Review Reports - Monomeric [CRP] and CRP-Controlled Stress and Pain Hypersensitization as Novel Predictors of Cognitive Disturbance and AD in Chronic Inflammatory Disease

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript entitled: “Monomeric [CRP] and CRP-controlled stress and pain hypersensitization as novel predictors of cognitive disturbance and AD in chronic inflammatory disease” by Slevin and Tero-Vescan (Manuscript ID: ijms-3967791) describes the potential role of two biomarkers of the systemic inflammation i.e. interleukin-6 (IL-6) and C-reactive protein (CRP) in the pathophysiology of neurodegenerative and stress-related disorders.

This is a rather well written MS which may be of interest to readers. I have no comments to authors.

Author Response

Dear Respected Reviewer 1,

We sincerely thank the reviewer for their positive evaluation of our manuscript entitled “Monomeric [CRP] and CRP-controlled stress and pain hypersensitization as novel predictors of cognitive disturbance and AD in chronic inflammatory disease.”
We greatly appreciate the reviewer’s time and encouraging comments, and we are pleased that the manuscript was found to be clearly written and of potential interest to the readership.

This is a rather well written MS which may be of interest to readers. I have no comments to authors.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript “Monomeric [CRP] and CRP-controlled stress and pain hypersensitization as novel predictors of cognitive disturbance and AD in chronic inflammatory disease” (ijms-3967791) by Slevin et al contains interesting perspectives, suggesting mCRP is not just a marker, but a mechanosensitive amplifier and effector in cognitive decline, is a fresh take. Manuscript is well written, and authors have shown in depth knowledge of subject. The article comprehensively integrates a broad swath of research, consistently referencing up-to-date peer-reviewed studies to support its central claims. Statements about the IL-6/CRP axis, pain sensitivity, stress amplification, and neurodegeneration are all backed by cited meta-analyses and large cohort studies. I have a few concerns listed below-

Major-

Briefly mention current mCRP assays, specificity issues, sample handling, and inter-lab variability, and state how this limits cross-study comparisons. This will pre-empt reviewer concerns and grounds your ratio proposal.
Therapeutic implications. Since this may go to a therapeutic special issue, expand the small therapy paragraph into a focused section; for e.g.-

Anti-IL-6 strategies; expected effect on hepatic CRP.
Emerging agents that may alter CRP isoform dynamics or mCRP signaling.
Non-pharmacologic methods that downshift CRP or mCRP, or its conversion.
Proposed enrichment biomarkers for trials (baseline mCRP/CRP,).

Minor-

There are too many grammar and spelling checks, authors are suggested to revise the manuscript and ensure that corrects spelling and words are being used.
The “NOTE” about CRP vs mCRP is valuable, but the tone is informal and there are typos. Fix spelling and add 1–2 references on assay specificity. Lines 114-119.
Figure 5, a good illustration. Consider adding a single sentence, in its caption, on shear-induced pCRP→mCRP conversion to connect mechanical stress with mCRP biology. Lines 605-616.

Typo-errors-

“Specificlly” → “Specifically.” Line 30.
“too date” → “to date.” Line 34-35.
Replace “could illicit an exaggerated…” with “could elicit an exaggerated…”. Lines 99-101
NF-κB not Nf- κB in line number 231.
Descrimination to discrimination in line number 115

Comments on the Quality of English Language

On page number 100, the word "illicit" doesn't fit to explain the context. It may be replaced with "elicit"

Typo-errors-

“Specificlly” → “Specifically.” Line 30.
“too date” → “to date.” Line 34-35.
Replace “could illicit an exaggerated…” with “could elicit an exaggerated…”. Lines 99-101
NF-κB not Nf- κB in line number 231.
Descrimination to discrimination in line number 115

Author Response

Dear Respected Reviewer 2,

We would like to sincerely thank you for your careful and thoughtful evaluation of our manuscript. We greatly appreciate the time and effort dedicated to providing constructive feedback, which has helped improve the quality and clarity of the article.

Kindly find the revisions made to the manuscript in accordance with your suggestions, highlighted using the track changes feature.

Major-

Briefly mention current mCRP assays, specificity issues, sample handling, and inter-lab variability, and state how this limits cross-study comparisons. This will pre-empt reviewer concerns and grounds your ratio proposal.

We thank the reviewer for this valuable suggestion. We have added a brief methodological note describing current mCRP assays, specificity considerations, key pre-analytic factors, and inter-laboratory variability, and we explicitly state how these issues limit cross-study comparisons and motivate our proposed mCRP/CRP ratio. The new text appears in the Limitations section.

The following paragraph was added to the article:

mCRP is currently measured using conformation-specific immunoassays that recognize epitopes exposed only on the monomeric isoform or by ligand-binding systems minimizing cross-reactivity with native pCRP. However, assay standardization remains limited, differences in antibody pairs, calibrators, and reporting units produce substantial variability across laboratories. Moreover, pre-analytic factors such as sample matrix, anticoagulant type, storage, and freeze–thaw cycles can promote artifactual pCRP→mCRP conversion or loss of vesicle-bound mCRP, confounding quantification. These analytical and pre-analytical inconsistencies restrict cross-study comparability of absolute mCRP levels. Therefore, use of isoform-selective assays and normalization through the mCRP/CRP ratio is recommended to mitigate methodological bias and better capture CRP isoform dynamics.

Reference

Fujita C, Sakurai Y, Yasuda Y, Homma R, Huang C-L and Fujita M (2022) mCRP as a Biomarker of Adult-Onset Still’s Disease: Quantification of mCRP by ELISA. Front. Immunol. 13:938173. doi: 10.3389/fimmu.2022.938173

Therapeutic implications. Since this may go to a therapeutic special issue, expand the small therapy paragraph into a focused section; for e.g.-

Anti-IL-6 strategies; expected effect on hepatic CRP.
Emerging agents that may alter CRP isoform dynamics or mCRP signaling.
Non-pharmacologic methods that downshift CRP or mCRP, or its conversion.
Proposed enrichment biomarkers for trials (baseline mCRP/CRP,).

We thank the reviewer for this valuable suggestion. In response, we have expanded the Therapeutic Implications paragraph, highlighting both pharmacologic and non-pharmacologic approaches that may modulate CRP/mCRP biology and its downstream inflammatory consequences.

The following phrases were added to the manuscript:

Pharmacologic interventions could target IL-6 as the principal hepatic driver of CRP synthesis. Monoclonal antibodies such as tocilizumab and sarilumab, which inhibit IL-6 receptor signaling, markedly reduce circulating CRP levels and have shown cognitive and fatigue benefits in inflammatory disorders including rheumatoid arthritis and post-COVID syndromes. Recent data also suggest that chronic IL-6 blockade can indirectly limit peripheral mCRP formation and endothelial activation by lowering substrate (pCRP) availability. Small molecules that stabilize pentameric CRP and prevent its dissociation (e.g., phosphocholine analogues) have demonstrated reduced vascular inflammation in preclinical models. In addition, novel compounds, including peptide inhibitors of mCRP–CD16 interaction and nanobody-based mCRP neutralizers are under investigation for limiting mCRP-driven microglial and endothelial activation ……………… Regular aerobic training and structured exercise programs (including HIIT) are associated with significant reductions in CRP, particularly in cardiometabolic risk groups………………… Quantifying baseline pCRP/mCRP ratios may help identify patients with disproportionate mCRP activity who are most likely to benefit from CRP-modulating therapies. Isoform-selective assays have become available: a validated mCRP ELISA has been applied in clinical cohorts, and recent translational studies have reported circulating mCRP measurements in patient populations. These tools can support isoform-resolved stratification in interventional trials.

Refereces:

Fujita C, Sakurai Y, Yasuda Y, Fujita M. Monomeric C-reactive protein as a therapeutic target in inflammatory diseases. RMD Open. 2025 Oct 15;11(4):e005379. doi: 10.1136/rmdopen-2024-005379. PMID: 41093630; PMCID: PMC12530421.
Fujita C, Sakurai Y, Yasuda Y, Homma R, Huang C-L and Fujita M (2022) mCRP as a Biomarker of Adult-Onset Still’s Disease: Quantification of mCRP by ELISA. Front. Immunol. 13:938173. doi: 10.3389/fimmu.2022.938173
Zeller J, Cheung Tung Shing KS, Nero TL, McFadyen JD, Krippner G, Bogner B, Kreuzaler S, Kiefer J, Horner VK, Braig D, Danish H, Baratchi S, Fricke M, Wang X, Kather MG, Kammerer B, Woollard KJ, Sharma P, Morton CJ, Pietersz G, Parker MW, Peter K, Eisenhardt SU. A novel phosphocholine-mimetic inhibits a pro-inflammatory conformational change in C-reactive protein. EMBO Mol Med. 2023 Jan 11;15(1):e16236. doi: 10.15252/emmm.202216236. Epub 2022 Dec 5. PMID: 36468184; PMCID: PMC9832874.
Camon, A.M., Alonso, R., Muñoz, F.J. et al. C-reactive protein cut-off for early tocilizumab and dexamethasone prescription in hospitalized patients with COVID-19. Sci Rep 12, 5250 (2022). https://doi.org/10.1038/s41598-022-08882-x
Nikseresht M, Nikseresht M. A novel periodized high‐repetition giant‐set resistance and high‐intensity interval training effects on the metabolic and pro‐inflammatory parameters in long‐ and short‐term overweight men. Eur J Sport Sci. 2024 Jan 30;24(1):127– doi: 10.1002/ejsc.12038. PMCID: PMC11235816.

Minor-

There are too many grammar and spelling checks, authors are suggested to revise the manuscript and ensure that corrects spelling and words are being used.

We thank the reviewer for this observation. The entire manuscript has been carefully revised for grammar, syntax, and spelling accuracy. All typographical errors have been corrected, and the text has been thoroughly edited for clarity and readability.

The “NOTE” about CRP vs mCRP is valuable, but the tone is informal and there are typos. Fix spelling and add 1–2 references on assay specificity. Lines 114-119.

We thank the reviewer for this helpful suggestion. The “NOTE” section has been revised to improve clarity and tone, to correct typographical errors, and to include supporting references on assay specificity distinguishing CRP and mCRP.

The text was revised as follows:

NOTE: In most published studies, no clear discrimination was made between circulating CRP and its monomeric form (mCRP), largely because the specific antibodies required to distinguish mCRP from native pentameric CRP (pCRP) were not yet available. Therefore, interpretations of CRP-associated pathology must be made with caution, as the relative contribution of each isoform to diagnostic or prognostic outcomes remains uncertain. Where mCRP was specifically quantified, it is clearly designated as such in the present text.

The following references were added:

Williams RD, Moran JA, Fryer AA, Littlejohn JR, Williams HM, Greenhough TJ and Shrive AK (2020) Monomeric C-Reactive Protein in Serum With Markedly Elevated CRP Levels Shares Common Calcium-Dependent Ligand Binding Properties With an in vitro Dissociated Form of C-Reactive Protein. Front. Immunol. 11:115. doi: 10.3389/fimmu.2020.00115
Fujita C, Sakurai Y, Yasuda Y, Homma R, Huang CL, Fujita M. mCRP as a Biomarker of Adult-Onset Still's Disease: Quantification of mCRP by ELISA. Front Immunol. 2022 Jul 1;13:938173. doi: 10.3389/fimmu.2022.938173. PMID: 35844576; PMCID: PMC9284222.

Figure 5, a good illustration. Consider adding a single sentence, in its caption, on shear-induced pCRP→mCRP conversion to connect mechanical stress with mCRP biology. Lines 605-616.

We thank the reviewer for this constructive suggestion. We have revised the caption of Figure 5 to include an explanatory sentence highlighting the shear-induced conversion of pentameric CRP (pCRP) to its monomeric form (mCRP) and its relevance to mechanical stress–mediated vascular inflammation and endothelial dysfunction. This addition strengthens the mechanistic link between hemodynamic shear and mCRP biology, as recommended.

The following phrase was added to the caption of Figure 5:

“Shear-induced mechanical stress promotes the dissociation of pentameric CRP (pCRP) into its monomeric form (mCRP), thereby linking vascular biomechanics with localized inflammatory signalling and endothelial activation.”

Comments on the Quality of English Language

On page number 100, the word "illicit" doesn't fit to explain the context. It may be replaced with "elicit"

We appreciate the reviewer’s attention to detail, which helped us improve the accuracy and clarity of the manuscript.

Typo-errors-

“Specificlly” → “Specifically.” Line 30.
“too date” → “to date.” Line 34-35.
Replace “could illicit an exaggerated…” with “could elicit an exaggerated…”. Lines 99-101
NF-κB not Nf- κB in line number 231.
Descrimination to discrimination in line number 115

We thank the reviewer for carefully identifying the typographical errors. All suggested corrections have been made.

Reviewer 3 Report

Comments and Suggestions for Authors

Novelty

This manuscript presents a novel integrative framework linking the mechanosensing function of monomeric C-reactive protein (mCRP) with inflammatory pain, stress responses, and dysregulation of the hypothalamic–pituitary–adrenal (HPA) axis. By incorporating the concept of shear-induced dissociation of mCRP into both mechanical and psychological stress models, it expands beyond the traditional IL-6–CRP correlation and offers a distinctive mechanistic perspective.

1. Scope and Focus Adjustment

The manuscript covers a wide range of interconnected topics, including inflammation, pain, stress, psychiatric disorders, genetics, epigenetics, and AI-based predictive modeling, all centered around the IL-6/CRP axis. However, this breadth slightly diffuses the main focus. If the core message is to present an integrated mechanistic model linking inflammation, pain, and neuropsychiatric disorders, the discussion should be streamlined toward this central theme. Genetic and AI-related aspects could then be presented more concisely as complementary mechanisms or potential applications. This adjustment would enhance the overall coherence and narrative focus of the manuscript.

2. Clarification of mCRP Evidence Limitations

The authors have commendably acknowledged in the Introduction that most previous studies did not distinguish between CRP and mCRP. Reiterating this limitation briefly in the Conclusion or Future Directions section would strengthen the manuscript’s transparency and consistency. Doing so will highlight that the authors are aware of the current methodological constraints and have not overstated their mechanistic interpretations.

3. Further Specification of the AI-Based Risk Model

The section on AI-based risk stratification currently remains conceptual. Providing a brief description of the model’s potential inputs (e.g., CRP, IL-6, cortisol, or genetic markers), structural approach (e.g., supervised learning, regression framework), or intended clinical application (e.g., early diagnosis, prognosis prediction) would significantly increase the manuscript’s practical relevance and credibility. Even a short paragraph outlining these aspects would make the proposed model more tangible and strengthen its translational potential.

Author Response

Dear Respected Reviewer 3,

Kindly find the revisions made to the manuscript in accordance with your suggestions, highlighted using the track changes feature.

Novelty

Scope and Focus Adjustment

Response:

We have written additional paragraphs at the end of sections 2 (2.1), 5 (5.2) and 7 with sub-titles focussed on these elements and these paragraphs provide a clarity and focus as to the impact within this inflammatory-pain sensitivity and neuropsychiatric system as follows…

2.1. Integrative focus and scope of the review:

Here we explicitly describe the IL-6/CRP–mCRP axis as the mechanistic spine that links systemic inflammation with pain hypersensitivity, stress responsivity, and neurocognitive vulnerability. In the pages that follow, we will prioritize evidence that clarifies causal pathways from IL-6–driven hepatocyte CRP production and context-dependent mCRP generation (at activated vascular and immune surfaces) to endothelial activation, BBB vulnerability, and central sensitization. By contrast, broader topics (e.g., multi-omic correlates, psychiatric comorbidity spectra) are treated selectively when they provide supportive evidence how this axis amplifies pain and stress signals into neurodegenerative risk. This focus ensures that downstream sections, on pain biology, HPA feedback, BBB integrity, and therapeutic levers, cohere around a single, testable model, that being inflammation → IL-6/CRP → mCRP effector signaling → vascular–neuroimmune dysregulation → cognitive decline.

5.2. Genetics, Epigenetics, and AI as Complementary Stratification Layers:

Genetic variation (e.g., IL6, CRP, NR3C1), stress-linked epigenetic marking, and machine-learning risk models are best viewed here as adjunct amplifiers rather than alternative mechanisms. Their value is to identify individuals with heightened inflammatory tone or impaired HPA feedback, and to flag those biased toward pCRP→mCRP conversion at vascular interfaces. They may also operationalize multimodal data (biomarkers, QST, imaging, stress physiology) into actionable risk tiers. Therefore, omics and AI whilst not replacing the mechanistic core, may refine who is most susceptible to the IL-6/CRP–mCRP cascade and when to intervene. Future work should therefore report isoform-aware metrics (absolute mCRP and mCRP/CRP ratio) alongside genetic/epigenetic panels and add these features in pre-registered predictive models with external validation, so that stratification directly serves mechanism-based prevention or treatment trials.

7.1. Mechanism-anchored therapeutic framing:

To maintain coherence with our mechanistic focus, we have interpreted interventions primarily through their impact on the IL-6/CRP–mCRP pathway focusssed upon upstream IL-6 blockade to reduce hepatic CRP drive, stabilization or neutralization strategies to limit mCRP effector signaling at activated endothelium/immune surfaces, and stress- and pain-modulating approaches (exercise, autonomic balance, cognitive therapies) that reduce the feed-forward coupling between nociception, HPA dysregulation, and inflammatory tone. This should help to clarifie why a therapy should work in this system and how its success ought to be measured (e.g., shifts in mCRP/CRP ratio, BBB-relevant markers, and pain sensitivity metrics), aligning trial design with the biology we propose.

Clarification of mCRP Evidence Limitations

We thank the reviewer for this thoughtful suggestion. We agree that restating this limitation enhances transparency and interpretative balance. Accordingly, we have added a brief statement in the Limitations section acknowledging that most prior studies did not differentiate between pCRP and mCRP, emphasizing the need for standardized isoform-specific assays and validation across laboratories.

The following phrase was added to the manuscript

mCRP is currently measured using conformation-specific immunoassays that recognize epitopes exposed only on the monomeric isoform or by ligand-binding systems minimizing cross-reactivity with native pCRP. However, assay standardization remains limited, differences in antibody pairs, calibrators, and reporting units produce substantial variability across laboratories. Moreover, pre-analytic factors such as sample matrix, anticoagulant type, storage, and freeze–thaw cycles can promote artifactual pCRP→mCRP conversion or loss of vesicle-bound mCRP, confounding quantification. These analytical and pre-analytical inconsistencies restrict cross-study comparability of absolute mCRP levels. Therefore, use of isoform-selective assays (doi: 10.3389/fimmu.2022.938173) and normalization through the mCRP/CRP ratio is recommended to mitigate methodological bias and better capture CRP isoform dynamics.

Reference

Fujita C, Sakurai Y, Yasuda Y, Homma R, Huang C-L and Fujita M (2022) mCRP as a Biomarker of Adult-Onset Still’s Disease: Quantification of mCRP by ELISA. Front. Immunol. 13:938173. doi: 10.3389/fimmu.2022.938173
Fuglestad AJ, Bousquet PA, Køstner AH, Meltzer S, Kleist B, Ree AH, Rajab IM, Potempa LA, Fernandez-Botran R, Kersten C. Circulating Levels of the Proinflammatory Monomeric Isoform of C-Reactive Protein (mCRP) Correlate with Intra-Tumoral mCRP Abundance in Stage II-III Colon Cancer Patients. J Inflamm Res. 2025;18:7115-7125
https://doi.org/10.2147/JIR.S485050

Further Specification of the AI-Based Risk Model

We added the following paragraphs to satisfy the reviewer within section 7

AI-component risk framework:

We propose a pragmatic, risk stratification framework that treats the IL-6 → CRP → mCRP cascade as the mechanistic core linking systemic inflammation with pain hypersensitivity, stress responsivity, and neurocognitive vulnerability. At first assessment, the model would estimate 12-month risk of (i) persistent high pain and functional impairment, (ii) clinically significant depressive/PTSD symptom burden, and (iii) cognitive decline. Candidate inputs prioritize biology that is both measurable and modifiable: hs CRP, mCRP and the mCRP/CRP ratio, IL-6 (± TNF-α), neural injury/astroglial markers (NfL, GFAP), AM cortisol and diurnal slope, simple wearables-derived sleep/HRV summaries, and, when available, kynurenine pathway indices (KYN/Trp, KYNA/QA). Brief psychometrics (pain interference, PHQ-9, GAD-7, PCL-5), cognition screens (e.g., MoCA), and context covariates (age, sex/menopausal status, BMI, comorbidities, medications, activity, smoking, socioeconomic factors) complete a clinically tractable feature set. Genetic/epigenetic markers (e.g., IL6, CRP, NR3C1, FKBP5, APOE) are positioned as optional refiners to identify individuals biased toward heightened inflammatory tone or pCRP→mCRP conversion.

Structurally, the approach favors transparent, validated models: a regularized logistic (or Cox) baseline for interpretability, complemented by gradient-boosted trees to capture nonlinearity and interactions; mixed time-series and text can be reduced to summary features and fused late. Performance should be demonstrated with temporal validation, calibration (Brier/reliability), AUPRC alongside AUROC given class imbalance, and decision-curve analysis to show net clinical benefit; external, sex-disaggregated testing is required. Outputs are delivered as risk tiers (low/intermediate/high) mapped to pre-specified actions, enabling mechanism-matched care: for example, escalation to anti-inflammatory strategies when mCRP/CRP and IL-6 dominate, or neuromodulatory/sleep-focused interventions when HRV/sleep and affective measures drive risk. Re-estimation after 4–8 weeks supports response monitoring and adaptive care. In summary, the model operationalizes the inflammation–pain–neurocognition axis into a practical, clinically interpretable tool that strengthens translational relevance without diluting mechanistic focus.

Author Response File: Author Response.docx