key: cord-0269379-ianswmwu
authors: Bekaddour, Nassima; Smith, Nikaïa; Beitz, Benoit; Llibre, Alba; Dott, Tom; Baudry, Anne; Korganow, Anne-Sophie; Nisole, Sébastien; Mouy, Richard; Breton, Sylvain; Bader-Meunier, Brigitte; Duffy, Darragh; Terrier, Benjamin; Schneider, Benoit; Quartier, Pierre; Rodero, Mathieu P; Herbeuval, Jean-Philippe
title: Targeting the chemokine receptor CXCR4 with histamine analogue to reduce inflammation in juvenile arthritis: a proof of concept for COVID-19 therapeutic approach
date: 2021-10-26
journal: bioRxiv
DOI: 10.1101/2021.10.24.465080
sha: 31657ac24422d0a174540fcc1103b44814a81272
doc_id: 269379
cord_uid: ianswmwu

Among immune cells, activated monocytes play a detrimental role in chronic and viral-induced inflammatory pathologies. The uncontrolled activation of monocytes and the subsequent excessive production of inflammatory factors damage bone-cartilage joints in Juvenile Idiopathic Arthritis (JIA), a childhood rheumatoid arthritis (RA) disease. Inflammatory monocytes also exert a critical role in the cytokine storm induced by SARS-CoV2 infection in severe COVID-19 patients. The moderate beneficial effect of current therapies and clinical trials highlights the need of alternative strategies targeting monocytes to treat RA and COVID-19 pathologies. Here, we show that targeting CXCR4 with small amino compound such as the histamine analogue clobenpropit (CB) inhibits spontaneous and induced-production of a set of key inflammatory cytokines by monocytes isolated from blood and synovial fluids of JIA patients. Moreover, daily intraperitoneal CB treatment of arthritic mice results in significant decrease in circulating inflammatory cytokine levels, immune cell infiltrates, joints erosion, and bone resorption leading to reduction of disease progression. Finally, we provide the prime evidence that the exposure of whole blood from hospitalized COVID-19 patients to CB significantly reduces levels of key cytokine-storm-associated factors including TNF-α, IL-6 and IL-1β. These overall data show that targeting CXCR4 with CB-like molecules may represent a promising therapeutic option for chronic and viral-induced inflammatory diseases.

Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with aggressive synovial hyperplasia causing destruction of articular joints. Joint inflammation is characterized by proliferation of macrophage-like [1] and fibroblast-like synoviocytes to form a pannus, which invades and destroys the cartilage [2] . Juvenile Idiopathic Arthritis (JIA) is a rare and complex form of RA occurring in children under the age of 16, characterized by a multifactorial disorder with heterogeneous manifestations that include all forms of chronic arthritis [3, 4] .

Over-production of TNF-α, IL-1β and IL-6 is strongly involved in most forms of JIA [5] [6] .

The release of such inflammatory factors by monocytes depends on several highly conserved families of pattern recognition receptors (PRR), one of them being the Toll-like receptor family (TLRs). The increase in circulating TNF-α levels in all forms of JIA argues for a major contribution of monocytes to disease progression [7] . The role of monocytes in JIA has been further highlighted in a work showing that stimulation of TLR-4 and TLR-8 in monocytes isolated from the blood of systemic JIA patients increased the proinflammatory responses compared to healthy donors [8] . Current antirheumatic drugs, including corticosteroids and antibodies-based biotherapies, target inflammatory macrophages to reduce synovial inflammation. However, not all patients respond to antibody therapy and chronic application of glucocorticoids leads to severe side effects, highlighting the need for novel therapeutic strategies.

Monocytes are also suspected to play a deleterious role during acute inflammation upon SARS-CoV-2 infection responsible for Coronavirus disease 2019 . Indeed, SARS-CoV2 infects the upper and lower respiratory tracts and causes mild or highly acute respiratory syndrome with consequent release of proinflammatory cytokines by hyper-activated monocytes [9, 10] . Recently, we measured over-production of proinflammatory cytokines in COVID-19 patients causing cytokine storm in lungs [10] . Furthermore, high levels of IL-6 and TNF- in bronchoalveolar lavage and plasma of those patients corelate with the severity of the pathology [10] . This SARS-CoV2 pandemic stressed the scientific and clinical communities to urgently develop novel anti-inflammatory strategies.

In the context of chronic and acute inflammatory diseases, the chemokine receptor CXCR4 could emerge a potential therapeutic target. We previously showed that histamine and the histamine analogue clobenpropit (CB) through their engagement of CXCR4 exhibited a broadspectrum inhibitory activity on the production of all subtypes of interferons (IFN) in TLR7activated human plasmacytoid dendritic cells (pDCs) [11] . Moreover, intranasal spray of CB resulted in drastic reduction of type I and III IFN secretions in broncho-alveolar lavages from Influenza A virus (IAV) infected mice [11] . Of note, the anti-IFN activity of CB was not mediated by histamine receptors but strictly dependent on CXCR4 [11] . As CXCR4 is highly expressed by all immune cells, including monocytes and macrophages, we hypothesize that CB could also downmodulate monocyte-driven inflammation in rheumatoid arthritis and SARS-CoV2 infection.

Here, we assessed whether CB tones down the spontaneous and induced production of proinflammatory cytokines by monocytes isolated from blood and synovial fluids from JIA patients. We further probed in vivo whether CB treatment would attenuate inflammation and impair disease progression in a model of collagen-induced arthritic mice. Finally, we tested the effect of CB on the production of inflammatory cytokines in whole blood from hospitalized COVID-19 patients.

To first assess the potential anti-inflammatory properties of histamine and the histamine analogue CB, we took advantage of the monocyte-derived THP-1 NF-κB reporter cell line.

Upon TLR7/8 activation by R848, the reporter gene SEAP is induced by activation of the NF-κB signaling pathway. We thus measured the production of SEAP by R848-activated THP-1 cells in the presence or absence of increasing concentration of histamine (Fig. 1A) or CB (Fig.   1B ) ranging from 1 to 100 µM. We showed that CB, and to a lesser extent histamine, reduced in a dose-dependent manner activation of NF-κB in THP-1 cells, without any obvious toxicity.

In addition, CB treatment (20 µM) prevented the transcription of TNF-α, IL-1β and IL-6 encoding genes in R848-activated THP-1 cells (Fig. 1C) .

To extend our results obtained with THP-1 cell line, we next tested the ability of CB to control cytokine production in R848-stimulated human blood mononuclear cells (PBMCs) from healthy donors (HD). We first tested the overall effect of CB by measuring the concentrations of multiple cytokines (i.e. IL-1β, -6, -8, -10, TNF-α…), chemokines (i.e. CCL2, CCL3, CCL4, CCL5…), growth factors (i.e. EGF, FGF, VEGF…) and interferons (IFNα/β/γ) in the cell culture medium of R848-stimulated PBMCs in presence or absence of CB (20 µM) (n=5) (Fig.   1D ). CB downmodulated the R848-induced production of chemokines, growth factors, IFN subtypes, and all proinflammatory cytokines, including TNF-α, IL-1β and IL-6 ( Fig. 1D and   1E ). Mass Cytometry (CyTOF) analysis revealed that among PBMCs monocytes represented the main producers of TNF-α, IL-1β and IL-6 upon TLR7/8 stimulation by R848. CB treatment drastically reduced the production of those inflammatory factors by R848-activated monocytes (Fig. 1F , G) (n=4). To better evaluated the effect of CB on the proinflammatory cytokines, we purified monocytes from the blood of HD. Accordingly, using a multiplex bead-based assay ( Fig. 1H) and intracellular flow cytometry staining of TNF-α and IL-1β, we showed in monocytes purified from HD that CB inhibited the production of TNF-α and IL-1β (IC50=2,8 μM and IC50=8 μM, respectively) with no observed toxicity (Fig. 1I) .

We next tested whether the anti-inflammatory properties of CB depend on CXCR4. We first used the well-known CXCR4 antagonist AMD3100 to modulate the anti-inflammatory effects of CB on activated PBMCs. Cells were cultured with increased concentrations of AMD3100 in presence of CB (20M) and R848 for 24h. Levels of IFN and pro-inflammatory factors produced by activated PBMC were indirectly quantified using the monocyte-derived IRF and NF-κB reporter cell line THP-1 dual. We showed that CB reduced activation of both IRF and NF-κB and that AMD3100 blocked the anti-inflammatory activity of CB in a concentration dependent manner ( Fig. 2A-B) .

We further quantified TNF-α, IL1 and IL-6 productions in R848-stimulated primary purified monocytes from HD in the presence of CB (20 µM) and increasing concentrations of AMD3100 (up to 50 µM) using flow cytometry. As expected, AMD3100 inhibited in a dose-dependent manner the ability of CB to reduce intracellular TNF-α, IL1 and IL-6 levels in monocytes without noticeable toxicity ( Fig. 2C-D) .

To firmly establish that CB immunoregulatory activity relates to CXCR4, the expression of CXCR4 was silenced in primary monocytes using small interfering RNA (siRNA). While the transfection of a siRNA control (siControl) had no impact on the CB-induced inhibition of proinflammatory cytokines, CB lost its ability to inhibit TNF-α, IL1 and IL-6 intracellular productions in CXCR4-silenced human monocytes (siCXCR4) (Fig. 2E-F) . Taking into account the 95% transfection efficiency in monocytes, we checked that CXCR4 expression was strongly reduced in siCXCR4-transfected monocytes compared to siControl (Fig. 2G-H) .

These overall data show that the immunoregulatory effects of CB strictly depends on CXCR4 engagement.

Our next attempt was to prob whether CB exerts an anti-inflammatory effect on immune cells isolated from AJI patients. Spontaneous inflammation is a well-known hallmark of JIA. While elevated levels of IL-6 are easily measurable in the plasma of RA patients [12] , the detection of circulating TNF-α in these patients remains a challenge. Thus, to capture TNF-α in the plasma, we developed a TNF-α digital ELISA (Simoa) that detects TNF-α for concentrations as low as 1 fg/mL. Using this technology, we measured detectable levels of TNF-α in patients' plasma and found higher of TNF-α in blood plasma from JIA patients than in HD (JIA patients' median = 3.4 pg/mL vs. HD' median = 1.07 pg/mL) (Fig. 3A) . To better capture the spontaneous inflammation in JAI patients, we performed a gene expression array of 579 inflammatory markers on purified monocytes of PBMC from HD and JAI patients. As compared to HD, the expression of 51 inflammatory genes significantly increased and 16 decreased in JIA patients (Fig. 3B) . Principal components analysis (PCA) showed that monocytes from each group clustered together (Fig. 3C) . Pathway analysis using DAVID [13, 14] showed enrichment in the rheumatoid arthritis pathway (Fig. 3D) . Since JIA is primarily a joint-related inflammatory disease [15] , this prompted us to examine the inflammatory status of synovial fluids. According to Simoa experiments, we measured TNF-α concentrations between 3 and 26 pg/mL (median = 10.8 pg/mL) in synovial fluid (SF) from eighteen JIA patients' knees with active arthritis (Fig. 3E) . When comparing circulating levels of TNF-α in plasma and SF from six matched patients, higher levels of TNF-α were systematically measured in SF compared to blood plasma (Fig. 3F) .

We next evaluated the impact of CB treatment on the spontaneous TNF-α production by human monocytes isolated from PBMCs ( Fig. 3G) and SFMCs (Fig. 3H ) from JIA patients. CB treatment significantly decreased TNF-α levels in the culture medium of both SFMCs and PBMCs monocytes. We also showed that CB exerted a dose-dependent anti-inflammatory effect in purified monocytes from SF of one patient without any significant toxicity (Fig. 3I) .

Altogether, these data provide evidence that CB inhibits the spontaneous production of inflammatory cytokines by blood and synovial human monocytes within an arthritic context.

We next evaluated whether CB would also inhibit cytokine production by stimulated immune cells from JAI patients. To this purpose, we first compared cytokine production by PBMCs from HD and JIA patients using the 45-plex bead-based immunoassay Luminex. We showed that PBMCs from three JIA patients out of five secreted more cytokines than those of HD (Fig.   4A ). This result confirms that cells from JIA patients are hyperresponsive to TLR-7/8 stimulation [8] . Furthermore, CB inhibited R848-induced production of inflammatory cytokines by PBMC from HD as well as the hypersecretion of inflammatory cytokines from JIA patients' cells (Fig. 4B) . As monocytes are the main producers of proinflammatory cytokines, we purified monocytes from blood patients' suffering from oligo articular (n=20), polyarticular (n=8) or systemic (n=5) JIA, or enthesitis-related arthritis (ERA) (n=3). For all subtypes of JIA, CB treatment drastically reduced levels of IL-1β, IL-6, and TNF-α in the culture medium of R848-activated patients' monocytes ( Fig. 4C) .

Finally, using purified synovial fluid mononuclear cells (SFMCs) from JIA patients we showed that CB inhibited the production of the 40 soluble factors induced by R848 activation (Fig. 4D) .

Overall data indicate that CB could reduce hyper secretion of inflammatory cytokines observed in JAI patients during flares.

The anti-inflammatory properties of CB in PBMCs, SFMCs and monocytes of JIA patients, suggest that CB represents a promising therapeutic option for arthritis. Thus, we tested the potential therapeutic effect of CB in a mouse model with an arthritis-like pathology, the collagen-induced arthritis (CIA) DBA/1J mice [16] . CIA mice share many clinical, histologic, and immunologic features with human RA [17] . This includes symmetric joint involvement, synovitis, cartilage, and bone erosions. Several studies reported a major role of IL-1β in disease development in CIA mouse model [18] , as well as a pathologic role of IL-6 in the effector phase of autoimmune arthritis by promoting bone destruction [19] .

CIA mice were daily intraperitoneally injected with CB (at 3 mg/kg (mpK), 10 mpK, and 30 mpK) over a two-week period. As a reference drug, the corticosteroid prednisolone (15 mpK) was also injected in CIA mice. CB treatment did not exert any effect on mouse body weight whatever the dose tested overtime (Fig. 5A) , indicating no major toxicity of CB in treated mice.

While CIA mice displayed elevated levels of circulating IL-1β and IL-6 two weeks after disease onset (Fig. 5B) , CB treatment resulted in a profound decrease of both cytokines (Fig. 5B) . Of note, the highest dose of CB (30 mpK) was as efficient as the referenced corticosteroid prednisolone.

We also investigated the effect of CB treatment on disease progression. The daily injection of CB attenuated the progression of the disease during the 14 days of treatment (Fig. 5C ). Mice treated with CB had thinner legs than mice treated with PBS (Fig.5D ). In arthritic mice, the histological analysis of the tissue injury revealed severe cartilage damage (yellow arrow), bone remodeling (black arrow) and tissue infiltration by immune cells (green arrow) (Fig. 5E) . Daily CB treatment attenuated all these pathological markers (Fig. 5E ). To further characterize the effect of CB treatment, individual paws were scored, reflecting the severity of the disease. CB treatment drastically reduced the terminal paw score (Fig. 5F) , which was associated with reduced inflammation, cartilage damage, bone remodeling on limbs and pannus ( Fig. 5G and   5H ).

In COVID-19 severe patients, lung infiltrated monocytes produce high levels of inflammatory cytokines such as IL-1β, IL-6 and TNF-α leading to uncontrolled inflammation [20] , "cytokine storm", and fever [21] . Moreover, COVID-19 patients requiring intensive care in hospitals, exhibit blood plasma levels of proinflammatory cytokines[10], like IL-1β, IL-6, IL-10 and TNF-α [22] higher than HD (Fig. 6A) . Thus, targeting proinflammatory cytokines and/or monocytes would represent therapeutic strategy for treatment of COVID-19 severe cases. Here, we provide the proof of concept that targeting CXCR4 with CB could represent a novel therapeutic approach for hospitalized COVID-19 patients. According to digital ELISA (Simoa) the treatment of whole blood from COVID-19 patients with CB at 20M for 16h decreased the spontaneous production of IL-1β, TNF-α, IL-6 and IL-10. (Fig. 6B ).

In this study, we reveal a broad-spectrum anti-inflammatory activity of the histamine analogue CB. We demonstrate that CB significantly reduces the spontaneous and induced productions of the key inflammatory cytokines by monocytes from blood and synovial fluids of JIA patients.

This immuno-modulatory activity of CB strictly depends on the engagement of CXCR4 chemokine receptor. In addition, CB displays high efficiency in vivo to reduce inflammation and subsequent tissue damage in arthritic mice, leading to reduction of disease progression. In addition, we provide evidence that targeting CXCR4 with CB also calms hyper inflammation associated with severe COVID-19. Put together, our results suggest that targeting CXCR4 with CB-like molecules represents a promising therapeutic strategy for inflammatory diseases in which hyper activation of monocytes plays a deleterious effect.

The pro-inflammatory cytokines TNF-α and IL-6 are major contributors to JIA and RA [23,24].

Among all pro-inflammatory factors produced during JIA flares, the hierarchical importance of TNF-α and IL-6 is strongly supported by the clinical benefits upon their inhibition. While the TNF-α signature has been clearly identified in JIA patients [25] , systemic TNF-α protein remains, however, difficult to detect and measure, notably because most classical ELISAs are not sensitive enough to detect TNF-α concentration below one pg/mL. To overcome this challenge, we developed an ultrasensitive digital TNF-α ELISA (Simoa) with attomolar sensitivity that permits to show that TNF-α concentration in plasma from JIA patients is sixfold higher than the one in healthy donors. Through Simoa experiments, we also measured that TNF-α concentration in synovial fluid JIA patients is higher in their synovial fluid than that in plasma. In most forms of RA, including JIA, blood monocytes are attracted to synovial fluids where they differentiate into inflammatory macrophages [1] and produce joint-degrading mediators. These immune cells emerge as being the major sources of TNF-α. Accordingly, we show that purified monocytes from JIA patients spontaneously produce more TNF-α than monocytes from healthy donors. As previously described by Cepika et al [1] , we also confirm that monocytes from JIA patients are hyperresponsive to TLR-7/8 activation compared to healthy individuals. In any case, we provide the prime evidence that CB is a potent inhibitor of the production of pro-inflammatory cytokines and chemokines in inflammatory monocytes originating from blood or synovial fluid of JIA patients. Interestingly, CB anti-inflammatory activity is not restricted to TNF- and IL-6, as the inflammatory signature of JIA patients is largely toned down under CB exposure. These ex vivo results suggest that CB potentially reduces hypersecretion of cytokines and chemokines observed in JIA patients during flares. In RA animal models, IL-6 has been reported to stimulate recruitment of leukocytes to inflammatory sites, synoviocyte proliferation and osteoclast activation, resulting in synovial pannus formation [19] . In synergy with IL-1β, IL-6 increases production of matrix metalloproteinases, which contributes to joint and cartilage destruction [26] . In collagen- In competition assays using the CXCR4 antagonist AMD3100 or siRNA-based experiments to down-regulate CXCR4 expression, we further demonstrated that CB anti-inflammatory activity on monocytes strictly depends on CXCR4. Beyond the impact of CXCR4 signaling on regulation of the IFN pathway [11] , this result highlights that CXCR4 also represents a broad spectrum regulator of inflammation in various cell types, including pDC and monocytes. This CXCR4-mediated dual anti-IFN and anti-inflammatory activity may provide clinical benefits as type I IFNs are widely detected and functionally active in RA [27] . Interestingly, a recent study shows that the levels of CXCR4 and its natural ligand, CXCL12 are significantly higher in the serum and joint synovial fluids of active RA patients compared to the control group [28] .

Moreover, CXCR4 and CXCL12 expression levels in the RA active group are higher compared to the remission group [28] . Higher accessibility to CXCR4 in RA patients makes targeting the CXCR4 anti-inflammatory pathway a particularly promising strategy for these pathologies.

For instance, drugs that target TNF-α, IL-6 and IL-1β cytokines or their receptors have shown beneficial effects in JIA patients [23] . However, these treatments are often associated with highly heterogeneous responses across patients in terms of efficacy and treatment resistance.

Consequently, it is not uncommon for a patient to change medication throughout the course of the disease. To some extent, this could be explained by the very high specificity of these treatments toward a single cytokine in a set of diseases characterized by a very broad inflammatory spectrum. By contrast to targeted therapies, corticosteroids have been used for decades to block overall pro-inflammation in RA patients, with, however, strong sideeffects [29] . To overcome these limitations, novel strategies such as Janus kinases (JAK) inhibitors, which are small molecules inhibiting the activity of JAK, showed reasonable success in adult form of arthritis [30] [31] [32] . As for strategies targeting JAK, we demonstrate that CB treatment exhibits wide-ranging cytokine inhibition in vitro, ex vivo and in vivo. CB acts, however, one step upstream of JAK inhibitors, by blocking the production of inflammatory cytokines rather than blocking cytokine-mediated signaling, which may confer some advantages in terms of therapies efficacies. This is in fact supported by the strong inhibition of disease progression in CB-treated RA mice, validating the concept of targeting CXCR4 with CB-like molecules to treat arthritic diseases. CB indeed holds all the required hallmarks for a promising new drug to treat RA: CB is a small molecule, which does not show any side effects in preclinical models in vivo, targets the widely expressed receptor CXCR4 on immune cells, and acts on cytokine production with a broad anti-inflammatory spectrum.

To illustrate the potential benefit of CB in another inflammatory disease context, we studied samples from COVID-19 patients. Hypersecretion of inflammatory cytokines by monocytes and macrophages in lung seems to be central in COVID-19 pathology. Studies of SARS-CoV2 infection have revealed highly inflammatory monocyte/macrophage [9, 10] , [33] population in the bronchoalveolar lavage of patients with severe but not mild COVID-19 [34, 35] . The lungs infiltrated with monocytes exhibit high levels of inflammatory cytokines such as IL1-β , IL-6 [9] and TNF-α at the root of an uncontrolled inflammation [20] , a cytokine storm, and fever [21] . We previously showed that patients requiring intensive care in hospitals displayed higher blood plasma levels of IL-6 and TNF-α, which continue to increase over time and correlate with bad prognostic[10]. Therefore, targeting pro-inflammatory cytokines and/or monocytes also represents a potential therapeutic strategy for the treatment of COVID-19 severe cases. Anti-inflammatory treatments have been proposed, based on the use of corticosteroids or JAK inhibitors [36] , or the humanized monoclonal anti-IL-6 antibody Tocilizumab. Tocilizumab is FDA-approved for the treatment of several inflammatory disorders, including RA[37]. However, up to now, these commercial drugs have shown conflicting results between studies. We show here, using Simoa, that CB controls the spontaneous productions of IL-1β, TNF-α, IL-6 and IL-10 in whole blood from severe COVID-19 patients. This suggests CB could reduce simultaneously the production of key proinflammatory cytokines involved in COVID-19 pathology. Although these results remain preliminary, they nevertheless open the door to the use of drugs targeting CXCR4 as a new therapeutic strategy to control pulmonary hyperinflammation in COVID-19 patients.

In conclusion, the very low cytotoxicity of CB in addition to its broad-spectrum inhibitory activity ex vivo on inflammatory cytokine production and its therapeutic activity in vivo, suggest that targeting CXCR4 with CB-like molecules could represent a novel promising therapeutic strategy for chronic inflammatory diseases such as rheumatoid arthritis and potentially to acute viral-mediated inflammation such as COVID-19 disease.

The blood from healthy donors was obtained from "Etablissement Français du Sang" was used to analyze data.

Monocytes were seeded at 10 5 cells/100 μl in 96-well plates and incubated at 37°C. Control siRNA (qiagen) and CXCR4 siRNA (SMARTPool, Dharmarcon) were diluted in DOTAP (1,2dioleoyl-3-trimethylammonium-propane; Roche Applied Sciences). The mix was gently mixed and incubated at room temperature for 15 min. After incubation, the mix was added to cells in culture at a final concentration of 160 nM. Last, cells were incubated at 37°C for 24 hours before adding treatments and stimulation.

Supernatants were tested for multiple cytokine production using the bead assay LEGENDplex

Antivirus Human panel (Biolegend, San Diego, USA) or with a commercial Luminex multianalyte assay (Biotechne, R&D systems) according to the manufacturer's instructions.

Simoa digital ELISA specific for TNF-α was developed using a Quanterix Homebrew Assay and two commercially available antibodies [38] . The 28401 antibody (R&D) clone was used as a capture antibody after coating paramagnetic beads (0.3 mg/mL), and the ab9635 polyclonal antibody (Abcam) was biotinylated (biotin/Ab ratio = 30:1) and used as the detector.

Recombinant TNF-α (R&D) was used as a standard curve after cross-reactivity testing. The limit of detection was calculated by the mean value of all blank runs ± 3 SDs and was 1 fg/mL.

Human THP1-Dual monocytes cells seeded at 1.6.10 6 cells/mL were pre-treated with The mice were anesthetized and exsanguinated into pre-chilled EDTA tubes. The blood was processed to plasma, frozen and then was thawed at RT, diluted 1:2, and assayed by ELISA for IL-1β (R&D Systems, Cat. MLB00C) and IL-6 (R&D Systems, Cat. M6000B). The limbs were individually removed to 10% neutral buffered formalin.

Formalin-fixed mouse paws were processed routinely, sectioned at approximately 8 microns, and stained with hematoxylin and eosin. Glass slides were evaluated using light microscopy by a board-certified veterinary pathologist. The severity of histologic findings was scored using the following scoring criteria as adapted from Crissman et al [39] . 

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. presented as median ± range. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. were pre-incubated with CB at 20 µM and then stimulated with R848 (5 µg/mL) for 16h.

Cytokine production was measured in the supernatant using a bead-based multiplexed immunoassay system Luminex. Kruskal-Wallis test with Dunn's post hoc correction. All data are presented as median ± range, ***P < 0.001, **P < 0.01, *P < 0.05. n = 8 mice per group 

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