Inflammation, fibrosis and E1 glycoprotein persistence in joint tissue of patients with post-Chikungunya chronic articular disease

ABSTRACT Introduction: Chikungunya chronic joint disease causes debilitating arthralgia, significantly impacting the quality of life of affected individuals. Methods: In this study, patients underwent clinical follow-ups, joint biopsies, and pre-biopsy and 24 months post-biopsy serum dosage of cytokines. Results: All participants were female and had pain in 12 joints on average, with 41.17% exhibiting moderate disease activity. Histopathological analysis revealed collagen deposition. Indirect immunofluorescence detected the CHIKV glycoprotein E1 antigen, and an increase in cytokines. Conclusions: Persistent inflammation and ineffective antiviral immune responses leading to antigen persistence may contribute to chronic CHIKV arthritis.

Chikungunya virus (CHIKV) is an arthritogenic Alphavirus belonging to the Martellivirales order, Togaviridae family, transmitted to humans by arthropod vectors from Aedes genus 1 .CHIKV causes acute febrile disease accompanied by arthralgia and joint edema.Subsequently, many patients develop persistent or recurrent polyarthralgia in the following weeks, evolving to subacute and chronic debilitating articular disease 2,3 .Three hypotheses currently elucidate the development of chronic arthritis.These include local persistence of viral RNA directly linked to virus replication, antigen persistence hindering viral clearance and subsequently triggering chronic inflammation, and the continuation of nonprotective immunemediated inflammation even after the resolution of CHIKV replication 4,5,6 .Histological studies enhance our understanding of Patients with chronic articular disease persisting for more than 12 months underwent joint biopsies.The selection of a specific joint was based on clinical manifestations and radiological findings.An orthopedist performed the procedure in an outpatient unit under aseptic conditions, beginning with local anesthesia using 1% lidocaine.
The extracted fragment was dehydrated in an ethanol solution, cleared with xylol, and embedded in paraffin within 24 h of extraction.Thin sections (3 mm; HIRAX M60 Carl Zeiss; Germany) were deparaffinized, rehydrated, and subjected to hematoxylineosin staining to identify cellular structures and to special Masson trichrome staining, evidencing the muscular tissue and collagen fibers 13 .A single analyzer performed histological analyses on an AxioScope A1 microscope (Carl Zeiss, GR).
The second joint fragment was cut into small pieces with scalpel blades, eluted in RNase free water (0.4 mL) and subjected to total RNA extraction (0.2 mL) with Trizol LS (Thermo Fischer) and viral nucleic acid extraction (0.2 mL) including MS2 phage RNA (Sigma Aldrich) extraction control with MagMax viral pathogen kit (Applied Biossystems) following manufacturer's instructions.
Total RNA was subjected to an RT-PCR test targeting the CHIKV E1 region (305 bp).The viral nucleic acid was examined using the TaqMan Zika Virus Triplex Screening RT-qPCR Kit, which screens for Zika, Dengue, and Chikungunya viruses (Applied Biossystems).This testing was conducted on a Quantistudio 5 platform (Applied Biosystems).
The inflammatory profile was assessed in the serum of 5 of 13 patients who continued to exhibit chronic disease on the day of the biopsy procedure and 11 of 13 patients 24 months post-biopsy.Venous blood was drawn into clot activator tubes, allowed to clot for 30 minutes, and then centrifuged for 15 minutes.
Clinically, patients presented a mean of 12 (1-28) painful and rigid articulations; 58.82% suffered arterial hypertension; 58.82% were using corticosteroids; 41.17% were taking nonsteroidal anti-inflammatory drugs (NSAIDs); and none were using immunomodulators at the time of admission to the rheumatology service.
On the day the biopsy was performed, all patients had normal inflammatory test results.With a mean of five painful joints, 47.05% had morning stiffness, and 5.88% were taking corticosteroids, 17.64% took NSAIDs, and 58.82% took immunomodulators.According to the CDAI, 17.64% of patients had high disease activity, 11.76% had moderate disease activity, 52.94% had low disease activity, and 17.64% were in remission.The mean value on CDAI at the time was 8.82 (Table 1).In the last clinical evaluation in October 2022 (24 months after biopsy), 12 out of 17 patients still had a mean of eight tender joints, 64.7% presented persisting matinal stiffness, 11.76% were using systemic corticosteroids, while 17.64% were taking NSAIDs.Furthermore, 41.17% of the patients were undergoing immunomodulatory therapy with methotrexate, hydroxychloroquine, sulfasalazine, or a combination of these drugs 11 .The CDAI showed that 41.17% patients presented low disease activity (mean: 12.52), 23.52% had high disease activity, 17.64% moderate disease activity, and 17.64% were in remission (Table 1).Histopathological findings in the joint fragments included in fibrosis (10 of 17) (Figure 1), associated with intense (3 of 17) or discrete (14 of 17) inflammation; one patient also had a periarticular inflammatory infiltrate (Figure 1).Four patients showed predominant synovitis, one had synovitis with the presence of histiocytes, and one patient presented fat necrosis (Figure 1).CHIKV E1 antigen was detected in 14 of 17 joint tissues, 57.14% with moderate staining (Figure 1).
All 17 fragments tested negative for viral RNA, as determined by both RT-PCR and RT-qPCR.
Table 2 displays the CDAI scores and average concentrations of various cytokines/chemokines measured in patient serum samples before and after biopsy.Notably, the levels of TNF-α, MCP-1, IP-10, IL-17, IL-4, and IL-2 were elevated in all patient samples, both pre-and post-biopsy.
Only one patient's post-biopsy sample showed increased levels of IL-10, which is an anti-inflammatory cytokine, and a second patient had post-biopsy increases in the levels of chemokine IL8.. Two patients showed increased levels of IL-12 before the biopsy.One of these patients had normal IL-12 levels after the biopsy.IFN-γ levels were increased in the serum of six patients, three pre-biopsy and three post-biopsy.One patient who had elevated IFN-γ levels prior to the biopsy had normal levels of this cytokine after the biopsy.
IFN-α was increased in the serum of four patients, only one patient had increased IFN-α pre-and postbiopsy, the same patient showed increased levels of IL-12 only before the biopsy.IL-6 and GM-CSF levels were within reference values in all patients.
Over 50% of CHIKV chronic patients attending to our service experienced ≥24 months of post-CHIKV chronic inflammatory joint disease.Bouquillard et al. reported 83.1% joint pain persistence over a 32-month follow-up but couldn't detect viral RNA using RT-PCR in some cases 14 .Similarly, we also failed to detect viral RNA in joint fragments using two molecular protocols.During the outpatient follow-up, we observed a gradual decrease in the number of affected joints, by the analog pain score, and the CDAI values.This was accompanied by the maintenance of C-reactive protein, ferritin, and erythrocyte sedimentation rate within normal limits.Additionally, we noted a decrease in the levels of inflammatory cytokines and chemokines, which corresponded with the patient's clinical improvement between the pre-and post-biopsy measurements (Table 2).
The collagen deposition observed in most joint samples in this study supports the notion that longterm intra-articular changes, induced by sustained inflammation, result in fibrosis.However, in our study, none of the patients tested positive for autoantibodies.
The epidemiological profile of these patients, predominantly middle-aged women with comorbidities, supports immunopathological studies indicating a reduction in viral clearance due to immune response dysregulation.This could partially account for the persistence of severe articular disease induced by CHIKV and the absence of E1 antigen clearance in the joints within this population 4 .

TABLE 2:
Clinical disease activity index (CDAI) and serum levels of cytokines and chemokines pre-and post-biopsy procedure in patients with chronic arthralgic disease induced by Chikungunya virus.The overrepresentation of female patients in our study could be attributed to their health-seeking behavior and the presence of risk factors for prolonged joint pain specific to this gender 15 .However, the study lacks supplementary data to elucidate the pathophysiological basis for the observed increase in chronic cases among females.

Patient
The chronic phase of the disease may be perpetuated by immune activation due to the persistent presence of viral RNA and E1 antigen in the synovial and muscle tissues.This results in the continuous production of inflammatory cytokines and chemokines 16 .Our study was unable to demonstrate whether the persistence of CHIKV RNA is due to viral replication or ineffective proinflammatory responses.While we detected persistent E1antigen in the joint tissues, we were unable to detect viral RNA.Chronic articular disease caused by the Chikungunya virus has been linked to elevated serum levels of MCP-1, IL-6, IL-10, IFN-α, and GM-CSF in several cohort studies 4 .In certain chronic patients, an increase in serum levels of IFN-α and IL-12 has been correlated with the persistence of the inflammatory process and disease progression 9 .Our patients exhibited significantly elevated serum levels of TNF-α, MCP-1, IP-10, IL-17, IL-4, and IL-2.The normal levels of GM-CSF and IL-6 observed in our study align with findings in chronic CHIKV cases, as increased circulating levels of this cytokine are typically present in acutely infected patients, with levels decreasing in individuals who have recovered 7 .
All our patients had increased levels of IFN-induced protein IP-10, with six of them also exhibiting heightened IFN-γ responses.However, IL-4 and IL-17, typically associated with Th2 and Th17 subtypes, were increased in the serum of all dosed patients.The introduction of immunomodulatory therapy during the patient's follow-up resulted in a decreased requirement for symptomatic drugs, including corticosteroids and NSAIDs.Our data revealed that, following a 30-month period, 43% of patients recovered from the disease, 35% exhibited mild disease activity, and 22% continued to display moderate disease activity.
The study had limitations due to a small number of patients undergoing joint biopsy and long-term evaluations.However, it revealed enduring inflammatory responses, intra-articular fibrosis signs, and viral E1 antigen persistence in patients with post-CHIKV articular complaints for ≥12 months.Elevated pro-inflammatory cytokine serum levels persisted even as CDAI indicated reduced pain complaints, over at least 29 months of follow-up.

ETHICS
Procedures involving human subjects and their samples received prior approval from the Health Ethics Council, Medicine School, Propeq/UFMT (CAEE 77593417.7.0000.8124approval 2.658.648).This study adhered to the National Health Council resolution 196/96.All patients were thoroughly briefed about the study procedures and subsequently provided their informed consent.

*
In years, when included in the study.** Duration of disease, in months, on the day biopsy was performed.*** Chikungunya E1 antigen detection by indirect immunofluorescence (IFI).**** fibrosis was defined by collagen deposition in the joint tissue.CDAI: clinical disease activity index; HBP: high blood pressure -hypertension; T2DM: diabetes mellitus type 2; NSAID: non-steroidal anti-inflammatory drugs.

*
When biopsy was collected.**Cytokines/chemokines dosed pre-biopsy (pre) and 24 months after biopsy in the last clinical evaluation (post).ND: not determined.de Brito MSG et al. • Pathophysiology of Chikungunya chronic disease