Abstract
Chikungunya virus (CHIKV) induces predominantly symptomatic infections, marked by fever, myalgia, rash and polyarthralgia that can last for up to 3 years after infection. Understanding the pathophysiology of CHIKV in the joints is challenging due to limited access to biological samples. Using a reporter virus expressing Nanoluciferase in a mouse model allowed us to monitor viral replication in real-time during acute and postacute phases. We showed viral replication in chondrocyte containing tissue in the metatarsi joints and confirmed with ex vivo analyses viral replication in leg bones and articular cartilages with histological evidence of focal erosive lesions and periarticular inflammation. Moreover, human chondrocytes prove susceptible to CHIKV infection, exhibiting viral production and bioluminescence activity. CHIKV induced apoptosis, the up-regulation of markers associated with cartilage remodeling and altered cytokine production. Our study provides insights into the ability of CHIKV to infect articular cartilages, shedding light on the mechanisms of alphaviral arthritis.
Keywords: chikungunya, bioluminescence, arthritis, chondrocyte, in vivo imaging
Polyarthralgia is a key feature of chikungunya virus (CHIKV) infection. Using a bioluminescent reporter virus, we show CHIKV replication in chondrocyte-containing tissues and articular cartilages. Human chondrocytes showed viral production, apoptosis, and altered cytokine profiles, elucidating alphaviral arthritis mechanisms.
Chikungunya virus (CHIKV) is an arthritogenic arbovirus belonging to the Togaviridae family, genus Alphavirus and responsible of incapacitating acute and chronic musculoskeletal disease [1].
Symptoms occur after an average incubation period of 3 days and usually consist of an abrupt onset of fever, followed by myalgia, polyarthralgia and macupapular rash. The arthralgia is often symmetrical and can affect any joint.
One remarkable feature of CHIKV infection is the proportion of patients presenting persistent symptoms [2]. Chronicity is characterized by muscle and joint pain in the wrists, ankles, knees, metacarpal and metatarsal joints lasting up to 3 years [3].
The pathophysiology of CHIKV infection remains poorly understood, one of the main reasons being the extreme scarcity of available biological samples. Only rare studies analyzed biopsy specimens of patients’ joint tissue [4, 5]. In the absence of joint samples, the use of animal models is necessary to understand this complex phenomenon.
Several animal models of alphaviral infection have been developed [6–15]. CHIKV, however, can cause severe and lasting disease, so models were also developed to investigate chronic infection [12, 16–19] as it occur in humans [7, 20, 21]. A study on partially immunodeficient mice [22] identified fibroblasts of the dermis, muscle and joint capsule, muscle satellite cells, and epithelial and endothelial cells of many organs, such as liver, brain, and spleen, as cellular targets during the acute phase. In a simian model, liver endothelial cells and macrophages have been found immunoreactive for CHIKV antigens during the chronic stage [23].
Little is known about the cellular targets of CHIKV in humans [24]. In vitro, human cell lines such as epithelial, fibroblastic and some endothelial cells were found susceptible to CHIKV infection as well as primary cells, such as macrophages, keratinocytes, synovial and dermal fibroblasts, osteoblasts, chondrocytes, and myoblasts [14–16]. The available in vivo data are very scarce, except for the demonstration of fibroblast infection in the dermis, joint capsule, and muscle facia from a single fatal neonatal case [22] and satellite muscle cells in biopsy specimens from 2 adult patients with myositis [16].
Very few studies have focused on cellular targets and infection characteristics in the joint. In murine models, only 2 studies have shown the presence of viral antigens and/or viral RNAs in the joint during the acute phase and viral RNA, without infectious particles, during the chronic phase [9, 11, 17, 25]. In infected patients with chronic arthritis, viral antigens could be detected in cells from joint tissue, without cell type characterization [4], and in synovial macrophages [5].
The objective of the current study was to characterize the mechanisms of transition to chronic arthritis related to CHIKV infection, using a published mouse model [6] and a bioluminescent reporter virus. Our hypothesis was that viral persistence takes place within a specific tissue, the articular cartilage. For this, we used a new tool generated in our laboratory, a reporter virus expressing Nanoluciferase (NLuc) to develop an in vivo imaging mouse model of acute and chronic CHIKV infection. Our aim was to precisely localize the sites of replication and persistence of the virus. Using this approach, we showed that metatarsi joints harbored viral replication after the end of the acute phase, and we identified chondrocytes as the major target of infection.
MATERIAL AND METHODS
Design of the Recombinant CHIKV–NLuc Virus
An NLuc gene was inserted in frame in a CHIKV infectious clone (based on the BNI-CHIKV 899 strain; accession no. FJ959103.1) [18] using a previously described strategy [19, 21]. RNA was in vitro transcribed (mMESSAGE mMACHINE Kit; Ambion) and electroporated into Vero E6 cells. Supernatants were collected 48 hours post inoculation (hpi) and used for production of viral stocks. The resulting virus was named CHIKV-NLuc; the reporter is expressed as part of the nonstructural polyprotein precursor and is cleaved by the viral non-structural protein 2 (nsP2) protease during the replication cycle (GMO agreement 2454 from the Ministère chargé de l'Enseignement et de la Recherche (MESR)). In some experiments, the wild-type CHIKV strain was used (CHIK 05-049; accession no. AM258994.1).
Quantification of NLuc Activity
Cells or tissues were lysed with Passive Lysis Buffer (Promega) before centrifugation and ×10 dilution in phosphate-buffered saline (PBS). Each sample was mixed volume to volume with a 1% furimazine solution before quantification luminescence with a Centro LB 960 luminometer (Berthold Technologies), expressed in relative luminescence units.
Assessment of CHIKV-NLuc Stability
Vero E6 cells were seeded and infected with CHIKV-NLuc at a multiplicity of infection (MOI) of 1. At 24 hpi, 100 µL of supernatant was used to infect new cells. The remainder of the supernatant was retained to assess the infectious titer. The cells were washed once with PBS, and quantification of NLuc activity was performed on the cell lysates. This operation was repeated for a total of 10 successive passages.
Cells
Vero E6 cells (American Type Culture Collection CRL-1586) were used for viral production and titration. Human chondrocytes (HCs; reference PB-402-05a) were cultured according to supplier's instructions (Cell Applications). The cells were incubated at 37°C and 5% carbon dioxide.
In Vitro Cell Infection
Aliquots of virus were diluted to obtain the targeted MOIs (0.1, 1, and 10) for inoculation on confluent cell monolayers. After an hour, the inoculum was removed and the cells washed before incubation until collection.
Viral Titration
Plaque assay titrations were performed on Vero E6 cells. Confluent monolayers of Vero E6 cells were seeded and inoculated with 1:10 serial dilutions of samples. After an hour of incubation, unadsorbed virus was removed, and Dulbecco modified Eagle's medium (DMEM) supplemented with 1.6% carboxymethyl cellulose and 2% fetal calf serum (FCS) was added. The supernatant was removed after 2 days, and the cells were washed and stained with cristal violet solution (PBS with 4% paraformaldehyde and 0.2% crystal violet) to visualize the plaques. The results were expressed as plaque-forming units (PFUs) per milliliter.
Mice
Animal experiments were approved by the Ethics Committee of the Institut Pasteur and authorized by the French Ministry of Higher Education and Research (reference CETEA 0762.02). The animal manipulations involving wild-type strains of CHIKV were performed on 4-week-old female C57Bl/6 mice (Charles River). To limit the absorption of the bioluminescent signal, 4-week-old albino female C57Bl/6N (B6N-TyrC-Brd/BrdCrCrl) mice (Charles River) were used for CHIKV-NLuc experiments.
Mice were anesthetized with an intraperitoneal ketamine and xylazine solution and inoculated subcutaneously with CHIKV (infectious dose of 103–106 PFUs) or vehicle only (Leibovitz L-15 medium; “control” condition) into the footpad of the right paw. The mice were observed daily, and weight, clinical signs, plantar edema, viremia, and blood parameters were assessed.
Blood samples were obtained by puncture at the distal end of the tail (volume strictly inferior to 30 µL). Larger blood volumes were obtained via intracardiac punctures during terminal sampling. For tissue sampling, mice were anesthetized and euthanized by intracardiac infusion of PBS. Samples intended for primary cell isolation or bioluminescent signal quantification were placed in PBS and kept at 4°C. Soft tissues (skin and muscle) were removed by dissection to expose bones and joints. The cartilages of the hip, knee, and metatarsi were isolated by dissection under a binocular loupe. For ex vivo bioluminescence quantification, the joints were placed in passive lysis buffer (Promega) and mechanically ground with ceramic beads (Precellys; Bertin Technologies).
Histopathology
After mice were euthanized, their legs were removed, fixed for 48–72 hours in 10% neutral buffered formalin, and embedded in paraffin; 4-μm-thick sections were obtained and stained with hematoxylin-eosin or safranin. Sections were analyzed by a trained veterinary pathologist using separate blinded scoring.
Viral Replication Assessment
Total RNA were extracted (Nucleospin RNA II; Macherey-Nagel) and stored at −80°C before analysis. Reverse-transcription quantitative polymerase chain reaction was performed using the Power SYBR Green RNA-to-CT 1-Step Kit (Applied Biosystems). Relative quantification was used for cellular RNAs, using the GAPDH housekeeping gene as reference. Viral RNA copy numbers were assessed using previously described primers [24] and dilutions of synthetic CHIKV RNA as standard. The signal was normalized relatively to the standard curve. For analysis of the change in cycle threshold (ΔCt), the normalized data were used to estimate the RNA copy number in each well.
Immunofluorescence
Cells were grown on coverslips, infected with different CHIKV MOIs (0.1, 1, and 10), fixed at different times with a 4% paraformaldehyde solution and permeabilized with 0.1% Triton X-100 solution. The cells were washed and incubated with the 3E4 recombinant anti-CHIKV E2 murine antibody (Creative Biolabs). When indicated, an anticollagen type II rabbit antibody (Novus Biologicals) was used. Anti-mouse and anti-rabbit immunoglobulin G, coupled with Cy5 and AlexaFluor 488 dyes, respectively, were used as secondary antibodies. The coverslips were mounted with ProLong gold antifade reagent (Life Technologies) with 4′,6-diamidino-2-phenylindole (DAPI) and were examined using a fluorescence microscope (EVOS; Thermo Fisher Scientific).
TUNEL Apoptotic and Caspase 3/7 Activity Assay
HCs were infected with CHIKV (MOIs of 1 and 10). Cells were processed at different times after inoculation to determine caspase 3/7 activity (Caspase-Glo 3/7 Assay kit; Promega). Luminescence was analyzed with a Centro LB 960 luminometer (Berthold Technologies) and expressed in relative luminescence units.
A nick-end labeling TUNEL (terminal deoxynucleotidal transferase–mediated biotin–deoxyuridine triphosphate nick-end labeling) assay was performed, using the In Situ Cell Death Detection Kit, Fluorescein (Roche). HCs were seeded on coverslips, infected with CHIKV (MOIs of 1 and 10) and fixed at 24 and 48 hpi with 4% paraformaldehyde solution. Endogenous peroxidase activity was blocked using a 3% hydrogen peroxide solution in methanol. Cells were then permeabilized with a 0.1% Triton-X and 0.1% sodium citrate solution. Negative labeling controls (without terminal deoxynucleotidal transferase) and mock-infected controls were included. Samples were labeled with the TUNEL reaction mixture before analysis. Coverslips were mounted with ProLong gold antifade reagent with DAPI and examined using a fluorescence microscope (EVOS Thermo Fisher Scientific).
Cytokine Relative Quantification in the Supernatant
Supernatants from infected and uninfected primary cells were collected and centrifuged. The relative levels of cytokines were assessed using the Proteome Profiler Human XLCytokine Array kit (R&D Systems). Membranes were analyzed using myECLImager software 1.0, and bioluminescence quantified with myImageAnalysis software, version 1.1 (Thermo Fisher Scientific). Signal from infected cells’ supernatants were normalized to negative controls and expressed as fold change.
In Vivo Imaging
Mice inoculated with virus or diluent alone were anesthetized and injected with 4 mg/kg of furimazine. In vivo imaging was performed using the IVIS Spectrum system, and the data obtained were analyzed using Living Image 4.5 software (PerkinElmer). Regions of interest of identical area and shape were manually defined to quantify bioluminescence. The results were expressed as total flux (photons per second per square centimeter per steradian).
Isolation of Primary Murine Chondrocytes
Cartilages isolated under binocular microscope were dilacerated using a sterile scalpel, then incubated in a 0.5-mg/mL trypsin solution (Sigma-Aldrich) under agitation at 37°C for 30 minutes. Following centrifugation, the solution was removed and replaced with 0.1% collagenase II in DMEM with 5% FCS, and samples were incubated for 4 hours at 37°C with agitation. Samples were placed on 40-µm filters (Cellstrainer; Thermo Fisher Scientific) and centrifuged at 500g for 5 minutes. The isolated cells were resuspended in DMEM supplemented with 5% FCS, immediately plated on glass coverslips using Cytofunnel (Thermo Fisher Scientific) and centrifuged at 1500g for 10 minutes with a Cytospin 4 (Thermo Fisher Scientific) before fixation with a 4% paraformaldehyde solution and labeling with anti-CHIKV 3E4 and anticollagen type II antibodies.
Statistical Analyses
Statistical analyses were performed using Prism software, version 6. Spearman correlation tests were used to investigate relationships between variables. Nonparametric Mann-Whitney tests and 2-way analysis of variance followed by Bonferroni correction were used to test for differences between groups. Differences were considered significant at P < .05.
RESULTS
Assessment of CHIKV-NLuc Stability
We first explored the stability of the viral construction by serial infections and assessment of the viral titer and luciferase activity. As shown in Figure 1, the bioluminescent signal is remarkably stable for 10 serial passages, with however a 1-log decrease in the luciferase activity at the last passage. Nevertheless, we observe a strong positive correlation between the bioluminescence and the viral titer (Spearman correlation test, r = 0.93; P < .001), indicating that CHIKV-NLuc is stable and can be used to monitor the infection.
Figure 1.
Generation of a chikungunya virus (CHIKV) expressing NanoLuc luciferase (NLuc). A, Genome organization of the recombinant CHIKV-NLuc. The NLuc gene was inserted in frame between the non-structural protein (nsP) 3 and nsp4 genes; arrows represent nsP2 cleavage sites. B, Stability of the recombinant CHIKV-NLuc. Vero E6 cells were serially infected for 10 passages with CHIKV-NLuc to assess the stability of the virus. At each passage, the viral titer of the supernatant was measured (curve), as well as the luciferase activity (bars), expressed as relative luminescence units (RLUs) in the infected cells. Curves and histograms show the median value and interquartile range of measurements from 4 replicates. Abbreviation: PFUs, plaque-forming units.
Visualization of CHIKV-NLuc Replication Sites During Infection in an Immunocompetent Mouse Model
We adapted the immunocompetent mouse model of infection previously described by Gardner et al [6], using the recombinant CHIKV-NLuc virus. Albino C57Bl/6 mice were subcutaneously infected in the footpad, and viral replication was assessed at different times. We reproduced the edema induced by CHIKV infection, quantified viremia as well as infectious particles in kidneys, and inoculated legs (Supplementary Figure 1). Moreover, we detected infectious viral particles in the blood (averaging 1000 PFUs/mL) at 3 days post inoculation (dpi).
As shown in Figure 2, a bioluminescent signal was detected in the inoculated footpad, as early as 1 dpi. During the acute phase, the signal increased until 5 dpi, extended from the paw to the hip joint, and gradually decreased at 7–9 dpi. Importantly, the bioluminescent signal was found throughout the body of infected mice, demonstrating the systemic nature of the infection in our model (Supplementary Figure 2). A weak signal persisted in the inoculated footpad during the postacute phase (12 and 15 dpi). Interestingly, a bioluminescent signal could still be observed at 34 dpi in 1 of 4 mice during the chronic phase (white arrow in Figure 2C). The quantified bioluminescent signal in the inoculated paw remained significantly higher in infected versus uninfected mice until 7 dpi (Figure 3).
Figure 2.
Plantar pad infections with chikungunya virus–NanoLuc luciferase (CHIKV-NLuc) generate sufficient bioluminescent signal for monitoring. Mice were subcutaneously inoculated into the plantar pad of the right hind paw with the L15 medium alone (control, on the top) or CHIKV-NLuc (103–106 plaque-forming units (PFUs) per mouse). Bioluminescent signal intensity is associated with a color code according to the scales provided to the left of the images; the dorsal view is presented here. A, Mice at 1, 3, 5, 7, and 9 days post inoculation (dpi; scale, 106–2.107 photons per second per square centimeter per steradian [p/s/cm2/sr]). B, Mice at 12 and 15 dpi (scale, 104–105 p/s/cm2/sr). C, Mice at 34 dpi; significant signal in the footpad of 1 mouse is indicated with a white arrow (scale, 103–104 p/s/cm2/sr). Data shown are representative of 2 experiments. Abbreviations: Max, maximum; Min, minimum.
Figure 3.
Assessment of bioluminescent signal in mice infected with chikungunya virus–NanoLuc luciferase virus (CHIKV-NLuc), in the whole body (A), inoculated paw (B), and contralateral paw (C) after subcutaneous inoculation (footpad) with 106 (dots), 105 (squares), 104 (upward-facing triangle), or 103 (downward-facing triangle) plaque-forming units (PFUs) of CHIKV-NLuc or noninfected control (rhombuses). Mice were imaged, regions of interest of the same area and location were defined, the signal was quantified at 1, 3, 5, 9, 12, 15, and 34 days post inoculation and expressed as total flux in photons per second (y-axis). Each point represents the value for a single mouse, and medians and interquartile ranges of measurements from 3 mice are also depicted. NS, not significant; *P < .05; **P < .01; ***P < .001 (2-way analysis of variance).
The bioluminescent signal observed in Figure 2 indicated viral replication in the ankle/metatarsus joints. To increase the specificity of the measure, ex vivo signal quantifications were performed. Leg bones of uninfected and infected animals (12 dpi) were isolated. Bioluminescence imaging revealed a significant signal in the leg of the infected animal (Figure 4).
Figure 4.
Ex vivo visualization and measurement of luciferase activity and infection in the leg bone and the articular cartilage of the ankle/metatarsi, knees and hips of infected mice. Mice were inoculated into the footpad of the right hind leg with chikungunya virus–NanoLuc luciferase (CHIKV-NLuc; 105 plaque-forming units [PFUs]) or the diluent alone (control). A, Leg bones of control and infected animals were dissected at 12 days post inoculation (dpi) and analyzed with bioluminescence imaging. Left, Infected mouse. Right, Control. Abbreviation: p/s/cm2/sr, photons per second per square centimeter per steradian. B, With use of a binocular magnifying glass, articular cartilages (hip, knee, and ankle/metatarsus) were isolated at 6, 12 and 30 dpi, and luciferase activity was quantified. Each point represents the luciferase activity of a cartilage normalized to uninfected controls and measured in 2 independent experiments; medians and interquartile ranges are also shown. Abbreviations: NS, not significant; RLU, relative luminescence unit. *P < .05; **P < .01; ***P < .001 (nonparametric Mann-Whitney test). C, D, Mice were euthanized at 6 dpi (C) and 30 dpi (D), and cells were isolated from metatarsal cartilage. Isolated cells were immediately fixed and stained. First line (blue) indicates DAPI; second line (green) collagen type II; and third line (red) CHIKV E2. Arrows indicate cells with double labeling of type II collagen and viral E2 protein, as shown in the merge image on the bottom right-hand corner. Scale bars represent 50 µm (C) and 20 µm (D).
To enable quantification, mice were euthanized at 6, 12, and 30 dpi and perfused with PBS to remove blood from tissues. With use of a binocular magnifier, articular cartilages from hips, knees and ankles/metatarsis were dissected and grinded. The NLuc activity was quantified and expressed as fold change relative to uninfected samples. The luciferase activity was significantly higher at 6 dpi in the cartilages of infected mice (up to 103 fold increase), indicating viral replication in the articular cartilage. Interestingly, while the knee and hip cartilages from infected mice show no difference compared with controls, those from the ankle displayed a significantly higher signal than uninfected cartilages.
Histological analysis revealed focal erosive lesions at 6 dpi in the cartilage of the metatarsophalangeal joints, indicating limited destruction of this tissue (Supplementary Figure 3). Interestingly, an inflammatory infiltrate of the periarticular connective tissue and the joint capsule was visible in the metatarsophalangeal joint. Persistent periarticular inflammation was visible at 12 dpi without marked destruction or regeneration of articular cartilage and mild periarticular inflammation was still visible at 30 dpi.
In Vivo Infection of Murine Chondrocytes
The in vivo bioluminescence imaging suggested active replication of CHIKV-NLuc in the articular cartilages. To characterize the infected cell type, infected mice were euthanized at 6 or 30 dpi, and metatarsal cartilages were dissected, isolated, and dissociated to isolate resident cartilage cells. To avoid postisolation infection, cells were immediately fixed with 4% PFA solution, and immunoreactivity to CHIKV E2 protein and type II collagen (chondrocyte marker) was assessed. At 6 dpi, most of the CHIKV E2–positive cells also showed immunoreactivity for type II collagen (Figure 4C). Interestingly, a limited number of infected chondrocytes (E2 and collagen II immunoreactive) could still be detected as late as 30 dpi in the metatarsal articular cartilage (Figure 4D).
HC Susceptibility to CHIKV
To investigate the susceptibility of primary HCs (pHCs) to CHIKV, we performed in vitro infections with CHIKV 05-049 strain or CHIKV-NLuc. Viral RNA quantity reached a plateau between 12 and 24 hpi (Figure 5), indicating active replication. Bioluminescence activity was observed in CHIKV-NLuc–infected pHCs, following similar kinetics as the wild-type strain. Moreover, plaque assay titration confirmed that infected pHCs produced infectious viral particles. Finally, CHIKV infection was also confirmed by fluorescence microscopy.
Figure 5.
Infection of primary human chondrocytes (pHCs) with chikungunya virus (CHIKV). A, Assessment of viral RNA levels in wild-type CHIKV–infected pHCs. Viral RNA was quantified in the cells lysates using reverse-transcription quantitative polymerase chain reaction after infection at 3 multiplicities of infection (MOIs). B, Bioluminescence activity, expressed in relative luminescence units (RLUs), after infection with CHIKV–NanoLuc luciferase (CHIKV-NLuc), at 3 MOIs. C, Numbers of infectious virus particles in the supernatant of CHIKV-infected pHCs, assessed by plaque assay. Curves show the median value and interquartile range obtained from 3 independent experiments in triplicate. Abbreviation: PFUs, plaque-forming units. D, In vitro cultured pHCs were inoculated with CHIKV (infected cells [right]; MOI, 1) or vehicle (uninfected cells [left]) and observed with fluorescence microscopy (scale bars, 200 μm).
Induction of Apoptosis and Cytokine Production by CHIKV Infection of pHCs
To explore the effect of CHIKV on the physiology of chondrocytes, we monitored the change in cytokine production after infection. Cytokines were quantified at 6 hpi using a broad-spectrum immunoenzymatic assay. Of the 102 cytokines analyzed, 8 were found at higher and 8 at lower levels than the control cells (Table 1).
Table 1.
Effect of Chikungunya Virus Infection of Primary Human Chondrocytes in Cytokine Production a
| Effect | Cytokine | Fold Change (Compared With Mock Infection) |
|---|---|---|
| Up-regulation | Serpin E1 (PAI1) | 13.8 |
| Chitinase 3–like 1 | 9 | |
| IGFBP-3 | 4.6 | |
| Complement factor D | 3.2 | |
| FGF-19 | 3 | |
| CD30 | 2.3 | |
| IL-6 | 2.1 | |
| EMMPRIN (CD147 or basigin) | 2.1 | |
| Down-regulation | TGF-α | −2.2 |
| TNF-α | −2.2 | |
| TfR | −2.3 | |
| VEGF | −2.6 | |
| uPAR | −3 | |
| IL-8 | −6.2 | |
| MCP-1 | −6.7 | |
| Thrombospondin-1 | −9.6 |
Abbreviations: EMMPRIN, extracellular matrix metalloproteinase inducer; FGF, fibroblast growth factor; IGFBP, insulin-like growth factor binding protein; IL-6, interleukin 6; IL-8, interleukin 8; MCP, monocyte chemoattractant protein; TfR, transferrin receptor; TGF, transforming growth factor; TNF, tumor necrosis factor; uPAR, urokinase plasminogen activator surface receptor; VEGF, vascular endothelial growth factor.
aSupernatants from chikungunya virus–infected and uninfected human chondrocytes were analyzed using a broad-spectrum enzyme-linked immunosorbent assay (Proteome Profiler Human XL Cytokine array kit; R&D Systems), 6 hours after inoculation from 1 experiment, in triplicate. The cytokines shown here displayed a minimum 2-fold change.
Some of the positively regulated cytokines are involved in cartilage remodeling (chitinase 3, CD147, or extracellular matrix metalloproteinase inducer [EMMPRIN]) or apoptosis (CD30, and insulin-like growth factor binding protein [IGFBP] 3). Since CD147 is known to activate matrix metalloproteases (MMPs), we performed an RNA quantification of 2 MMPs involved in cartilage catabolism (MMP-3 and MMP-9). The RNA levels of both MMPs were higher in infected cells than in controls (Figure 6). As IGFBP-3 has been reported to induce chondrocyte apoptosis, we also investigated this feature and showed that infected cells underwent significant morphological changes and destruction of the cell layer increased massively between 24 and 72 hours.
Figure 6.
Virus-induced alterations in human chondrocyte (HC) biology. A, Increased matrix metalloprotease (MMP) 3 and MMP-9 messenger RNA (mRNA) synthesis after chikungunya virus (CHIKV) infection of HCs. HCs were infected with CHIKV (multiplicity of infection [MOI], 1). At 48 hours post inoculation, MMP-3 and MMP-9 mRNAs were assessed by means of reverse-transcription quantitative polymerase chain reaction. The amount of mRNA is expressed relative to uninfected controls, after normalization against a housekeeping gene (GAPDH). Histograms show the median value and interquartile range obtained in 3 independent experiments performed in triplicate. **P < .01 (nonparametric Mann-Whitney test). B, TUNEL (terminal deoxynucleotidal transferase–mediated biotin–deoxyuridine triphosphate nick-end labeling) labeling was performed, and TUNEL-positive cells were visualized using a fluorescence microscope. Displayed are the average numbers of TUNEL-positive cells over 3 randomly observed fields (as percentages of the total). ***P < .001 (nonparametric Mann-Whitney test). C, Caspase 3 and 7 activity following CHIKV infection of primary HCs (pHCs) (multiplicity of infection, 1). At different times after infection, the cell monolayer was lysed, and caspase 3 and 7 activity was measured (Caspase Glo 3/7 Assay Kit; Promega); the bioluminescent signal is expressed in relative light units (RLUs). Curves show the median value and interquartile range obtained in 2 independent experiments performed in triplicate.
In situ cell death was confirmed by labeling with the TUNEL technique. At 48 hpi, 16.1% of the cells show positive TUNEL labeling. To further investigate the cell death mechanism involved, we measured the caspase 3/7 activity and observed an important activation after infection.
DISCUSSION
The initial stage of our work involved adapting the adult murine model developed in 2010 by Gardner et al [6] to our own experimental conditions, using a new recombinant virus expressing NLuc. The bioluminescence live imaging approach has been shown valuable for studies of experimental CHIKV infection [20] and other arthritogenic alphaviruses, such as Ross River and Sindbis viruses [19, 26]. Using the recombinant CHIKV-NLuc, we were able to observe a bioluminescent signal in vivo during the acute phase, which persisted beyond, suggesting that complete replicating virus remained in the paws, in line with previous observations of Teo et al [20]. Over time, the signal was localized mainly in the metatarsi, similarly to observations in humans showing a preferential impairment of distal joints [27, 28].
Moreover, we identified CHIKV-infected tissues within the joints after isolation of cartilage from mice infected with the wild-type virus. We were able to isolate infected cells displaying dual immunoreactivity for the viral protein E2 and the chondrocyte marker type II collagen. We also identified metatarsal joints as the main site of infection as late as 30 dpi, but this might be a consequence of the inoculation site. While other arthritogenic alphaviruses, such as Ross River virus, have previously been shown to infect chondrocytes in vivo [29, 30], to the best of our knowledge, our findings provide, the first evidence of in vivo CHIKV infection within chondrocyte-containing tissue. We acknowledge the limitations of our current model, however, as high-dose footpad inoculation may not accurately represent natural infection routes. Therefore, we recommend additional investigations using alternative methods and diverse animal models to validate and expand on these observations.
We also acknowledge that our model, despite demonstrating in vitro stability and detectable bioluminescent signals in vivo, may have limitations for studying long-term infections. This is due to the potential selection pressure favoring viral genomes with enhanced fitness, which may include variants that do not express NLuc. Next, we showed, using an in vitro model of pHCs isolated from 2 healthy patients, that CHIKV is able to replicate efficiently in HCs.
As previous studies have suggested that Ross River virus–infected chondrocytes may serve as a significant source of inflammatory cytokines, potentially contributing to the onset and persistence of arthritis [31], we also assessed cytokines production and observed significant changes in several cytokines’ secretion levels at 6 hpi. Interestingly 7 overexpressed cytokines are found at high levels in patients with joint pathology [32–37], with possible involvement in CHIKV pathogenesis. E1 serpin, chitinase 3–like 1, IGFBP-3, complement factor D, CD30, interleukin 6, and EMMPRIN are highly expressed cytokines, potentially contributing to tissue degradation and immune response activation and indicating a disruption in cartilage metabolism regulation, favoring catabolism. We further explored the effect of CHIKV infection on HCs and showed an activation of matrix metalloproteinases, in favor of cartilage breakdown. Finally, we showed CHIKV-induced apoptosis in chondrocytes, confirming the effect of factors like IGFBP-3 on cell physiology after infection.
In vitro observations and especially the rapid cell death following chondrocytes infection do not align with in vivo outcomes, hinting at potential protective factors within the joint environment. One could be the cartilage's relative paucity in cells, which could limit viral replication, contrary to the replication observed in vitro. While in vitro observations provide valuable insights, the complex in vivo environment may also involve paracrine regulatory mechanisms, highlighting the intricate dynamics within the joint.
In conclusion, our study provides compelling evidence for CHIKV infection of chondrocytes both in vitro and in vivo, revealing potential mechanisms of joint pathology and highlighting the complex interplay between viral infection, host response, and the joint microenvironment in CHIKV-induced arthritis, while also emphasizing the need for further research to fully elucidate the long-term consequences of CHIKV infection on joint health.
Supplementary Material
Contributor Information
Vincent Legros, Unité Epidémiologie et Physiopathologie des Virus Oncogènes, Institut Pasteur, Université Paris Cité, UMR CNRS 3569, Paris, France; Unité Recherche et Expertise Environnement et Risques Infectieux, Institut Pasteur, Université Paris Cité, Paris, France.
Essia Belarbi, Unité Recherche et Expertise Environnement et Risques Infectieux, Institut Pasteur, Université Paris Cité, Paris, France; Center for Immunology of Viral, Autoimmune, Hematological and Bacterial Diseases (IMVA-HB/IDMIT), Université Paris-Saclay, INSERM, CEA, Fontenay-aux-Roses, France.
Patricia Jeannin, Unité Epidémiologie et Physiopathologie des Virus Oncogènes, Institut Pasteur, Université Paris Cité, UMR CNRS 3569, Paris, France.
Virginie Geolier, Unité Recherche et Expertise Environnement et Risques Infectieux, Institut Pasteur, Université Paris Cité, Paris, France.
Beate M Kümmerer, Institute of Virology, Medical Faculty, University of Bonn, Bonn, Germany; German Centre for Infection Research, Partner Site Bonn-Cologne, Bonn, Germany.
David Hardy, Plate-Forme d’Histopathologie, Institut Pasteur, Université Paris Cité, Paris, France.
Philippe Desprès, Processus Infectieux en Milieu Insulaire Tropical, Université de La Réunion, INSERM U1187, CNRS 9192, IRD 249, Plateforme Technologique CYROI, Sainte-Clotilde, La Réunion, France.
Antoine Gessain, Unité Epidémiologie et Physiopathologie des Virus Oncogènes, Institut Pasteur, Université Paris Cité, UMR CNRS 3569, Paris, France.
Pierre Roques, Center for Immunology of Viral, Autoimmune, Hematological and Bacterial Diseases (IMVA-HB/IDMIT), Université Paris-Saclay, INSERM, CEA, Fontenay-aux-Roses, France; Unité de Virologie, Institut Pasteur de Guinée, Conakry, Guinea.
Pierre-Emmanuel Ceccaldi, Unité Epidémiologie et Physiopathologie des Virus Oncogènes, Institut Pasteur, Université Paris Cité, UMR CNRS 3569, Paris, France.
Valérie Choumet, Unité Recherche et Expertise Environnement et Risques Infectieux, Institut Pasteur, Université Paris Cité, Paris, France.
Supplementary Data
Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org/). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.
Notes
Disclaimer. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.
Financial support. This work was supported by Region "Ile-de-France" (grant DIM-MALINF 130053), Agence Nationale de la Recherche (Projet Kerarbo ANR-12-BSV3-0004; Programme d’Investissements d’Avenir ANR-11-INBS-0008 and ANR-10-EQPX-02-01), and the Fondation Pour la Recherche Médicale (fellowship to V. L.).
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