAMP has led to a new field in inflammatory disease research, while many knowledge gaps still exist and need to be addressed. As a stabilizing RNA-binding protein, eCIRP is pro-inflammatory in most inflammatory conditions but also reduces inflammation by unknown mechanisms and pathways. On one hand, it remains to be investigated in other cell types such as B lymphocytes and NK cells since current studies have only focused on eCIRPs role in macrophages, neutrophils, and T cell biology. On the other hand, only macrophages have been identified as the source of exosomal CIRP, while other cell types as sources of exosomal CIRP remain to be studied.

Both in-vivo and in-vitro evidence support the notion that eCIRP can be considered as a novel target for the research and development of innovative drugs for the prevention or treatment of inflammatory diseases. Future studies should be aimed at clarifying the pivotal roles eCIRP plays in other potential pathways of inflammatory diseases in the hope that more therapeutic targets can be identified. Peptides and natural products targeting exosomal CIRP may facilitate the clinical translation of these new therapeutic strategies against inflammation.

The proinflammatory role and the plausible protective effects of CIRP blockage using neutralizing antibody or proteins/peptides in sepsis and other inflammatory diseases suggested a pivotal role of CIRP in inflammation-related diseases and has offered a strong basis for the exploration of therapeutic potential of neutralizing antibody or peptide in tackling various inflammatory diseases. Therefore, elucidating the role of CIRP in these diseases will have a significant impact on our understanding of physiopathological of diseases and may provide the rationale for the design of novel therapeutics.

Conceptualization, CX, HC; writing—original draft preparation, JH, YZ, PG, TD, HW, ST, YL, QY, BH and GZ. All authors have edited the published version of the manuscript.