C-Reactive Protein: The Most Familiar Stranger.

C-reactive protein (CRP) is a highly conserved pentraxin with pattern recognition receptor-like activities. However, despite being used widely as a clinical marker of inflammation, the in vivo functions of CRP and its roles in health and disease remain largely unestablished. This is, to certain extent, due to the drastically different expression patterns of CRP in mice and rats, raising concerns about whether the functions of CRP are essential and conserved across species and how these model animals should be manipulated to examine the in vivo actions of human CRP. In this review, we discuss recent advances highlighting the essential and conserved functions of CRP across species, and propose that appropriately designed animal models can be used to understand the origin-, conformation-, and localization-dependent actions of human CRP in vivo. The improved model design will contribute to establishing the pathophysiological roles of CRP and facilitate the development of novel CRP-targeting strategies.

Identification of a distal enhancer that determines the expression pattern of acute phase marker C-reactive protein.

C-reactive protein (CRP) is a major acute phase protein and inflammatory marker, the expression of which is largely liver specific and highly inducible. Enhancers are regulatory elements critical for the precise activation of gene expression, yet the contributions of enhancers to the expression pattern of CRP have not been well defined. Here, we identify a constitutively active enhancer (E1) located 37.7 kb upstream of the promoter of human CRP in hepatocytes. By using chromatin immunoprecipitation, luciferase reporter assay, in situ genetic manipulation, CRISPRi, and CRISPRa, we show that E1 is enriched in binding sites for transcription factors STAT3 and C/EBP-ß and is essential for the full induction of human CRP during the acute phase. Moreover, we demonstrate that E1 orchestrates with the promoter of CRP to determine its varied expression across tissues and species through surveying activities of E1-promoter hybrids and the associated epigenetic modifications. These results thus suggest an intriguing mode of molecular evolution wherein expression-changing mutations in distal regulatory elements initiate subsequent functional selection involving coupling among distal/proximal regulatory mutations and activity-changing coding mutations.

[Platelet adhesion mediated by von Willebrand factor in patients with premature coronary artery disease].

Aim    To study platelet adhesion mediated by von Willebrand factor (VWF) in patients with premature ischemic heart disease (IHD).Material and methods    This study enrolled 58 patients with stable IHD, including 45 men younger than 55 years with the first manifestation of IHD at the age of <50 years and 13 women younger than 65 years with the first manifestation of IHD at the age of <60 years. The control group consisted of 33 patients, 13 men younger than 55 years and 20 women younger than 65 years without IHD. Platelet adhesion to the collagen surface at the shear rate of 1300 s-1 was studied by evaluating the intensity of scattered laser light from the collagen-coated optical substrate in a flow chamber of a microfluidic device after 15-min circulation of whole blood in the chamber. Decreases in platelet adhesion after addition to the blood of monoclonal antibodies (mAb) to platelet receptors glycoproteins Ib (GPIb) to inhibit the receptor interaction with VWF were compared for patients of both groups. Results    In patients with premature IHD, the decrease in platelet adhesion following the platelet GPIb receptor inhibition was significantly less than in patients of the control group (74.8 % (55.6; 82.7) vs. 28.9 % (-9.8; 50,5), p <0.001). For the entire sample, the median decrease in platelet adhesion following the GPIb receptor inhibition was 62.8 % (52.2; 71.2). With an adjustment for traditional risk factors of IHD, a decrease in platelet adhesion of >62.8% after blocking GPIb receptors increased the likelihood of premature IHD (OR=9.84, 95 % CI: 2.80-34.59; p <0.001).Conclusion    Blocking the interaction of GPIb receptors with VWF in patients with premature IHD and increased shear rate induced a greater decrease in platelet adhesion than in patients without this disease. This suggested that an excessive interaction of VWF with platelets might contribute to the pathogenesis of premature IHD.

C-reactive protein exacerbates epithelial-mesenchymal transition through Wnt/ß-catenin and ERK signaling in streptozocin-induced diabetic nephropathy.

Previous studies have reported the pathogenic role of C-reactive protein (CRP) during diabetic kidney disease (DKD) in human CRP transgenic and Crp-/- mice. However, because humans and mice have inverse acute phase expression patterns of CRP and serum amyloid P component, this could lead to the inaccurate evaluation of CRP function with the above-mentioned CRP transgenic mouse. But different from mice, rats have the same acute phase protein expression pattern as human, which might avoid this problem and be a better choice for CRP function studies. To dispel this doubt and accurately define the role of CRP during diabetic nephropathy, we created the first Crp-/- rat model, which we treated with streptozocin to induce DKD for in vivo studies. Moreover, an established cell line (human kidney 2) was used to further investigate the pathologic mechanisms of CRP. We found that CRP promotes epithelial-mesenchymal transition (EMT) through Wnt/ß-catenin and ERK1/2 signaling, which are dependent on CRP binding to FcγRII on apoptotic cells. By promoting EMT, CRP was demonstrated to accelerate the development of DKD. We thus present convincing evidence demonstrating CRP as a therapeutic target for DKD treatment.-Zhang, L., Shen, Z.-Y., Wang, K., Li, W., Shi, J.-M., Osoro, E. K., Ullah, N., Zhou, Y., Ji, S.-R. C-reactive protein exacerbates epithelial-mesenchymal transition through Wnt/ß-catenin and ERK signaling in streptozocin-induced diabetic nephropathy.

An Intrinsically Disordered Motif Mediates Diverse Actions of Monomeric C-reactive Protein.

Most proinflammatory actions of C-reactive protein (CRP) are only expressed following dissociation of its native pentameric assembly into monomeric form (mCRP). However, little is known about what underlies the greatly enhanced activities of mCRP. Here we show that a single sequence motif, i.e. cholesterol binding sequence (CBS; a.a. 35-47), is responsible for mediating the interactions of mCRP with diverse ligands. The binding of mCRP to lipoprotein component ApoB, to complement component C1q, to extracellular matrix components fibronectin and collagen, to blood coagulation component fibrinogen, and to membrane lipid component cholesterol, are all found to be markedly inhibited by the synthetic CBS peptide but not by other CRP sequences tested. Likewise, mutating CBS in mCRP also greatly impairs these interactions. Functional experiments further reveal that CBS peptide significantly reduces the effects of mCRP on activation of endothelial cells in vitro and on acute induction of IL-6 in mice. The potency and specificity of CBS are critically determined by the N-terminal residues Cys-36, Leu-37, and His-38; while the versatility of CBS appears to originate from its intrinsically disordered conformation polymorphism. Together, these data unexpectedly identify CBS as the major recognition site of mCRP and suggest that this motif may be exploited to tune the proinflammatory actions of mCRP.

C-reactive protein directly suppresses Th1 cell differentiation and alleviates experimental autoimmune encephalomyelitis.

Human C-reactive protein (CRP) is a serum-soluble pattern recognition receptor that serves as a marker of inflammation and directly contributes to innate immunity. In this study, we show that human CRP also directly contributes to adaptive immunity, that is, native CRP binds specifically to human Jurkat T cells and to mouse naive CD4(+) T cells and modulates their Th1 and Th2 responses. In vitro both exogenously added (purified) and endogenously expressed (via transfection) human CRP inhibited Th1 differentiation and augmented Th2 differentiation of naive CD4(+) T cells. In vivo for human CRP transgenic compared with wild-type mice, a lesser proportion of the T cells recovered from the spleens of healthy animals were Th1 cells. Moreover, in both CRP transgenic mice and in wild-type mice treated with human CRP, during myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis both the Th1 cell response and disease severity were inhibited. These pattern recognition-independent actions of CRP directly on T cells highlights the potential for this soluble pattern recognition receptor to act as a tonic regulator of immunity, shaping global adaptive immune responses during both homeostasis and disease.

Topological localization of monomeric C-reactive protein determines proinflammatory endothelial cell responses.

The activation of endothelial cells (ECs) by monomeric C-reactive protein (mCRP) has been implicated in contributing to atherogenesis. However, the potent proinflammatory actions of mCRP on ECs in vitro appear to be incompatible with the atheroprotective effects of mCRP in a mouse model. Because mCRP is primarily generated within inflamed tissues and is rapidly cleared from the circulation, we tested whether these discrepancies can be explained by topological differences in response to mCRP within blood vessels. In a Transwell culture model, the addition of mCRP to apical (luminal), but not basolateral (abluminal), surfaces of intact human coronary artery EC monolayers evoked a significant up-regulation of MCP-1, IL-8, and IL-6. Such polarized stimulation of mCRP was observed consistently regardless of EC type or experimental conditions (e.g. culture of ECs on filters or extracellular matrix-coated surfaces). Accordingly, we detected enriched lipid raft microdomains, the major surface sensors for mCRP on ECs, in apical membranes, leading to the preferential apical binding of mCRP and activation of ECs through the polarized induction of the phospholipase C, p38 MAPK, and NF-κB signaling pathways. Furthermore, LPS and IL-1ß induction of EC activation also exhibited topological dependence, whereas TNF-α did not. Together, these results indicate that tissue-associated mCRP likely contributes little to EC activation. Hence, topological localization is an important, but often overlooked, factor that determines the contribution of mCRP and other proinflammatory mediators to chronic vascular inflammation.

Cataract-linked mutation R188H promotes ßB2-crystallin aggregation and fibrillization during acid denaturation.

Cataract is characterized by the formation of light-scattering protein aggregates in the lens. ß/γ-Crystallins are the predominant structural proteins in the cytosol of lens fiber cells, and more than fifty ß/γ-crystallin mutations have been linked to autosomal dominant congenital cataract. However, the structural role of these mutations in the formation of the core structures of amorphous aggregates or amyloid-like fibrils has not been elucidated yet. In this research, we studied the effects of the V187M and R188H mutations on the aggregation and fibrillization of ßB2-crystallin during acid denaturation. The behavior of V187M was the same as the WT protein, suggesting that the residue at position 187 contributed little to the aggregation/fibrillization process. R188H promoted the formation of amorphous aggregates at pH above 3 and accelerated fibrillization at pH 3. The distinct behaviors of the mutants suggested that the residue at position 188 might play a regulatory role in ßB2-crystallin aggregation/fibrillization but not reside in the core of the aggregates/fibrils.

Intra-membrane oligomerization and extra-membrane oligomerization of amyloid-ß peptide are competing processes as a result of distinct patterns of motif interplay.

Soluble oligomers of amyloid-ß peptide (Aß) are emerging as the primary neurotoxic species in Alzheimer disease, however, whether the membrane is among their direct targets that mediate the downstream adverse effects remains elusive. Herein, we show that multiple soluble oligomeric Aß preparations, including Aß-derived diffusible ligand, protofibril, and zinc-induced Aß oligomer, exhibit much weaker capability to insert into the membrane than Aß monomer. Aß monomers prefer incorporating into membrane rather than oligomerizing in solution, and such preference can be reversed by the aggregation-boosting factor, zinc ion. Further analyses indicate that the membrane-embedded oligomers of Aß are derived from rapid assembly of inserted monomers but not due to the insertion of soluble Aß oligomers. By comparing the behavior of a panel of Aß truncation variants, we demonstrate that the intra- and extra-membrane oligomerization are mutually exclusive processes that proceed through distinct motif interplay, both of which require the action of amino acids 37-40/42 to overcome the auto-inhibitory interaction between amino acids 29-36 and the N-terminal portion albeit via different mechanisms. These results indicate that intra- and extra-membrane oligomerization of Aß are competing processes and emphasize a critical regulation of membrane on the behavior of Aß monomer and soluble oligomers, which may determine distinct neurotoxic mechanisms.

Combination of anti-C1qA08 and anti-mCRP a.a.35-47 antibodies is associated with renal prognosis of patients with lupus nephritis.

Objective: The aim of this study is to explore the prevalence and clinicopathological associations between anti-C1qA08 antibodies and anti-monomeric CRP (mCRP) a.a.35-47 antibodies and to explore the interaction between C1q and mCRP. Methods: Ninety patients with biopsy-proven lupus nephritis were included from a Chinese cohort. Plasma samples collected on the day of renal biopsy were tested for anti-C1qA08 antibodies and anti-mCRP a.a.35-47 antibodies. The associations between these two autoantibodies and clinicopathologic features and long-term prognosis were analyzed. The interaction between C1q and mCRP was further investigated by ELISA, and the key linear epitopes of the combination of cholesterol binding sequence (CBS; a.a.35-47) and C1qA08 were tested by competitive inhibition assays. The surface plasmon resonance (SPR) was used to further verify the results. Results: The prevalence of anti-C1qA08 antibodies and anti-mCRP a.a.35-47 antibodies were 50/90 (61.1%) and 45/90 (50.0%), respectively. Levels of anti-C1qA08 antibodies and anti-mCRP a.a.35-47 antibodies were negatively correlated with serum C3 concentrations ((0.5(0.22-1.19) g/L vs. 0.39(0.15-1.38) g/L, P=0.002) and (0.48(0.44-0.88) g/L vs. 0.41(0.15-1.38) g/L, P=0.028), respectively. Levels of anti-C1qA08 antibodies were correlated with the score of fibrous crescents and tubular atrophy (r=-0.256, P=0.014 and r=-0.25, P=0.016, respectively). The patients with double positive antibodies showed worse renal prognosis than that of the double negative group (HR 0.899 (95% CI: 0.739-1.059), P=0.0336). The binding of mCRP to C1q was confirmed by ELISA. The key linear epitopes of the combination were a.a.35-47 and C1qA08, which were confirmed by competitive inhibition experiments and SPR. Conclusion: The combination of anti-C1qA08 and anti-mCRP a.a.35-47 autoantibodies could predict a poor renal outcome. The key linear epitopes of the combination of C1q and mCRP were C1qA08 and a.a.35-47. A08 was an important epitope for the classical pathway complement activation and a.a.35-47 could inhibit this process.

Monomeric C-reactive protein evokes TCR Signaling-dependent bystander activation of CD4+ T cells.

Bystander activation of T cells is defined as induction of effector responses by innate cytokines in the absence of cognate antigens and independent of T cell receptor (TCR) signaling. Here we show that C-reactive protein (CRP), a soluble pattern-recognition receptor assembled noncovalently by five identical subunits, can instead trigger bystander activation of CD4 + T cells by evoking allosteric activation and spontaneous signaling of TCR in the absence of cognate antigens. The actions of CRP depend on pattern ligand-binding induced conformational changes that result in the generation of monomeric CRP (mCRP). mCRP binds cholesterol in plasma membranes of CD4 + T cells, thereby shifting the conformational equilibrium of TCR to the cholesterol-unbound, primed state. The spontaneous signaling of primed TCR leads to productive effector responses manifested by upregulation of surface activation markers and release of IFN-γ. Our results thus identify a novel mode of bystander T cell activation triggered by allosteric TCR signaling, and reveal an interesting paradigm wherein innate immune recognition of CRP transforms it to a direct activator that evokes immediate adaptive immune responses.

A redox switch in C-reactive protein modulates activation of endothelial cells.

C-reactive protein (CRP) has been implicated in the regulation of inflammation underlying coronary artery disease; however, little is known about the molecular mechanisms responsible for the expression of its pro- or anti-inflammatory activities. Here, we have identified the intrasubunit disulfide bond as a conserved switch that controls the structure and functions of CRP. Conformational rearrangement in human pentameric CRP to monomeric CRP (mCRP) is the prerequisite for this switch to be activated by reducing agents, including thioredoxin. Immunohistochemical analysis revealed 36-79% colocalization of thioredoxin and mCRP in human advanced coronary atherosclerotic lesions. Nonreduced mCRP was largely inert in activating human coronary artery endothelial cells (HCAECs), whereas reduced or cysteine-mutated mCRP evoked marked release of IL-8 and monocyte chemoattractant protein-1 from HCAECs, with ~50% increase at a concentration of 1 µg/ml. Reduced mCRP was ~4 to 40-fold more potent than mCRP in up-regulating adhesion molecule expression, promoting U937 monocyte adhesion to HCAECs, and inducing cytokine release from rabbit arteries ex vivo and in mice. These actions were primarily due to unlocking the lipid raft interaction motif. Therefore, expression of proinflammatory properties of CRP on endothelial cells requires sequential conformational changes, i.e., loss of pentameric symmetry followed by reduction of the intrasubunit disulfide bond.

Purification of Recombinant Mouse C-Reactive Protein from Pichia Pastoris GS115 by Nickel Chelating Sepharose Fast-Flow Affinity Chromatography and P-Aminophenyl Phosphoryl Choline Agarose Resin Affinity Chromatography in Tandem.

C-reactive protein (CRP) is a circulating marker of inflammation yet with ill-defined biological functions. This is partly due to the uncharacterized activities of endogenous CRP in mice, the major animal model used to define protein function. The hurdles for purification and characterization of mouse CRP are its low circulating levels and the lack of specific antibodies. To clear these hurdles, here we developed an efficient expression system by constructing recombinant Pichia pastoris cells for secretion of native conformation mouse CRP. The recombinant expression of mouse CRP in Escherichia coli failed to yield sufficient amount of native protein, reflecting the importance of post-translational modification of glycosylation in aiding proper folding. By contrast, sufficient amount of native mouse CRP was successfully purified from P. pastoris. Preliminary purification was performed by Nickel Chelating Sepharose Fast-Flow affinity chromatography with 6 × His tags attached to the protein. Subsequently, p-Aminophenyl Phosphoryl Choline Agarose resin affinity chromatography was used for tandem purification. The purified mouse CRP showed native pentamer and capabilities of PC binding. Moreover, the 6 × His tag provides a convenient tool for detecting the interactions of mouse CRP with ligands.

C-Reactive Protein Protects Against Acetaminophen-Induced Liver Injury by Preventing Complement Overactivation.

BACKGROUND AND AIMS: C-reactive protein (CRP) is a hepatocyte-produced marker of inflammation yet with undefined function in liver injury. We aimed to examine the role of CRP in acetaminophen-induced liver injury (AILI). METHODS: The effects of CRP in AILI were investigated using CRP knockout mice and rats combined with human CRP rescue. The mechanisms of CRP action were investigated in vitro and in mice with Fcγ receptor 2B knockout, C3 knockout, or hepatic expression of CRP mutants defective in complement interaction. The therapeutic potential of CRP was investigated by intraperitoneal administration at 2 or 6 hours post-AILI induction in wild-type mice. RESULTS: CRP knockout exacerbated AILI in mice and rats, which could be rescued by genetic knock-in, adeno-associated virus-mediated hepatic expression or direct administration of human CRP. Mechanistically, CRP does not act via its cellular receptor Fcγ receptor 2B to inhibit the early phase injury to hepatocytes induced by acetaminophen; instead, CRP acts via factor H to inhibit complement overactivation on already injured hepatocytes, thereby suppressing the late phase amplification of inflammation likely mediated by C3a-dependent actions of neutrophils. Importantly, CRP treatment effectively alleviated AILI with a significantly extended therapeutic time window than that of N-acetyl cysteine. CONCLUSION: Our results thus identify CRP as a crucial checkpoint that limits destructive activation of complement in acute liver injury, and we argue that long-term suppression of CRP expression or function might increase the susceptibility to AILI.

Secretory quality control constrains functional selection-associated protein structure innovation.

Biophysical models suggest a dominant role of structural over functional constraints in shaping protein evolution. Selection on structural constraints is linked closely to expression levels of proteins, which together with structure-associated activities determine in vivo functions of proteins. Here we show that despite the up to two orders of magnitude differences in levels of C-reactive protein (CRP) in distinct species, the in vivo functions of CRP are paradoxically conserved. Such a pronounced level-function mismatch cannot be explained by activities associated with the conserved native structure, but is coupled to hidden activities associated with the unfolded, activated conformation. This is not the result of selection on structural constraints like foldability and stability, but is achieved by folding determinants-mediated functional selection that keeps a confined carrier structure to pass the stringent eukaryotic quality control on secretion. Further analysis suggests a folding threshold model which may partly explain the mismatch between the vast sequence space and the limited structure space of proteins.

Membrane localization of beta-amyloid 1-42 in lysosomes: a possible mechanism for lysosome labilization.

beta-Amyloid peptide (Abeta42) is the core protein of amyloid plaque in Alzheimer disease. The intracellular accumulation of Abeta42 in the endosomal/lysosomal system has been under investigation for many years, but the direct link between Abeta42 accumulation and dysfunction of the endosomal/lysosomal system is still largely unknown. Here, we found that both in vitro and in vivo, a major portion of Abeta42 was tightly inserted into and a small portion peripherally associated with the lysosomal membrane, whereas its soluble portion was minimal. We also found that the Abeta42 molecules inserted into the membrane tended to form multiple oligomeric aggregates, whereas Abeta40 peptides formed only dimers. Neutralizing lysosomal pH in differentiated PC12 cells decreased the lysosomal membrane insertion of Abeta42 and moderated Abeta42-induced lysosomal labilization and cytotoxicity. Our findings, thus, suggest that the membrane-inserted portion of Abeta42 accumulated in lysosomes may destabilize the lysosomal membrane and induce neurotoxicity.

Monomeric C-reactive protein activates endothelial cells via interaction with lipid raft microdomains.

Emerging evidence indicates that in addition to native pentameric C-reactive protein (CRP), monomeric CRP (mCRP) also plays an active role in inflammation associated with cardiovascular diseases. mCRP activates endothelial cells, one of the critical events in cardiovascular diseases; however, the underlying molecular mechanisms are incompletely understood. Here we report that association of mCRP with human aortic and coronary artery endothelial cells is predominantly due to membrane insertion rather than binding to the surface proteins Fc gammaRs and proteoglycans. We identify lipid rafts as the preferential membrane microdomains for mCRP anchorage. mCRP binding depends on membrane cholesterol content and is synergistically mediated by the putative cholesterol binding consensus sequence of CRP (aa 35-47) and the C-terminal octapeptide (aa 199-206). Conversely, disrupting lipid rafts with methyl-beta cyclodextrin or nystatin abrogated mCRP-induced cytokine release, reactive oxygen species generation, and adhesion molecule expression in endothelial cells. Furthermore, ex vivo treatment of rabbit thoracic aorta and carotid artery segments with nystatin prevented mCRP-induced IL-8 release. Our data identify mCRP-lipid raft interaction as an important mechanism in mediating cellular responses to mCRP and lend further support to the notion of mCRP regulation of endothelial cell function during inflammation.

Reversible promoter methylation determines fluctuating expression of acute phase proteins.

Acute phase reactants (APRs) are secretory proteins exhibiting large expression changes in response to proinflammatory cytokines. Here we show that the expression pattern of a major human APR, that is C-reactive protein (CRP), is casually determined by DNMT3A and TET2-tuned promoter methylation status. CRP features a CpG-poor promoter with its CpG motifs located in binding sites of STAT3, C/EBP-ß and NF-κB. These motifs are highly methylated at the resting state, but undergo STAT3- and NF-κB-dependent demethylation upon cytokine stimulation, leading to markedly enhanced recruitment of C/EBP-ß that boosts CRP expression. Withdrawal of cytokines, by contrast, results in a rapid recovery of promoter methylation and termination of CRP induction. Further analysis suggests that reversible methylation also regulates the expression of highly inducible genes carrying CpG-poor promoters with APRs as representatives. Therefore, these CpG-poor promoters may evolve CpG-containing TF binding sites to harness dynamic methylation for prompt and reversible responses.

Corrigendum: Monomeric C-Reactive Protein Binds and Neutralizes Receptor Activator of NF-κB Ligand-Induced Osteoclast Differentiation.

[This corrects the article DOI: 10.3389/fimmu.2018.00234.].

A Functional Genetic Variant at the C-Reactive Protein Promoter (rs3091244) Is Not Associated With Cancer Risk in a Chinese Population.

Background: The association of genetically elevated levels of circulating C-reactive protein (CRP) with cancer risk has been extensively investigated in European populations; however, there are conflicting conclusions. The tri-allelic rs3091244 is a functionally validated genetic variant, and its allelic frequencies differ significantly between European and Asian populations. Here, we examined the association of rs3091244 with cancer risk in a Chinese population. Methods: rs3091244 was genotyped by Sanger sequencing in 4,971 cancer cases and 2,485 controls. The rs1205 and rs2794521 gene variants were also genotyped using TaqMan assays in subgroups. Results: No association was detected between the genotyped CRP variants and cancer risk, with or without distinguishing cancer types, suggesting that circulating CRP is not causally involved in tumorigenesis in Chinese populations.