Chronic Inflammatory Disorders (Cids) Are A Heterogeneous Group Of Debilitating Conditions Characterized By Persistent And Dysregulated Inflammation, Leading To Tissue Damage And Organ Dysfunction. Prominent Examples Include Rheumatoid Arthritis (Ra), A Systemic Autoimmune Disease Primarily Affecting Synovial Joints, And Inflammatory Bowel Disease (Ibd), Which Encompasses Crohn’s Disease And Ulcerative Colitis, Characterized By Chronic Inflammation Of The Gastrointestinal Tract (1). While Their Clinical Manifestations Are Distinct, These Disorders Are Thought To Share Common Underlying Molecular Pathways Driving Their Pathogenesis (2).

The Innate Immune System Serves As The First Line Of Defense Against Pathogens And Cellular Stress. A Central Player In This System Is The Inflammasome, A Multiprotein Cytosolic Complex Responsible For Activating Inflammatory Caspases And Processing The Pro-Inflammatory Cytokines Interleukin-1β (Il-1β) And Interleukin-18 (Il-18) (3). Among The Various Inflammasomes Identified, The Nlrp3 Inflammasome Is The Most Extensively Studied. It Is Activated By A Wide Array Of Stimuli, Including Pathogen-Associated Molecular Patterns (Pamps) And Damage-Associated Molecular Patterns (Damps), Such As Extracellular Atp, Uric Acid Crystals, And Amyloid-Β Aggregates (4). Upon Activation, The Nlrp3 Sensor Protein Recruits The Adaptor Protein Asc (Apoptosis-Associated Speck-Like Protein Containing A Card), Which In Turn Recruits And Activates Pro-Caspase-1. Active Caspase-1 Then Cleaves Pro-Il-1β And Pro-Il-18 Into Their Mature, Biologically Active Forms, Potent Mediators Of Inflammation (5).

Recent Literature Has Increasingly Implicated Nlrp3 Inflammasome Dysregulation In The Pathophysiology Of Various Cids. Studies In Ra Have Shown That Synovial Fluid From Patients Contains Damps Capable Of Activating The Nlrp3 Inflammasome, Leading To Elevated Il-1β Levels That Promote Synovial Inflammation And Cartilage Destruction (6, 7). Similarly, In Ibd, Microbial Dysbiosis And Epithelial Barrier Dysfunction Are Thought To Provide The Signals For Nlrp3 Activation In Intestinal Macrophages, Contributing To Mucosal Inflammation (8, 9). However, A Significant Research Gap Exists (1, 2). Most Studies Have Focused On The Role Of Nlrp3 Within A Single Disease Context. There Is A Lack Of Direct, Comparative Studies That Simultaneously Investigate The Extent Of Nlrp3 Activation Across Different Cids Using Standardized Methodologies. Such A Comparison Is Crucial To Determine Whether Nlrp3 Hyperactivation Is A Universal Hallmark Of Chronic Inflammation Or If Its Contribution Varies Significantly Between Diseases.

This Study Aims To Address This Gap By Posing The Following Research Question: Is The Level Of Systemic Nlrp3 Inflammasome Activation Elevated In Patients With Ra And Ibd Compared To Healthy Individuals, And Does This Level Of Activation Correlate With Clinical Disease Severity? Our Central Hypothesis Is That Nlrp3 Inflammasome Activation Is Significantly Upregulated In Patients With Both Ra And Ibd Compared To Healthy Controls, And That The Concentration Of Its Downstream Effector Cytokines, Il-1β And Il-18, Directly Correlates With Established Clinical Scores Of Disease Activity (3). The Significance Of This Research Lies In Its Potential To Validate The Nlrp3 Pathway As A Common Pathogenic Driver Across Different Cids, Which Could Pave The Way For The Development Of Broad-Spectrum Anti-Inflammatory Therapies And Establish Nlrp3-Related Molecules As Reliable Biomarkers For Monitoring Disease Progression

Study Design

A Cross-Sectional, Case-Control Study Was Conducted At The Tertiary Care General Hospital In India.

Participants And Samples

The Study Enrolled 150 Participants, Divided Into Three Groups: 50 Patients With A Confirmed Diagnosis Of Ra, 50 Patients With A Confirmed Diagnosis Of Ibd (28 With Crohn's Disease, 22 With Ulcerative Colitis), And 50 Age- And Sex-Matched Healthy Controls (Hc).

• Inclusion Criteria: For The Ra Group, Patients Had To Fulfill The 2010 American College Of Rheumatology/European League Against Rheumatism (Acr/Eular) Classification Criteria. For The Ibd Group, Patients Required A Diagnosis Confirmed By Endoscopic And Histological Findings. All Participants Were Between 18 And 65 Years Of Age. Healthy Controls Had No History Of Chronic Inflammatory, Autoimmune, Or Malignant Disease.
• Exclusion Criteria: Common Exclusion Criteria For All Groups Included Acute Infection Within Four Weeks Of Enrollment, Severe Renal Or Hepatic Dysfunction, Pregnancy, And Current Malignancy. For Patient Groups, Exclusion Criteria Included The Use Of Biologic Agents Specifically Targeting The Il-1 Pathway (E.G., Anakinra, Canakinumab) Within The Past Three Months To Avoid Direct Confounding Effects On The Study's Primary Outcomes.

Data Collection Procedures

Demographic Information (Age, Sex) And Clinical Data Were Collected From All Participants. For Ra Patients, Disease Activity Was Quantified Using The Disease Activity Score 28-C-Reactive Protein (Das28-Crp). For Ibd Patients, The Harvey-Bradshaw Index (Hbi) Was Used To Assess Disease Activity. For Each Participant, 20 Ml Of Peripheral Blood Was Collected Via Venipuncture Into Edta-Containing Tubes.

Laboratory Procedures And Instruments

Blood Samples Were Processed Within Two Hours Of Collection.

1. Pbmc And Plasma Isolation: Peripheral Blood Mononuclear Cells (Pbmcs) Were Isolated By Density Gradient Centrifugation Using Ficoll-Paque Plus (Ge Healthcare). The Plasma Supernatant Was Collected, Aliquoted, And Stored At -80°C Until Analysis. Pbmc Pellets Were Lysed Using Ripa Buffer Containing Protease Inhibitors For Protein Extraction.
2. Western Blot Analysis: Total Protein Concentration In Pbmc Lysates Was Determined Using A Bca Protein Assay Kit (Thermo Fisher Scientific). Equal Amounts Of Protein (30 Μg) Were Separated By Sds-Page And Transferred To A Polyvinylidene Difluoride (Pvdf) Membrane. Membranes Were Blocked And Incubated Overnight At 4°C With Primary Antibodies Against Nlrp3 (1:1000, Cell Signaling Technology), Asc (1:1000, Adipogen), Caspase-1 P20 (Cleaved Form) (1:1000, Cell Signaling Technology), And Β-Actin (1:5000, Santa Cruz Biotechnology) As A Loading Control. After Incubation With Hrp-Conjugated Secondary Antibodies, Protein Bands Were Visualized Using An Enhanced Chemiluminescence (Ecl) Detection System (Bio-Rad), And Band Densities Were Quantified Using Imagej Software.
3. Elisa: Plasma Concentrations Of Il-1β And Il-18 Were Measured Using Commercially Available High-Sensitivity Enzyme-Linked Immunosorbent Assay (Elisa) Kits (R&D Systems) According To The Manufacturer's Instructions. All Samples Were Run In Duplicate. The Lower Limits Of Detection Were 0.1 Pg/Ml For Il-1β And 12.5 Pg/Ml For Il-18.

Statistical Analysis

All Statistical Analyses Were Performed Using Spss Version 26.0 (Ibm Corp.). Data Were First Assessed For Normality Using The Shapiro-Wilk Test. Demographic And Clinical Characteristics Were Summarized Using Means And Standard Deviations (Sd) For Continuous Variables And Frequencies (Percentages) For Categorical Variables. Differences In Nlrp3 Inflammasome Protein Expression And Cytokine Levels Among The Three Groups (Ra, Ibd, Hc) Were Analyzed Using One-Way Analysis Of Variance (Anova) Followed By Tukey's Post-Hoc Test For Multiple Comparisons. The Relationship Between Cytokine Levels (Il-1β, Il-18) And Disease Activity Scores (Das28-Crp, Hbi) Was Assessed Using The Pearson Correlation Coefficient (R). A P-Value Of < 0.05 Was Considered Statistically Significant.

Participant Characteristics

A Total Of 150 Participants Were Included In The Final Analysis. The Demographic And Clinical Characteristics Of The Study Population Are Summarized In Table 1. The Three Groups Were Well-Matched For Age And Sex. As Expected, Patients With Ra And Ibd Had Significantly Higher C-Reactive Protein (Crp) Levels And Disease Activity Scores Compared To The Negligible Values In Healthy Controls.

Table 1: Demographic And Clinical Characteristics Of Study Participants

HBI (mean ± SD) N/A       N/A       7.8 ± 2.4               N/A

Upregulation of NLRP3 Inflammasome Components in RA and IBD Patients

Western blot analysis of PBMC lysates revealed a significant upregulation of NLRP3 inflammasome components in both patient cohorts compared to healthy controls (Figure 1A). Quantitative densitometry confirmed that the relative protein expression of NLRP3, ASC, and the active (cleaved) form of Caspase-1 were markedly higher in both RA and IBD groups than in the HC group (p<0.001 for all comparisons) (Figure 1B). There was no statistically significant difference in the expression levels of these proteins between the RA and IBD groups.

Elevated Plasma Levels of IL-1β and IL-18 in RA and IBD Patients

Consistent with the increased expression of inflammasome components, ELISA results showed that plasma concentrations of the downstream cytokines IL-1β and IL-18 were significantly elevated in patients with RA and IBD compared to healthy controls (Table 2). The mean IL-1β level in the RA group was 12.4 pg/mL and 10.8 pg/mL in the IBD group, both substantially higher than the 1.1 pg/mL observed in the HC group (p<0.001). A similar pattern was observed for IL-18.

Table 2: Plasma Levels of NLRP3 Inflammasome-Related Cytokines

Correlation Between Inflammasome Activation and Disease Severity

To determine if the degree of NLRP3 inflammasome activation was associated with disease progression, we performed a correlation analysis. A strong, positive, and statistically significant correlation was found between plasma IL-1β levels and the DAS28-CRP score in RA patients (r = 0.72, p<0.001) (Figure 2A). Similarly, IL-1β levels showed a strong positive correlation with the HBI score in IBD patients (r = 0.68, p<0.001) (Figure 2B). Plasma IL-18 levels also correlated significantly with disease activity in both RA (r=0.65, p<0.001) and IBD (r=0.61, p<0.001) groups (data not shown).

This study provides compelling comparative evidence for the central role of NLRP3 inflammasome hyperactivation in the systemic inflammation underlying both RA and IBD. Our primary finding is that key components and downstream products of the NLRP3 inflammasome are significantly elevated in the peripheral circulation of patients with these distinct CIDs compared to healthy individuals. Furthermore, we established a strong positive correlation between the levels of inflammasome-derived cytokines and validated markers of clinical disease activity, supporting our initial hypothesis (6).

The observation of increased NLRP3, ASC, and cleaved Caspase-1 protein in PBMCs from both RA and IBD patients aligns with and extends previous research. Prior studies have demonstrated elevated NLRP3 expression in specific disease-relevant tissues, such as the synovial tissue of RA patients and the colonic mucosa of IBD patients (7, 10). Our work confirms that this activation is also reflected systemically in circulating immune cells, suggesting a widespread inflammatory state. By analyzing two different CIDs concurrently with a standardized methodology, our study overcomes a limitation of previous single-disease investigations and demonstrates that NLRP3 hyperactivation may be a common denominator in the pathology of CIDs affecting different organ systems (2). This finding supports the concept of shared pathogenic pathways that could be targeted by broad-spectrum therapeutics.

The strong correlation between plasma IL-1β levels and disease activity scores (DAS28-CRP and HBI) is a particularly significant finding. It suggests that the NLRP3 inflammasome is not merely an initiator of inflammation but is actively involved in its perpetuation and progression (11). This positions IL-1β and other inflammasome-related molecules as plausible biomarkers for monitoring disease activity and response to treatment. While CRP is a widely used marker of systemic inflammation, it is non-specific. Measuring IL-1β or IL-18 could provide a more direct readout of the specific inflammatory cascade driven by the NLRP3 pathway, potentially offering greater precision in patient management (12).

The practical implications of our findings are substantial. The central role of the NLRP3 inflammasome in driving inflammation in both RA and IBD reinforces its potential as a high-value therapeutic target. Specific NLRP3 inhibitors are currently in clinical development, and our data suggest they could have therapeutic efficacy across a spectrum of CIDs, not just in rare autoinflammatory syndromes (13, 14, 15). By targeting a common upstream node in the inflammatory cascade, such therapies may offer a more fundamental approach to treatment than downstream cytokine blockade alone.

Despite the robustness of our findings, this study has several limitations. First, its cross-sectional design precludes any inference of causality; we can demonstrate association but cannot prove that NLRP3 activation causes disease progression. Longitudinal studies are needed to track these markers over time and in response to therapy. Second, our analysis was based on PBMCs, which may not fully represent the inflammatory microenvironment within the target tissues (e.g., synovium or gut mucosa). However, using peripheral blood enhances the clinical applicability of our findings for developing accessible biomarkers. Third, although we excluded patients on IL-1 inhibitors, other medications such as methotrexate or TNF inhibitors could have confounding effects on inflammasome activity, an area that warrants further investigation. Finally, our sample size was relatively modest, and the findings should be validated in larger, multi-center cohorts.

In conclusion, this study demonstrates that systemic activation of the NLRP3 inflammasome is a prominent and shared feature of both Rheumatoid Arthritis and Inflammatory Bowel Disease. The extent of this activation correlates strongly with clinical disease severity, highlighting the pathway's integral role in the pathogenesis and progression of these disorders. These findings contribute to a deeper understanding of the common molecular mechanisms underlying CIDs and underscore the potential of the NLRP3 inflammasome as both a valuable biomarker for disease monitoring and a promising target for novel therapeutic interventions. Future research should focus on longitudinal studies to establish causality and on clinical trials of NLRP3 inhibitors to assess their therapeutic efficacy in these patient populations.

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