Beyond the Biomarker: Monomeric CRP as a Driver of Multisystem Pathology in Rheumatoid Arthritis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear editors:   

 It is a great honor and pleasure for me to be invited as the reviewer for this important work entitled " Beyond the Biomarker: Monomeric CRP as a Driver of Multi-system Pathology in Rheumatoid Arthritis ". Andreea Lazarut-Nistor and Mark Slevin comprehensively reviewed the signaling-mechanistic insights into the monomeric form of CRP (mCRP), particularly its intricate relationship between systemic and neuroinflammation, to identify new interventional strategies for RA patients at elevated risk of neurodegenerative and vascular complications. This study topic is interesting, attributing to their team’s long-term efforts and contributions in this scientific field. I have a number of comments concerning this study:

The main findings and strengths of the study:

1.The concept of mCRP as an active effector molecule, rather than a passive biomarker, is both novel and clinically relevant, particularly in the context of RA-AD comorbidity.

2.The authors successfully integrated synovial inflammation, neuroinflammation, BBB disruption, and complement activation, offering an interdisciplinary biology perspective that extends beyond organ-specific pathology.

3. The emphasis on CRP isoform conversion, CD32/CD64 signaling, and complement activation provides mechanistic depth, rather than simply cataloging associations.

Call for Future Research:

The manuscript highlights critical gaps and calls for mechanistic and translational studies, which is appropriate for a narrative review aiming to shape future directions. Major concerns:

1. What types of experimental designs (e.g., randomized controlled trials, crossover studies, acute feeding trials) were predominantly included in the review, and how were these distinguished in the analysis?

 

2. How do the authors account for variability in dosing regimens, timing of protein intake, and duration of intervention across the studies discussed?

 

3. Were studies with both resistance training and protein supplementation included? If so, how do the authors isolate the effect of whey protein alone on muscle protein synthesis?

 

4. Is there consistency in the biomarkers or methodologies used to assess muscle protein synthesis (e.g., stable isotope tracers, fractional synthesis rate) across the cited studies?

 

5. How do the authors address the heterogeneity of participant characteristics (e.g., age, sex, training status, baseline muscle mass) in the studies reviewed?

 

6. Were the included studies powered sufficiently to detect changes in muscle mass or protein synthesis, and was statistical significance consistently reported?

 

7. What criteria were used to evaluate the quality or methodological rigor of the studies included in the review?

 

8. How do acute protein feeding studies compare to long-term supplementation trials in terms of reported effects on muscle protein synthesis in the review?

 

9. Were any studies included that directly compared whey protein to other protein sources using controlled experimental conditions? If so, how was the effect size quantified?

 

10. Do the authors distinguish between studies using fasted vs. fed-state measurements of muscle protein synthesis, and how does this impact the interpretation of the results?

 

11. Lack of Methodological Transparency in Literature Selection might be a concern. As a narrative review, the authors should clarify the criteria for literature inclusion to minimize selection bias. Even a brief statement about the scope and sources (e.g., databases, key terms) would improve reproducibility and credibility.

 

12. Overextension of Pathophysiological Links is another issue.
    While the shared inflammatory pathways between RA and AD are intriguing, the manuscript at times overstates causality without sufficient evidentiary support (e.g., presence of mCRP in Aβ plaques does not confirm a pathogenic role). This should be more clearly framed as hypothesis-generating rather than conclusive.

 

13. Discussion of contradictory evidence is insufficient. There is minimal consideration of studies that fail to support a pathogenic role of mCRP, or those that question its significance compared to pentameric CRP (pCRP). Addressing such perspectives would balance the narrative.

 

14. Terminological Clarity is needed. The manuscript uses terms such as “driver,” “mediator,” and “amplifier” somewhat interchangeably in describing mCRP. It would be helpful to define these roles more precisely within different disease contexts.

 

15. All clinical therapeutic implications need caution. While mCRP is proposed as a therapeutic target, the lack of clinical data on mCRP-targeted interventions should be acknowledged more directly, to temper the translational enthusiasm.

 

        Minor Concerns

1. Include a comparative discussion of CRP isoforms (pCRP vs. mCRP) in terms of function, localization, and detection challenges.
2. Explicitly discuss existing limitations in detecting mCRP in vivo, including specificity of antibodies and the stability of isoforms.
3. Reference key longitudinal studies or biobank data (if any) that assess CRP isoforms and long-term neurocognitive outcomes in RA patients.

 

Thank you for allowing me to review this interesting article. After appropriate revision, this important review article should be published as soon as possible.

Author Response

Dear Respected Reviewer 1,

We sincerely appreciate your careful and thoughtful review of our manuscript. Your constructive feedback and the time you devoted to the evaluation have been invaluable in enhancing the quality and clarity of our work.

Please find the revised manuscript, with all the changes as per your suggestions.

Reviewer’s overview:

It is a great honor and pleasure for me to be invited as the reviewer for this important work entitled " Beyond the Biomarker: Monomeric CRP as a Driver of Multi-system Pathology in Rheumatoid Arthritis ". Andreea Lazarut-Nistor and Mark Slevin comprehensively reviewed the signaling-mechanistic insights into the monomeric form of CRP (mCRP), particularly its intricate relationship between systemic and neuroinflammation, to identify new interventional strategies for RA patients at elevated risk of neurodegenerative and vascular complications. This study topic is interesting, attributing to their team’s long-term efforts and contributions in this scientific field. I have a number of comments concerning this study:

The main findings and strengths of the study:

1.The concept of mCRP as an active effector molecule, rather than a passive biomarker, is both novel and clinically relevant, particularly in the context of RA-AD comorbidity.

2.The authors successfully integrated synovial inflammation, neuroinflammation, BBB disruption, and complement activation, offering an interdisciplinary biology perspective that extends beyond organ-specific pathology.

3.The emphasis on CRP isoform conversion, CD32/CD64 signaling, and complement activation provides mechanistic depth, rather than simply cataloging associations.

Call for Future Research:

The manuscript highlights critical gaps and calls for mechanistic and translational studies, which is appropriate for a narrative review aiming to shape future directions. Major concerns:

1. What types of experimental designs (e.g., randomized controlled trials, crossover studies, acute feeding trials) were predominantly included in the review, and how were these distinguished in the analysis?
2. How do the authors account for variability in dosing regimens, timing of protein intake, and duration of intervention across the studies discussed?
3. Were studies with both resistance training and protein supplementation included? If so, how do the authors isolate the effect of whey protein alone on muscle protein synthesis?
4. Is there consistency in the biomarkers or methodologies used to assess muscle protein synthesis (e.g., stable isotope tracers, fractional synthesis rate) across the cited studies?
5. How do the authors address the heterogeneity of participant characteristics (e.g., age, sex, training status, baseline muscle mass) in the studies reviewed?
6. Were the included studies powered sufficiently to detect changes in muscle mass or protein synthesis, and was statistical significance consistently reported?
7. What criteria were used to evaluate the quality or methodological rigor of the studies included in the review?
8. How do acute protein feeding studies compare to long-term supplementation trials in terms of reported effects on muscle protein synthesis in the review?
9. Were any studies included that directly compared whey protein to other protein sources using controlled experimental conditions? If so, how was the effect size quantified?
10. Do the authors distinguish between studies using fasted vs. fed-state measurements of muscle protein synthesis, and how does this impact the interpretation of the results?
11. Lack of Methodological Transparency in Literature Selection might be a concern. As a narrative review, the authors should clarify the criteria for literature inclusion to minimize selection bias. Even a brief statement about the scope and sources (e.g., databases, key terms) would improve reproducibility and credibility.

 The points 1-11 above we believe have been mistakenly added to the reviewer’s report as we dont see they are connected to our paper please confirm Thank you.

Additions made to the points below are seen in RED highlight in the new manuscript.

12. Overextension of Pathophysiological Links is another issue.
While the shared inflammatory pathways between RA and AD are intriguing, the manuscript at times overstates causality without sufficient evidentiary support (e.g., presence of mCRP in Aβ plaques does not confirm a pathogenic role). This should be more clearly framed as hypothesis-generating rather than conclusive.

We thank the reviewer for this insightful suggestion. We added the following paragraph as a clarification on the issue: “While the shared inflammatory mechanisms between RA and AD are scientifically compelling, the current body of evidence does not support a direct causal relationship between the two conditions. For instance, the presence of mCRP within Aβ plaques and its co-localization with complement proteins such as C1q in AD brain tissue suggest a potential role in local immune activation and neuroinflammatory processes [1]. However, these findings are primarily associative and do not establish mCRP as a pathogenic driver of AD. Similarly, elevated systemic inflammation in RA may contribute to blood–brain barrier dysfunction and heightened neuroinflammatory states, but it remains unclear whether this directly initiates or accelerates AD pathology. These observations should therefore be framed as hypothesis-generating rather than conclusive. Additional studies are needed to determine whether these shared inflammatory features represent overlapping but independent pathways or reflect a true pathogenic bridge between RA and AD.”

13. Discussion of contradictory evidence is insufficient. There is minimal consideration of studies that fail to support a pathogenic role of mCRP, or those that question its significance compared to pentameric CRP (pCRP). Addressing such perspectives would balance the narrative.

We thank the reviewer for this pertinent information. We will add the following paragraph for clarity: “Several studies have questioned the pathogenic role of mCRP and its significance compared to pCRP. Experimental work has shown that many pro-inflammatory effects attributed to mCRP may be artefactual, resulting from contamination with endotoxin or sodium azide in CRP preparations [2]. For instance, a study comparing native and denatured CRP found no pro-inflammatory activity from mCRP in endothelial cells, contradicting earlier claims and attributing previous findings to impurities [3]. Similarly, reviews have emphasized that the conditions used to generate mCRP in vitro often do not reflect physiological processes, and many findings may lack in vivo relevance [4]. Animal studies using CRP transgenic models have yielded conflicting results, with several showing no proatherogenic effect, and in some cases, mCRP even exerting anti-inflammatory activity via upregulation of IL-10. Furthermore, large-scale Mendelian randomization studies have demonstrated that genetically elevated CRP levels do not associate with increased cardiovascular disease risk, undermining the case for CRP (in either form) as a causal factor [5]. Collectively, this body of work suggests that mCRP may not be as pathogenic as previously thought and that its biological significance relative to pCRP remains unresolved.”

14. Terminological Clarity is needed. The manuscript uses terms such as “driver,” “mediator,” and “amplifier” somewhat interchangeably in describing mCRP. It would be helpful to define these roles more precisely within different disease contexts.

 We thank the reviewer for this helpful suggestion. We will address those terms and clarify exactly what is implied, highlighted in red.

15. All clinical therapeutic implications need caution. While mCRP is proposed as a therapeutic target, the lack of clinical data on mCRP-targeted interventions should be acknowledged more directly, to temper the translational enthusiasm.

 We thank the reviewer for pointing this out. We will add a few sentences where needed in order to be more realistic about the real-life therapeutic implications of mCRP.

 “While targeting mCRP holds promise as a therapeutic strategy due to its pro-inflammatory and tissue-damaging roles in various diseases, translating these findings into clinical treatments remains challenging. The development of specific inhibitors that can selectively target mCRP without disrupting the beneficial functions of native pentameric CRP is still in early stages. Furthermore, variability in mCRP expression across different tissues and disease states complicates its use as a universal therapeutic target. More research is needed to determine optimal delivery methods, specificity, and long-term safety of potential mCRP-targeted therapies.”

        Minor Concerns

1. Include a comparative discussion of CRP isoforms (pCRP vs. mCRP) in terms of function, localization, and detection challenges.

CRP exists in two main isoforms: pentameric CRP and monomeric CRP , which differ markedly in their structure, localization, biological activity, and detection. pCRP is the native, circulating form composed of five identical subunits, primarily synthesized by the liver in response to IL-6 [6], [7]. It serves as a widely used systemic marker of inflammation, activating the classical complement pathway and binding to phosphocholine on microbial surfaces and apoptotic cells. In contrast, mCRP is generated at sites of inflammation through the dissociation of pCRP, often triggered by oxidative stress, activated cell membranes, or bioactive lipids [8]. This monomeric form is structurally and functionally distinct, exerting potent pro-inflammatory effects by promoting endothelial dysfunction, inducing cytokine and adhesion molecule expression, and enhancing leukocyte and platelet activation. While pCRP is readily measured using standard immunoassays, the detection of mCRP poses significant challenges due to its conformational differences and the lack of standardized assays, often leading to its underrepresentation in clinical studies. Importantly, mCRP is increasingly recognized not just as a byproduct but as an active mediator of local inflammation in various pathological conditions, including atherosclerosis, neurodegenerative diseases, and autoimmune disorders, underscoring the need to distinguish between CRP isoforms in both research and clinical settings [9], [10].

2. Explicitly discuss existing limitations in detecting mCRP in vivo, including specificity of antibodies and the stability of isoforms.

Detecting mCRP in vivo remains challenging due to several key limitations, particularly regarding antibody specificity and isoform stability. Major obstacles include the lack of antibodies that can reliably distinguish mCRP from pCRP and the absence of standardized, clinically validated assays for mCRP detection. Many commercially available antibodies exhibit cross-reactivity, recognizing shared epitopes between the isoforms, especially under conditions where pCRP partially dissociates or denatures [8], [11]. This lack of specificity can lead to inaccurate localization or quantification of mCRP in tissues. Additionally, mCRP is structurally unstable under physiological conditions; unlike the stable, circulating pCRP, mCRP is typically generated at sites of inflammation and rapidly associates with membranes or extracellular components, making it poorly soluble and difficult to detect in plasma or serum [12]. Moreover, since mCRP is primarily localized at inflammatory sites rather than circulating freely in blood, conventional biofluid-based assays may not reflect its true pathological burden, necessitating invasive tissue sampling or advanced imaging for accurate assessment [13].

3. Reference key longitudinal studies or biobank data (if any) that assess CRP isoforms and long-term neurocognitive outcomes in RA patients.

Several large-scale longitudinal studies and biobanks have investigated the relationship between systemic inflammation and neurocognitive outcomes in RA, though few have specifically assessed CRP isoforms (mCRP and pCRP). The UK Biobank, which includes over 500,000 participants with extensive cognitive, imaging, and inflammatory marker data, has shown that hsCRP is associated with increased risk of cognitive decline and dementia in RA patients, though it does not differentiate CRP isoforms [14], [15]. Similarly, the Brigham and Women’s Rheumatoid Arthritis Sequential Study (BRASS), an RA-specific cohort, reported associations between higher systemic inflammation (including total CRP) and subjective cognitive complaints. The Mayo Clinic Biobank and Rochester Epidemiology Project also offer longitudinal RA data with cognitive outcomes, though only total CRP has been measured to date. While the Alzheimer’s Disease Neuroimaging Initiative (ADNI) provides robust imaging and cognitive data, RA patients are underrepresented, and CRP isoform data are lacking [16]. Experimental studies, however, have increasingly implicated mCRP in neuroinflammatory processes, showing its colocalization with amyloid plaques and its capacity to disrupt the blood-brain barrier and activate microglia [17], [18]. These findings suggest that mCRP could serve as a mechanistic link between chronic RA-related inflammation and neurodegeneration. Although no longitudinal cohort has yet measured CRP isoforms in this context, stored biospecimens in biobanks such as UK Biobank or BRASS could potentially be reanalyzed using isoform-specific assays to clarify these relationships.

            References

[1]        F. Strang et al., “Amyloid Plaques Dissociate Pentameric to Monomeric C‐Reactive Protein: A Novel Pathomechanism Driving Cortical Inflammation in Alzheimer’s Disease?,” Brain Pathol., vol. 22, no. 3, pp. 337–346, Nov. 2011, doi: 10.1111/j.1750-3639.2011.00539.x.

[2]        M. B. Pepys et al., “Pro-inflammatory Effects of Bacterial Recombinant Human C-Reactive Protein are Caused by Contamination with Bacterial Products not by C-Reactive Protein Itself,” Circ. Res., vol. 97, no. 11, pp. e97-103, Nov. 2005, doi: 10.1161/01.RES.0000193595.03608.08.

[3]        K. E. Taylor and C. W. van den Berg, “Structural and functional comparison of native pentameric, denatured monomeric and biotinylated C-reactive protein,” Immunology, vol. 120, no. 3, pp. 404–411, Mar. 2007, doi: 10.1111/j.1365-2567.2006.02516.x.

[4]        J. R. Thiele et al., “A Conformational Change in C-Reactive Protein Enhances Leukocyte Recruitment and Reactive Oxygen Species Generation in Ischemia/Reperfusion Injury,” Front. Immunol., vol. 9, p. 675, Apr. 2018, doi: 10.3389/fimmu.2018.00675.

[5]        “C-reactive protein concentration and risk of coronary heart disease, stroke, and mortality: an individual participant meta-analysis,” Lancet, vol. 375, no. 9709, pp. 132–140, Jan. 2010, doi: 10.1016/S0140-6736(09)61717-7.

[6]        S. Black, I. Kushner, and D. Samols, “C-reactive Protein *,” J. Biol. Chem., vol. 279, no. 47, pp. 48487–48490, Nov. 2004, doi: 10.1074/jbc.R400025200.

[7]        N. R. Sproston and J. J. Ashworth, “Role of C-Reactive Protein at Sites of Inflammation and Infection,” Front. Immunol., vol. 9, p. 754, Apr. 2018, doi: 10.3389/fimmu.2018.00754.

[8]        J. R. Thiele et al., “Dissociation of Pentameric to Monomeric C-Reactive Protein Localizes and Aggravates Inflammation,” Circulation, vol. 130, no. 1, pp. 35–50, Jul. 2014, doi: 10.1161/CIRCULATIONAHA.113.007124.

[9]        M. B. Pepys and G. M. Hirschfield, “C-reactive protein: a critical update,” J. Clin. Invest., vol. 111, no. 12, pp. 1805–1812, Jun. 2003, doi: 10.1172/JCI18921.

[10]      J. R. Thiele, J. Zeller, H. Bannasch, G. B. Stark, K. Peter, and S. U. Eisenhardt, “Targeting C-Reactive Protein in Inflammatory Disease by Preventing Conformational Changes,” Mediators Inflamm., vol. 2015, p. 372432, 2015, doi: 10.1155/2015/372432.

[11]      R. Lill, “From the discovery to molecular understanding of cellular iron-sulfur protein biogenesis,” Biol. Chem., vol. 401, no. 6–7, pp. 855–876, May 2020, doi: 10.1515/hsz-2020-0117.

[12]      S. U. Eisenhardt et al., “Dissociation of Pentameric to Monomeric C-Reactive Protein on Activated Platelets Localizes Inflammation to Atherosclerotic Plaques,” Circ. Res., vol. 105, no. 2, pp. 128–137, Jul. 2009, doi: 10.1161/CIRCRESAHA.108.190611.

[13]      D. Thompson, M. B. Pepys, and S. P. Wood, “The physiological structure of human C-reactive protein and its complex with phosphocholine,” Structure, vol. 7, no. 2, pp. 169–177, Feb. 1999, doi: 10.1016/S0969-2126(99)80023-9.

[14]      I. Lourida et al., “Association of Lifestyle and Genetic Risk With Incidence of Dementia,” JAMA, vol. 322, no. 5, pp. 430–437, Aug. 2019, doi: 10.1001/jama.2019.9879.

[15]      K. A. Walker et al., “Association of Midlife to Late-Life Blood Pressure Patterns With Incident Dementia,” JAMA, vol. 322, no. 6, pp. 535–545, Aug. 2019, doi: 10.1001/jama.2019.10575.

[16]      C. Holmes et al., “Systemic inflammation and disease progression in Alzheimer disease,” Neurology, vol. 73, no. 10, pp. 768–774, Sep. 2009, doi: 10.1212/WNL.0b013e3181b6bb95.

[17]      M. Slevin et al., “Modified C‐Reactive Protein Is Expressed by Stroke Neovessels and Is a Potent Activator of Angiogenesis In Vitro,” Brain Pathol., vol. 20, no. 1, pp. 151–165, Jan. 2009, doi: 10.1111/j.1750-3639.2008.00256.x.

[18]      M. Slevin et al., “Monomeric C-reactive protein-a key molecule driving development of Alzheimer’s disease associated with brain ischaemia?,” Sci. Rep., vol. 5, p. 13281, Sep. 2015, doi: 10.1038/srep13281.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript reviews the ole of C-reactive protein (CRP), particularly its monomeric form (mCRP), as a central mediator linking systemic autoimmune inflammation with neuroinflammatory and vascular pathology.

A few concerns for the authors.

1. A lot more references will be needed throughout the manuscript. As a review paper, the authors will need to provide many more references to support their claims. Most of the time, only 1 reference is provided for each paragraph, which is not acceptable.
2. The authors really haven’t stated clearly about the pro-inflammatory mechanisms of mCPR in RA, which should include activation of immune cells, complement system activation, endothelial dysfunction and leukocyte recruitment and autoantigen modification and autoimmunity.
3. The authors spend a huge amount of time discussing the role of mCRP in CVD like CHF and Atherosclerosis, as well as neurodegenerative disorders like AD and dementia. And the title seems to focus on RA. All the mentioned conditions have shared inflammatory pathways which involve mCRP for sure. But will too much discussion on the other conditions deviate the theme from RA? Would suggest the authors provide more detailed, referenced information on the mechanisms of mCRP on RA.

Author Response

Dear Respected Reviewer 2,

We sincerely appreciate your careful and thoughtful review of our manuscript. Your constructive feedback and the time you devoted to the evaluation have been invaluable in enhancing the quality and clarity of our work.

Please find the revised manuscript, with all the changes as per your suggestions.

This manuscript reviews the role of C-reactive protein (CRP), particularly its monomeric form (mCRP), as a central mediator linking systemic autoimmune inflammation with neuroinflammatory and vascular pathology.

1. A lot more references will be needed throughout the manuscript. As a review paper, the authors will need to provide many more references to support their claims. Most of the time, only 1 reference is provided for each paragraph, which is not acceptable.

 

We thank the reviewer for this insightful suggestion. We added a significant number of references to support our review and adjusted the paragraphs to include more than 1 reference. The manuscript now contains almost 100 references. Additions have been marked with yellow highlight (and also red in response to reviewer-2 additional evidence).

 

2. The authors really haven’t stated clearly about the pro-inflammatory mechanisms of mCRP in RA, which should include activation of immune cells, complement system activation, endothelial dysfunction and leukocyte recruitment and autoantigen modification and autoimmunity.
3. And point 3. The authors spend a huge amount of time discussing the role of mCRP in CVD like CHF and Atherosclerosis, as well as neurodegenerative disorders like AD and dementia. And the title seems to focus on RA. All the mentioned conditions have shared inflammatory pathways which involve mCRP for sure. But will too much discussion on the other conditions deviate the theme from RA? Would suggest the authors provide more detailed, referenced information on the mechanisms of mCRP on RA.

 

 

We thank the reviewer for this pertinent information. We added further insights into the pro-inflammatory mechanisms of mCRP  through complement activation and immune cells- also focusing specifically in RA and have inserted the following paragraphs into the relevant positions in the manuscript:  

The monomeric form of CRP is primarily responsible for pro-inflammatory activity, while the pentameric form of CRP generally exhibits anti-inflammatory properties. The transition from pCRP to mCRP is triggered by binding to damaged membranes or inflammatory microenvironments. This process produces an intermediate (pCRP*) that activates the classical complement cascade and ultimately converts to mCRP, which enhances immune responses such as platelet aggregation, neutrophil migration, cytokine release, and NK cell activation. Conversely, pCRP can inhibit platelet and neutrophil activity, reflecting its regulatory role. CRP isoforms interact with different Fc receptors, contributing to their opposing effects. The transition from pCRP to mCRP is fast and localized, but slows to prevent systemic inflammation, with detectable pCRP levels rising in serum 6–12 hours after tissue injury [1].

CRP's conformational change from pCRP to mCRP enhances leukocyte recruitment and reactive oxygen species generation in ischemia/reperfusion injury. This transition occurs when pCRP dissociates into monomers, typically in response to tissue damage or other inflammatory stimuli. The structural shift is significant as mCRP exhibits pro-inflammatory properties, such as platelet activation, leukocyte recruitment, and endothelial dysfunction, which contribute to the pathogenesis of various diseases, including RA and cardiovascular conditions [2].

The complement (C) system is a critical component of the innate immune response, consisting of approximately 60 proteins found in serum and on cell membranes. It functions alongside antibody-mediated reactions to defend against microbial infections and helps clear damaged tissues and cellular debris. The system operates through three activation pathways: the classical (antibody-triggered), lectin (recognizing microbial carbohydrate patterns), and alternative (continuously active, antibody-independent) pathways, all converging on the activation of C3 and deposition of C3b. Tight regulation of complement activity is essential, as dysregulation can contribute to various diseases including infections, cancer, renal disorders, and autoimmune conditions. In RA, evidence suggests that inappropriate activation of the complement system plays a role in disease pathogenesis. Given the complement system’s involvement in inflammation and tissue damage, it represents a promising area for biomarker discovery and therapy development. A study by Rodríguez-González et al. comprehensively evaluated all three complement (C) pathways: classical, alternative, and lectin in 430 patients with RA, using both functional assays and measurements of individual complement proteins. The findings show that RA disease activity is primarily associated with upregulation of the terminal pathway, particularly through the classical cascade. Higher disease activity of RA correlates with increased functional test values, indicating reduced consumption and increased hepatic production of complement components. CRP demonstrated strong correlations with the classical and terminal pathways but weaker associations with the lectin pathway, supporting its role in activating the classical cascade via C1q. Lectin pathway deficiency, a common condition affecting 5-30% of the general population, has been linked to more severe or erosive RA. The presence of RA and ACPA was associated with lower levels of classical pathway components, possibly reflecting local complement consumption in inflamed joints. Although complete inhibition of the C system has not shown efficacy in RA, the findings in this study suggest that targeted or partial complement modulation may offer therapeutic benefit [3]. Complement activation appears to be localized during the preclinical phase and becomes systemic with the onset of clinical RA, suggesting it contributes to disease progression rather than initiation [4].

A study which looked at 107 patients with active RA and 177 patients with inactive RA displayed strong evidence that CRP contributes directly to complement activation in RA, as demonstrated by elevated levels of CRP-complement complexes (C3d-CRP and C4d-CRP), particularly in patients with active disease. The significant correlation between these complexes and disease activity scores (DAS28) supports the idea that CRP-mediated complement activation is not only present but also linked to disease severity. This suggests that CRP is more than just a marker of inflammation in RA and that it may actively participate in the disease’s pathogenesis through its role in complement activation [5]. Another study identified CRP as a potential contributor to complement activation on the surface of microparticles, particularly in plasma. Microparticles isolated from the plasma of both RA patients and healthy individuals showed a strong correlation between CRP binding and C1q binding, implicating CRP in the initiation of the classical complement pathway. CRP is known to bind to phosphorylcholine and oxidized phosphatidylcholine on cell membranes, especially in the presence of lysophosphatidylcholine, which may be present on microparticles due to oxidative stress and increased enzymatic activity. Interestingly, although fluid-phase CRP levels were elevated in both plasma and synovial fluid of RA patients, complement activation was associated specifically with microparticle-bound CRP, suggesting that its role in complement activation is localized to surfaces rather than driven by soluble CRP levels. These findings underscore CRP's potential function as a surface-bound activator of complement on microparticles and highlight its possible contribution to systemic inflammation in RA [6].

CRP plays a key role in innate immunity by binding to phosphocholine (PC) on apoptotic cells and pathogens and interacting with complement factor C1q and Fcγ receptors. In its native pentameric form, CRP is not inherently pro-inflammatory. However, under certain conditions (e.g., heat, acidic environments, or phospholipase A2 activity), pCRP can dissociate into mCRP or an intermediate form (pCRP*), both of which expose neoepitopes and exhibit strong pro-inflammatory properties. Dissociation alters the structure and solubility of CRP, making mCRP largely tissue-bound. Microparticles from activated cells can transport dissociated CRP forms to distant sites, including injured or inflamed tissues. While pCRP is mostly inactive, both mCRP and pCRP* are potent activators of inflammation and thrombosis. Plasma CRP levels, which can rise dramatically during inflammation, serve as a widely used clinical marker of systemic inflammation [7] and are used to monitor disease activity in RA patients.

Looking at the bigger picture, other autoimmune conditions related to RA, such as systemic lupus erythematosus (SLE), were investigated. To emphasize mCRP’s role in autoimmunity, a study by Karlsson et al. investigated EV-bound CRP isoforms and anti-CRP autoantibodies in SLE, revealing that EVs carrying mCRP are elevated in patients with active disease. Unlike pCRP, which showed no correlation with disease activity whether in serum or on EVs, mCRP+ EVs were associated with heightened disease activity, particularly in cases of lupus nephritis (LN), and contributed to inflammation and the generation of anti-CRP autoantibodies. The study by Karlsson et al. found an inverse relationship between mCRP+ EV abundance and disease duration, especially in patients with active disease and LN, suggesting their relevance in early disease stages. Interestingly, patients with organ damage had lower mCRP+ EV levels, possibly reflecting reduced disease activity over time or redistribution of CRP to other sites such as the endothelium. The study underscored a potential pathogenic role of mCRP+ EVs in SLE through their involvement in inflammation and autoantibody generation [8].

A recent study by Thomé et al. explored the interaction between CRP isoforms, particularly mCRP, and T cells, revealing a complex, context-dependent relationship. While both mCRP and pCRP bound to T cells, only mCRP induced increased proliferation and reduced apoptosis, particularly in activated T cells. However, mCRP did not directly activate T cells, as evidenced by unchanged expression of activation markers CD69 and CD25, and even reduced activation under CD3/CD28 co-stimulation. Notably, the proinflammatory effects of mCRP on T cells only emerged in the presence of monocytes, indicating a monocyte-dependent activation mechanism, likely mediated by mCRP-induced upregulation of CD80 on monocytes, leading to T cell activation via the CD80/CD28 co-stimulatory pathway. This mechanism was confirmed by the use of Belatacept, which blocked the pathway and suppressed T cell activation. These findings suggest that mCRP indirectly promotes T cell activation through monocyte interaction, with significant implications for immune responses in settings like allograft rejection and autoimmune diseases [9].

Apoptotic cells, when properly cleared by macrophages, generally induce anti-inflammatory cytokines like TGF-β, while necrotic cells trigger proinflammatory responses. A study by Gershov et al. demonstrated that CRP binds specifically to the membranes of apoptotic and not necrotic cells in a calcium-dependent manner, likely targeting altered membrane lipids such as lysophospholipids. This binding enhances classical complement pathway activation (notably C1q and C3b deposition) without leading to formation of the membrane attack complex (MAC), thereby promoting phagocytosis without cell lysis. CRP achieves this by recruiting factor H, which inhibits late complement components. Elevated CRP levels boost macrophage uptake of apoptotic cells and sustain anti-inflammatory TGF-β expression. However, in the absence of C1q, CRP fails to suppress proinflammatory TNF-α and may even exacerbate inflammation, emphasizing the importance of C1q and CRP working together to ensure non-inflammatory clearance of dying cells and suggesting a mechanism linking deficiencies in early complement components and CRP to autoimmune diseases such as SLE. Inadequate clearance of apoptotic cells, especially in tissues with ongoing inflammation, may result in secondary necrosis, self-antigen presentation, and breakdown of immune tolerance, contributing to autoimmune disease progression [10].

An experimental study demonstrated that mCRP triggers inflammation in cartilage cells, in both human primary chondrocytes and the ATDC5 murine chondrocyte cell line. In human chondrocytes from healthy donors, mCRP promoted a pro-inflammatory environment, with effects comparable to those induced by LPS, a known trigger of inflammation. Similarly, in ATDC5 cells, mCRP significantly increased nitrite production and upregulated several inflammatory and catabolic markers. Also, by using chondrocytes from OA patients and healthy individuals, mCRP exerted persistent, multigenic inflammatory effects across different conditions, highlighting the broad and sustained activity of mCRP in promoting inflammation, even in chondrocytes not previously exposed to a diseased environment [11]. Another experimental study investigated whether CRP directly contributes to the development of metabolic OA, using a human CRP -transgenic mouse model fed a high-fat diet. Compared to wild-type mice, human CRP-transgenic males developed more severe OA, with greater cartilage damage and osteophyte formation. Although both groups showed similar levels of systemic inflammation and synovitis, human CRP-transgenic mice had increased activation of monocytes, suggesting enhanced immune cell recruitment as a possible mechanism. The findings support the idea that CRP plays an active role in worsening metabolic OA and could be a target for therapeutic intervention [12].

A report by Rajab et al. introduced a new perspective on interpreting diagnostic CRP levels by highlighting the link between CRP and inflammation-related tissue damage. pCRP is a serum-soluble precursor protein that holds considerable potential energy which is released when the pentamer dissociates, triggering a spontaneous conformational change that initiates and amplifies acute inflammation. The monomeric form of CRP is the true "prototypical acute phase reactant," characterized by strong, localized, and short-lived proinflammatory effects. During the early stages of the host defense response to tissue injury, any available pCRP is rapidly converted into mCRP. As the production of mCRP declines or the protein is degraded by proteases, the acute inflammatory response transitions into a chronic phase. pCRP begins to accumulate in the bloodstream only after the conversion to mCRP slows, typically 6 to 10 hours after the initiating event. Therefore, elevated blood levels of pCRP are more indicative of a chronic inflammatory state. If this chronic response is sustained or severe, it can lead to further tissue damage through prolonged neutrophil activity and the release of reactive oxygen species and hydrolytic enzymes into affected tissues [13].

Conformational change in CRP leads to the exposure of neo-epitopes that drive leukocyte activation, oxidative stress through reactive oxygen species (ROS), and leukocyte–endothelial interactions, particularly via lipid raft-mediated signaling pathways [14]. Furthermore, mCRP exhibits unique structural and functional properties that influence neutrophil behavior. Unlike nCRP, mCRP up-regulates neutrophil CD11b/CD18 expression and enhances their adhesion to activated endothelial cells, promoting inflammatory responses at vascular injury sites [15]. Another study took a different viewpoint on the way mCRP influences endothelial cell (EC) responses. Since ECs are more responsive to apical (blood-facing) stimulation than basolateral (tissue-facing) exposure, tissue-resident mCRP, often produced in inflamed sites, may not efficiently activate ECs, especially during chronic local inflammation like atherosclerosis and it might even have protective effects early in plaque development by promoting non-inflammatory clearance of damaged cells and inhibiting foam cell formation. The explanation comes from the polarized EC response being due to an uneven distribution of lipid rafts across the cell membrane, which are enriched apically. In chronic settings, mCRP may act mainly as a pattern recognition molecule, whereas in acute inflammation, circulating mCRP may amplify responses through endothelial, platelet, and neutrophil activation [16].

Eisenhardt et al. examined how mCRP and pCRP affect inflammation in immune cells, showing that both forms of CRP can trigger pro-inflammatory responses, but mCRP has a much stronger and distinct effect compared to pCRP. mCRP appeared to play a more active role in promoting inflammation, especially in processes linked to atherosclerosis, such as monocyte activation and adhesion. While pCRP has sometimes been associated with anti-inflammatory effects, this study suggests it may also contribute to inflammation under certain conditions. The results support the idea that mCRP and pCRP have separate and important roles in the development of vascular disease [17].

References:

[1]       M. E. Olson et al., “A biofunctional review of C-reactive protein (CRP) as a mediator of inflammatory and immune responses: differentiating pentameric and modified CRP isoform effects,” Front. Immunol., vol. 14, p. 1264383, Sep. 2023, doi: 10.3389/fimmu.2023.1264383.

[2]       E. Karasu et al., “A conformational change of C-reactive protein drives neutrophil extracellular trap formation in inflammation,” BMC Biol., vol. 23, no. 1, p. 4, Jan. 2025, doi: 10.1186/s12915-024-02093-8.

[3]       D. Rodríguez-González et al., “Complete Description of the Three Pathways of the Complement System in a Series of 430 Patients with Rheumatoid Arthritis,” Int. J. Mol. Sci., vol. 25, no. 15, Art. no. 15, Jan. 2024, doi: 10.3390/ijms25158360.

[4]       E. A. Bemis et al., “Complement and its Environmental Determinants in the Progression of Human Rheumatoid Arthritis,” Mol. Immunol., vol. 112, pp. 256–265, Aug. 2019, doi: 10.1016/j.molimm.2019.05.012.

[5]       E. T. Molenaar, A. E. Voskuyl, A. Familian, G. J. van Mierlo, B. A. Dijkmans, and C. E. Hack, “Complement activation in patients with rheumatoid arthritis mediated in part by C-reactive protein,” Arthritis Rheum., vol. 44, no. 5, pp. 997–1002, May 2001, doi: 10.1002/1529-0131(200105)44:5<997::AID-ANR178>3.0.CO;2-C.

[6]       É. Biró et al., “Activated complement components and complement activator molecules on the surface of cell‐derived microparticles in patients with rheumatoid arthritis and healthy individuals,” Ann. Rheum. Dis., vol. 66, no. 8, pp. 1085–1092, Aug. 2007, doi: 10.1136/ard.2006.061309.

[7]       J. D. McFadyen et al., “Dissociation of C-Reactive Protein Localizes and Amplifies Inflammation: Evidence for a Direct Biological Role of C-Reactive Protein and Its Conformational Changes,” Front. Immunol., vol. 9, p. 1351, Jun. 2018, doi: 10.3389/fimmu.2018.01351.

[8]       J. Karlsson et al., “Extracellular vesicles opsonized by monomeric C-reactive protein (CRP) are accessible as autoantigens in patients with systemic lupus erythematosus and associate with autoantibodies against CRP,” J. Autoimmun., vol. 139, p. 103073, Sep. 2023, doi: 10.1016/j.jaut.2023.103073.

[9]       J. Thomé et al., “C-reactive protein induced T cell activation is an indirect monocyte-dependent mechanism involving the CD80/CD28 pathway,” Front. Immunol., vol. 16, Jul. 2025, doi: 10.3389/fimmu.2025.1622865.

[10]     D. Gershov, S. Kim, N. Brot, and K. B. Elkon, “C-Reactive Protein Binds to Apoptotic Cells, Protects the Cells from Assembly of the Terminal Complement Components, and Sustains an Antiinflammatory Innate Immune Response,” J. Exp. Med., vol. 192, no. 9, pp. 1353–1364, Nov. 2000, doi: 10.1084/jem.192.9.1353.

[11]     C. Ruiz-Fernández et al., “Monomeric C reactive protein (mCRP) regulates inflammatory responses in human and mouse chondrocytes,” Lab. Invest., vol. 101, no. 12, pp. 1550–1560, Dec. 2021, doi: 10.1038/s41374-021-00584-8.

[12]     A. E. Kozijn et al., “Human C-reactive protein aggravates osteoarthritis development in mice on a high-fat diet,” Osteoarthritis Cartilage, vol. 27, no. 1, pp. 118–128, Jan. 2019, doi: 10.1016/j.joca.2018.09.007.

[13]     I. M. Rajab, P. C. Hart, and L. A. Potempa, “How C-Reactive Protein Structural Isoforms With Distinctive Bioactivities Affect Disease Progression,” Front. Immunol., vol. 11, p. 2126, Sep. 2020, doi: 10.3389/fimmu.2020.02126.

[14]     J. R. Thiele et al., “A Conformational Change in C-Reactive Protein Enhances Leukocyte Recruitment and Reactive Oxygen Species Generation in Ischemia/Reperfusion Injury,” Front. Immunol., vol. 9, p. 675, Apr. 2018, doi: 10.3389/fimmu.2018.00675.

[15]     C. Zouki, B. Haas, J. S. Chan, L. A. Potempa, and J. G. Filep, “Loss of pentameric symmetry of C-reactive protein is associated with promotion of neutrophil-endothelial cell adhesion,” J. Immunol. Baltim. Md 1950, vol. 167, no. 9, pp. 5355–5361, Nov. 2001, doi: 10.4049/jimmunol.167.9.5355.

[16]     J. G. Filep and L. A. Potempa, “Topological Localization of Monomeric C-reactive Protein Determines Proinflammatory Endothelial Cell Responses,” J. Biol. Chem., vol. 289, no. 20, pp. 14283–14290, May 2014, doi: 10.1074/jbc.M114.555318.

[17]     S. U. Eisenhardt et al., “A proteomic analysis of C-reactive protein stimulated THP-1 monocytes,” Proteome Sci., vol. 9, no. 1, p. 1, Jan. 2011, doi: 10.1186/1477-5956-9-1.

 

 

4. The authors spend a huge amount of time discussing the role of mCRP in CVD like CHF and Atherosclerosis, as well as neurodegenerative disorders like AD and dementia. And the title seems to focus on RA. All the mentioned conditions have shared inflammatory pathways which involve mCRP for sure. But will too much discussion on the other conditions deviate the theme from RA? Would suggest the authors provide more detailed, referenced information on the mechanisms of mCRP on RA.

 

We thank the reviewer for this helpful suggestion. The title does imply that RA is a multi-systemic condition and the review highlights the cardiovascular and neurocognitive implications this autoimmune condition has. We looked at mCRP, through shared inflammatory pathways, as an explanation for these co-morbidities associated with RA, including CVD, CHF, atherosclerosis, AD, dementia and even depression. There might be other links between these conditions but other explanations might deviate from the purpose of this review. We further support the mechanisms of mCRP (but mostly CRP, since mCRP in RA has not been investigated in detail yet) in RA through the addition of the following paragraph:

The reason we give such importance to CRP levels in autoimmune dieseases such as RA was underscored by a study which investigated whether sustained suppression of inflammation, as measured by CRP levels, more effectively prevents the development of new joint damage than the progression of existing damage in RA. Over a 5-year period, 359 patients with active RA were monitored, with CRP levels and radiographic joint damage assessed regularly. Results showed a strong correlation between higher time-averaged CRP levels and increased joint damage, particularly in patients with early RA. Notably, new joint involvement rose sharply with higher CRP (a 5.4-fold increase), whereas progression in already damaged joints increased to a lesser extent (1.6-fold). These findings suggest that early and sustained control of inflammation is critical to preventing the spread of joint damage in RA [18]. In another study, radiographic progression after one year was associated with severe initial joint damage, elevated CRP levels, and the presence of IgM rheumatoid factor at baseline [19]. To further support this, in a group of 109 patients with normalized CRP, functional improvement persisted unless CRP levels rose again, concluding that CRP suppression is associated with improved function and that elevated CRP can serve as a useful short-term predictor of functional outcomes and a therapeutic guide in early RA [20].

References:

[18]     M. J. Plant, A. L. Williams, M. M. O’Sullivan, P. A. Lewis, E. C. Coles, and J. D. Jessop, “Relationship between time-integrated C-reactive protein levels and radiologic progression in patients with rheumatoid arthritis,” Arthritis Rheum., vol. 43, no. 7, pp. 1473–1477, Jul. 2000, doi: 10.1002/1529-0131(200007)43:7<1473::AID-ANR9>3.0.CO;2-N.

[19]     L. Jansen, I. E. van der Horst-Bru..., D. van Schaardenburg, P. Bezemer, and B. Dijkmans, “Predictors of radiographic joint damage in patients with early rheumatoid arthritis,” Ann. Rheum. Dis., vol. 60, no. 10, pp. 924–927, Oct. 2001, doi: 10.1136/ard.60.10.924.

[20]     J. Devlin et al., “The acute phase and function in early rheumatoid arthritis. C-reactive protein levels correlate with functional outcome,” J. Rheumatol., vol. 24, no. 1, pp. 9–13, Jan. 1997.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Overall, I recognize the authors' effort to complete the revision and endorse the publication.

Reviewer 2 Report

Comments and Suggestions for Authors

No more issues except that it would be better to have more references.