Abstract

Introduction

   Among immune cells, activated monocytes play a detrimental role in
   chronic and viral-induced inflammatory pathologies, particularly in
   Juvenile Idiopathic Arthritis (JIA), a childhood rheumatoid arthritis
   (RA) disease. The uncontrolled activation of monocytes and excessive
   production of inflammatory factors contribute to the damage of
   bone-cartilage joints. Despite the moderate beneficial effect of
   current therapies and clinical trials, there is still a need for
   alternative strategies targeting monocytes to treat RA.

Methods

   To explore such an alternative strategy, we investigated the effects of
   targeting the CXCR4 receptor using the histamine analog clobenpropit
   (CB). Monocytes were isolated from the blood and synovial fluids of JIA
   patients to assess CB's impact on their production of key inflammatory
   cytokines. Additionally, we administered daily intraperitoneal CB
   treatment to arthritic mice to evaluate its effects on circulating
   inflammatory cytokine levels, immune cell infiltrates, joints erosion,
   and bone resorption, as indicators of disease progression.

Results

   Our findings demonstrated that CXCR4 targeting with CB significantly
   inhibited the spontaneous and induced-production of key inflammatory
   cytokines by monocytes isolated from JIA patients. Furthermore, CB
   treatment in a mouse model of collagen-induce arthritis resulted in a
   significant decrease in circulating inflammatory cytokine levels,
   immune cell infiltrates, joints erosion, and bone resorption, leading
   to a reduction in disease progression.

Discussion

   In conclusion, targeting CXCR4 with the small amino compound CB shows
   promise as a therapeutic option for chronic and viral-induced
   inflammatory diseases, including RA. CB effectively regulated
   inflammatory cytokine production of monocytes, presenting a potential
   targeted approach with potential advantages over current therapies.
   These results warrant further research and clinical trials to explore
   the full therapeutic potential of targeting CXCR4 with CB-like
   molecules in the management of various inflammatory diseases.

   Keywords: monocytes, cytokines, arthritis, inflammation, treatment

Introduction

   Rheumatoid arthritis (RA) is a long inflammatory condition that results
   to the aggressive synovial hyperplasia causing destruction of articular
   joints. Joint inflammation is characterized by proliferation of
   macrophage-like ([55]1) and fibroblast-like synoviocytes to form a
   pannus, which invades and destroys the cartilage ([56]2). Juvenile
   Idiopathic Arthritis (JIA) is a rare and complex form of RA affects
   children younger than 16 years old, characterized by a multifactorial
   disorder with heterogeneous manifestations that include all forms of
   chronic arthritis ([57]3, [58]4). Over-production of TNF-α, IL-1β and
   IL-6 is strongly involved in most forms of JIA ([59]5, [60]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 ([61]7). This is
   supported by the observation that monocytes for systemic JIA patients
   produced more inflammatory cytokines than monocytes from healthy donors
   in response to TLR4 and TLR8 stimulation ([62]8). Current antirheumatic
   drugs, including corticosteroids and antibodies-based biotherapies,
   target inflammatory macrophages or macrophages secreted cytokines 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.

   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 analog
   clobenpropit (CB) through their engagement of CXCR4 exhibited a
   broad-spectrum inhibitory activity on the production of all subtypes of
   interferons (IFN) in TLR7-activated human plasmacytoid dendritic cells
   (pDCs) ([63]9). 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 ([64]9). Of note, the
   anti-IFN activity of CB was not associated with histamine receptors but
   strictly dependent on CXCR4 ([65]9). 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.

   In this study, we explored the potential of CB to decrease the
   production of proinflammatory cytokines by monocytes obtained from the
   blood and synovial fluids of individuals diagnosed with Juvenile
   Idiopathic Arthritis (JIA). In these monocytes, we evaluated both the
   spontaneous and induced cytokine production. Finally, we further probed
   in vivo whether CB treatment would attenuate inflammation and impair
   disease progression in a model of collagen-induced arthritic mice.

Results

CB down-regulates TNF-α, IL-1β, and IL-6 productions in TLR-7/8-activated
monocytes

   To first assess the potential anti-inflammatory properties of histamine
   and the histamine analog 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 varying concentration of
   histamine ([66] Figure 1A ) or CB ([67] Figure 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 ([68] Figure 1C ).

Figure 1.

   [69]Figure 1
   [70]Open in a new tab

   CB inhibited TLR-7/8-mediated TNF-α, IL-1β and IL-6 production by human
   monocytes. (A, B) THP1 NF-κB reporter cells were treated with
   increasing concentration of Histamine (A) or CB (B) ranging from 1 to
   100 µM then stimulated with R848 (5 μg/ml) for 24 hours. NF-κB reporter
   activity was measured using QUANTI-Blue, a SEAP detection reagent. (C)
   mRNA levels of TNF-α, IL-1β and IL-6 from THP1-dual pre-incubated with
   increased doses of CB and stimulated overnight with R848 (5 μg/ml),
   were measured by RT-qPCR and normalized to RPL13A. Kruskal-Wallis with
   Dunn’s multiple comparisons test. (D-G) PBMCs from healthy donors (HD)
   were preincubated with CB (20 μM) then stimulated with R848 (5 μg/mL)
   overnight. (D, E) Cytokine production was measured in the supernatant
   using a bead-based multiplexed immunoassay system Luminex. (D) Heatmap
   representation of statistically different cytokines (P<0.05) between
   the different conditions (Null, R848, R848+CB), ordered by hierarchical
   clustering. Up-regulated cytokines are shown in orange, and
   down-regulated in blue. P values were determined with the
   Kruskal-Wallis test. (E) Individual cytokines from the Luminex are
   represented. Mann-Whitney test. (F) PBMCs were analyzed by mass
   cytometry (CyTOF) and tSNE analysis were performed using CD56, CD3,
   CD11c, BDCA4, CD14, HLADR, CD123, CD4, CD8, and CD19 markers.
   Intracellular levels of TNF-α, IL-1β, IL-6, IFNγ, and IL-17were
   evaluated. (G) Mean Metal Intensity (MMI) of TNF-α, IL-1β, and IL-6 was
   evaluated on gated monocytes from four different donors. (H) Cytokine
   production was measured in the supernatants of purified monocytes from
   nine HD using the multiplex bead-based immunoassay LEGENDplex.
   Mann-Whitney test. (I) Purified monocytes from five HD were
   preincubated with increased doses of CB then stimulated with R848
   during 5 h. Intracellular levels of TNF-α and IL-1β as well as
   viability were evaluated by flow cytometry. SSC-A, side scatter. FSC-A,
   forward scatter. 2-way ANOVA. All data are presented as median ± range.
   ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.

   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 with or without of CB (20 µM) (n=5) ([71]
   Figure 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 ([72] Figures 1D, E ). 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 ([73]
   Figures 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 ([74] Figure 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 (IC[50 = ]2,8 μM and IC[50 = ]8 μM, respectively) with no
   observed toxicity ([75] Figure 1I ).

CB controls activation of monocytes from healthy individuals in a
CXCR4-dependent manner

   We next tested whether the anti-inflammatory properties of CB depend on
   CXCR4. In our initial approach, we employed the widely recognized CXCR4
   antagonist AMD3100 to regulate the anti-inflammatory effects of CB on
   activated PBMCs. Cells were cultured with increased concentrations of
   AMD3100 in presence of CB (20μM) and R848 for 24h. The supernatants
   were transferred to reporter THP1-Dual cells allowing simultaneous
   quantification of the activity of IRF and NF-κB promoters. 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 ([76] Figures 2A, B ).

Figure 2.

   [77]Figure 2
   [78]Open in a new tab

   CB immunoregulatory activity on primary monocytes is CXCR4 dependent.
   (A) and (B) PBMCs from two healthy donors were cultured in presence of
   increased concentrations of AMD3100, then treated with CB (20 µM) and
   activated with R848 (5 µg/mL) for 24 hours. The level of (A)
   interferons and (B) inflammatory cytokines in the culture supernatants
   was measured using the reporter cell line THP1-dual. Two-way ANOVA with
   Dunnett’s multiple comparisons test. (C) and (D) Isolated monocytes
   from five healthy donors were cultured in presence of increased
   concentrations of AMD3100, then treated with CB (20 µM) and activated
   with R848 (1 µg/mL) for 6 hours. Intracellular levels of TNF-α, IL-1β
   and IL-6 were evaluated by flow cytometry, (C) dot plot representation
   from a donor and (D) histogram representation from 5 donors. Friedman
   with Dunn’s multiple comparisons test. (E, F) Monocytes were treated
   for 24 hours with CXCR4 siRNA (siCXCR4) or Control siRNA (siControl) at
   160 nM. Cells were then pre-incubated with CB (20 μM) and stimulated
   with R848 (1µg/mL) for 6 hours. Intracellular levels of TNF-α, IL-1β
   and IL-6 were evaluated by flow cytometry: (E) dot plot representation
   from a donor and (F) histogram representation from 3 donors. Data shown
   as the median +/-s.d. Krustal-Wallis with Dunn’s multiple comparisons
   test. (G) the efficiency of transfection with dotap on monocytes was
   evaluated with GLO siRNA in flow cytometry. (H) The expression of CXCR4
   on monocytes treated with siControl and siCXCR4 was assessed by flow
   cytometry. **P < 0.01, *P < 0.05. NS, nonstimulated.

   We further quantified TNF-α, IL-1β 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, IL-1β and IL-6
   levels in monocytes without noticeable toxicity ([79] Figures 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
   pro-inflammatory cytokines, CB lost its ability to inhibit TNF-α, IL-1β
   and IL-6 intracellular productions in CXCR4-silenced human monocytes
   (siCXCR4) ([80] Figures 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 ([81] Figures 2G, H ). These overall data show that the
   immunoregulatory effects of CB strictly depends on CXCR4 engagement.

CB inhibits spontaneous proinflammatory cytokine production in JIA patients’
monocytes

   Our next attempt was to prob whether CB exerts an anti-inflammatory
   effect on immune cells isolated from JIA 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 ([82]10), 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 concentration 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) ([83] Figure 3A ). To better capture the
   spontaneous inflammation in JIA patients, we conducted a gene
   expression analysis on a set of 579 markers associated with
   inflammation 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 oligoJIA patients ([84] Figure 3B ). The
   results of the principal components analysis (PCA) revealed that
   monocytes in each group grouped together ([85] Figure 3C ). Pathway
   analysis using DAVID ([86]11, [87]12) showed enrichment in the
   rheumatoid arthritis pathway ([88] Figure 3D ). Since JIA is primarily
   a joint-related inflammatory disease ([89]13), this prompted us to
   examine the inflammatory status of synovial fluids. Using Simoa
   technology, 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 ([90] Figure 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 ([91] Figure 3F ).

Figure 3.

   [92]Figure 3
   [93]Open in a new tab

   CB controls spontaneous inflammation in cells from JIA patients. (A)
   TNF-α was measured in the plasma of blood from HD and JIA patients by
   digital ELISA (Simoa). Box and whisker plots with median ± range.
   Mann-Whitney test. (B) Monocytes from blood from three healthy donors
   (HD) and three oligoJIA patients were isolated and total RNA extracted
   from the cells was isolated and subsequently analyzed using the
   NanoString nCounter system. Heat map illustrates the fold increase in
   mRNA expression levels of genes that exhibited significant differential
   expression between the two groups. (P < 0.05, fold change > 1.5). (C)
   PCA was performed based on genes from the array differentially
   expressed between the HD (gray) and the JIA patients (red). (P < 0.05,
   fold change > 1.5). (D) David pathway analysis was used for pathway
   enrichment analysis of differentially upregulated genes in JIA patients
   compared to the HD. (E) TNF-α was dosed in the plasma from blood or
   synovial fluids (SF) of JIA patients by Simoa. Mann-Whitney test. (F)
   TNF-α levels in paired plasma and SF from six different donors were
   dosed by Simoa. Wilcoxon test. TNF-α was measured in the supernatant of
   monocytes by digital ELISA (Simoa) purified from PBMCs of twenty JIA
   patients (G) or SFMCs of seventeen JIA patients (H). Average baseline
   of HD is represented by the red dashed line. Mann-Whitney test. (I)
   Monocytes from one JIA patient were treated with various concentration
   of CB for 16h and TNF-α was measured in the supernatant by digital
   ELISA (Simoa). ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.

   We next assessed the effect of CB treatment on the spontaneous TNF-α
   production by human monocytes isolated from PBMCs ([94] Figure 3G ) and
   synovial fluid mononuclear cells (SFMCs) ([95] Figure 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 ([96]
   Figure 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.

CB attenuates R848-induced cytokine production in JIA patients’ cells

   We next evaluated whether CB would also inhibit cytokine production by
   stimulated immune cells from JIA 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 (P11, P16 and P28) of five secreted more
   cytokines than those of HD ([97] Figure 4A ). This result confirms that
   cells from JIA patients are hyperresponsive to TLR-7/8 stimulation
   ([98]8). Furthermore, CB reduced R848-induced inflammatory cytokine
   production by HD PBMCs as well as inflammatory cytokine hypersecretion
   by cells from JIA patients, and most drastically CCL20, CCL2 and CCL3
   ([99] Figure 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 and
   TNF-α and reduced level of IL-6 in the culture medium of R848-activated
   patients’ monocytes ([100] Figure 4C ).

Figure 4.

   [101]Figure 4
   [102]Open in a new tab

   CB controls R848-induced inflammation in cells from JIA patients. (A)
   PBMCs from HD and JIA patients were incubated with R848 (5 µg/mL) for
   16h. P2 P12 P18 and P19 patients are oligoJIA, P10 is an polyJIA.
   Cytokine production was measured in the supernatant using a bead-based
   multiplexed immunoassay system Luminex. Heatmap representation of
   statistically different cytokines (P<0.05) in PBMCs supernatant upon
   R848 stimulation between HD and JIA patients, ordered by hierarchical
   clustering. Mann-Whitney test. (B) PBMCs from HD and JIA patients 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. Individual cytokines
   are represented. Two-way RM ANOVA with Tukey post hoc correction. (C)
   Monocytes from JIA patients were pre-incubated with CB at 20 µM and
   then stimulated with R848 (5 µg/mL) for 16h. IL-6, IL-1β and TNF-α
   production was measured in the supernatant using the multiplex
   bead-based immunoassay LEGENDplex. Mann-Whitney test. (D) SFMCs were
   pre-incubated with CB at 20 µM and then stimulated with R848 (5 µg/mL)
   for 16h. P5 P11 and P28 patients are oligoJIA, P16 is a polyJIA and P36
   an sJIA. Cytokine production was measured in the supernatant using a
   bead-based multiplexed immunoassay system Luminex. ****P < 0.0001, ***P
   < 0.001, **P < 0.01, *P < 0.05.

   Finally, using purified SFMCs from JIA patients we showed that CB
   inhibited the production of the 40 soluble factors induced by R848
   activation ([103] Figure 4D ). Overall data indicate that CB could
   reduce hyper secretion of inflammatory cytokines observed in JIA
   patients during flares.

CB inhibits inflammatory cytokine and reduces disease progression in
collagen-induced arthritis mouse model

   The anti-inflammatory properties of CB in PBMCs, SFMCs and purified
   monocytes of JIA patients, suggest that CB represents a promising
   therapeutic option for arthritis. Consequently, we proceeded to
   evaluate the potential therapeutic impact of CB in DBA/1J mice, which
   serve as a mouse model for arthritis-like pathology known as
   collagen-induced arthritis (CIA) ([104]14). CIA mice exhibit numerous
   clinical, histological, and immunological similarities to human RA
   ([105]15). 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 ([106]16), as well as a
   pathologic role of IL-6 in the effector phase of autoimmune arthritis
   by promoting bone destruction ([107]17).

   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 ([108] Figure 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 ([109]
   Figure 5B ), CB treatment resulted in a profound decrease of both
   cytokines ([110] Figure 5B ). Of note, the highest dose of CB (30 mpK)
   was as efficient as the referenced corticosteroid prednisolone.

Figure 5.

   [111]Figure 5
   [112]Open in a new tab

   CB controls inflammation and disease onset in collagen induced
   arthritic mice. (A) The body weight of collagen induced arthritic mice
   treated with prednisolone (Pred, 15 mpK) or 3, 10, or 30 mpK of CB was
   measured every other day. (B) IL-1β and IL-6 were measured by ELISA in
   the serum after 2 weeks of treatment. Kruskal-Wallis test with Dunn’s
   post hoc correction. (C) the progression of the disease was assessed
   according to several markers in the legs. (D) The thickness of the legs
   was measured. (E) Representative hematoxylin and eosin staining of paw
   sections from collagen induced arthritic mice treated with prednisolone
   (Pred, 15 mpK) or 3, 10, or 30 mpK of CB: joints with pannus and
   inflammation (green arrow), loss of articular cartilage (yellow arrow)
   and bone remodeling (black arrow). (F) Impact of the treatment was
   evaluated by a paw score, signs of arthritis in all paws according to a
   0-4 scale of ascending severity, after 2 weeks of treatments. (G)
   Effects of the treatment on the scoring for inflammation, cartilage
   damage, bone remodeling, and pannus after 2 weeks of treatment. (H)
   Combined disease score after 2 weeks of treatment. 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.

   Additionally, we conducted an investigation to assess the impact of CB
   treatment on the progression of the disease.The daily injection of CB
   attenuated the progression of the disease during the 14 days of
   treatment ([113] Figure 5C ). Mice treated with CB had thinner paws
   than mice treated with PBS ([114] Figure 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) ([115] Figure 5E ). Daily CB
   treatment attenuated all these pathological markers ( [116]Figure 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 ([117] Figure 5F ), which
   was associated with reduced inflammation, cartilage damage, bone
   remodeling on limbs and pannus ([118] Figures 5G, H ).

Discussion

   In this study, we reveal a broad-spectrum anti-inflammatory activity of
   the histamine analog CB. Our findings reveal a notable decrease in the
   production of key inflammatory cytokines when monocytes derived from
   the blood and synovial fluids of individuals with JIA are exposed to
   CB. This reduction is observed both in spontaneous and induced cytokine
   production. 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 summary, our results strongly indicate that the targeting of CXCR4
   using CB-like molecules holds great potential as a therapeutic approach
   for inflammatory diseases characterized by detrimental hyperactivation
   of monocytes.

   The pro-inflammatory cytokines TNF-α and IL-6 are major contributors to
   JIA and RA ([119]18, [120]19). The clinical benefits observed upon
   inhibiting TNF-α and IL-6 strongly support the hierarchical
   significance of these two pro-inflammatory factors among all the
   factors produced during JIA flares. While the TNF-α signature has been
   clearly identified in JIA patients ([121]20), 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 six-fold 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 ([122]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. ([123]1), we also confirm that monocytes from JIA
   patients are hyperresponsive to TLR-7/8 activation compared to healthy
   individuals. Our study provides evidence that CB is a powerful
   inhibitor of pro-inflammatory cytokine and chemokine production in
   inflammatory monocytes derived from the blood or synovial fluid of
   individuals with Juvenile Idiopathic Arthritis (JIA). Interestingly, CB
   anti-inflammatory activity is not restricted to TNF-α 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 shown to promote
   osteoclast activation, synoviocyte proliferation, and recruitment to
   inflammatory areas, leading to the development of synovial pannus
   ([124]17). In conjunction with IL-1β, IL-6 enhances the production of
   matrix metalloproteinases, thereby contributing to the degradation of
   joints and cartilage ([125]21). In collagen-induced arthritis (CIA)
   mice, daily treatment of CB exhibits strong anti-inflammatory
   properties by blocking both IL-1β and IL-6 secretions. According to
   massive reduction of cartilage damage, bone remodeling, immune cell
   tissue infiltration and pannus CB treatment results in reduced disease
   progression and paw thickness in arthritic mice similarly to the
   referenced corticosteroid prednisolone. These in vivo experiments
   validate the concept of the immunomodulatory activity of CB observed in
   vitro and ex vivo on synovial monocytes from JIA patients.

   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 ([126]9), this result highlights that CXCR4 also represents a
   broad spectrum regulator of inflammation in various cell types,
   including pDC and monocytes. The concurrent anti-IFN and
   anti-inflammatory effects exerted by CXCR4 can have substantial
   clinical advantages, particularly considering the frequent presence and
   functional activity of type I interferons (IFNs) in RA ([127]22). It is
   worth noting that a recent study has revealed significantly elevated
   levels of CXCR4 and its natural ligand, CXCL12, in both the serum and
   joint synovial fluids of individuals with active RA when compared to a
   control group ([128]23). Furthermore, the expression levels of CXCR4
   and CXCL12 in the active RA group were found to be higher compared to
   the group in remission ([129]23). 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
   ([130]18). 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 side-effects
   ([131]24). In order to address these limitations, innovative approaches
   such as Janus kinase (JAK) inhibitors have emerged as promising
   strategies. These small molecules effectively inhibit the activity of
   JAK, and they have demonstrated reasonable success in treating the
   adult form of arthritis ([132]25–[133]27). Regarding strategies
   targeting JAK, our findings demonstrate that CB treatment effectively
   inhibits a broad range of cytokines in vitro, ex vivo, and in vivo.

   In contrast, CB exerts its effects at an earlier stage compared to JAK
   inhibitors. Instead of directly targeting cytokine-mediated signaling,
   CB acts one step upstream by inhibiting the production of inflammatory
   cytokines. This approach may offer certain advantages in terms of
   therapy efficacy compared to JAK inhibitors. The significant
   suppression of disease progression observed in mice with RA following
   treatment with CB provides strong validation for the concept of
   targeting CXCR4 using CB-like molecules as a potential therapeutic
   strategy for arthritic conditions. Indeed, CB exhibits all the
   essential characteristics of a promising new drug for the treatment of
   rheumatoid arthritis (RA). It is a small molecule that has demonstrated
   an absence of side effects in in vivo preclinical models. CB
   specifically targets the widely expressed immune cell receptor CXCR4,
   and it exerts a broad anti-inflammatory effect by modulating cytokine
   production. These attributes collectively highlight CB as a potential
   candidate for RA therapy.

   In summary, the minimal cytotoxicity of CB, coupled with its
   wide-ranging inhibitory effects on ex vivo production of inflammatory
   cytokines and its therapeutic efficacy demonstrated in vivo, indicates
   that the use of CB-like molecules to target CXCR4 could serve as a
   novel and promising therapeutic strategy for chronic inflammatory
   diseases, including rheumatoid arthritis.

Materials and methods

Blood samples isolation and culture of blood leukocytes

   The blood samples obtained from donors in good health were sourced from
   “Etablissement Français du Sang” (agreement # 07/CABANEL/106), located
   in Paris, France. The use of materials from JIA patients was conducted
   with the approval of the Comité de Protection des Personnes (N°
   EudraCT: 2018-A01358-47) in France. The experimental procedures
   involving human blood adhered to the guidelines set by the European
   Union and the Declaration of Helsinki. Informed consent was obtained
   from all donors, including both healthy individuals and patients.
   [134]Table 1 provides a summary of the clinical data pertaining to the
   JIA patients. In vitro experiments were conducted using human
   mononuclear cells obtained from peripheral blood or synovial fluid (SF)
   through centrifugation using density gradient medium (STEMCELL
   Technologies). Human monocytes were isolated by positive selection
   using Human CD14 microbeads (Miltenyi). SFMC (Synovial fluid
   mononuclear cells), PBMC (Peripheral blood mononuclear cells), and
   monocytes were cultured in RPMI 1640 medium (Invitrogen, Gaithersburg,
   MD) (R10) supplemented with 10% heat-inactivated fetal bovine serum and
   1mM glutamine (Hyclone, Logan, UT). The JIA patients were identified as
   P1 to P36 (refer to [135]Figure 2 ).

Table 1.

   Clinical information of recruited patients.
   Patients Age Gender Disease Treatment TNF-α (pg.ml) - Blood TNF-α
   (pg.ml) - SF
   plasma Null CB plasma Null CB
   P1 18 F oligoJIA apranax 1,9 – – 25,6 – –
   P1’ 18 F oligoJIA apranax 0,7 – – 22,3 – –
   P2 5 F oligoJIA ibuprofen – – 26,5 331,1 83,3
   P3 5 F oligoJIA ibuprofen 18,1 – – 16,4 2,6 0,5
   P4 13 F oligoJIA naproxen – – 3,5 – –
   P5 10 F oligoJIA ANA+ voltaren + inexium 2,4 1,7 0,3 11,1 – –
   P6 16 M ERA celecoxib – – – 1,6 2,0 0,1
   P7 17 M polyJIA RF- Tocilizumab – 13,2 1,1 16,1 19,1 4,1
   P8 15 F polyJIA RF- tocilizumab + abatacept – – – 13,8 0,6 0,0
   P9 5 F polyJIA tocilizumab + cortansyl 1,2 3,4 0,2 – 94,8 4,8
   P9’ 5 F polyJIA tocilizumab + cortansyl 1,0 0,0 9,3 – –
   P10 17 M polyJIA RF- methotrexate – – – 11,6 2,9 0,1
   P11 15 F oligoJIA untreated 1,4 4,9 0,1 5,3 2,3 2,6
   P12 11 F oligoJIA untreated – – – 7,5 18,9 2,0
   P13 15 M oligoJIA untreated 3,7 0,3 0,3 5,2 1,0 21,6
   P14 16 F oligoJIA untreated – – – 10,5 57,4 0,1
   P15 5 F oligoJIA ANA+ untreated – 7,7 14,7 14,4 – –
   P16 17 M polyJIA RF- ANA+ untreated 2,1 3,4 1,4 3,6 – –
   P16’ 17 M polyJIA RF- ANA+ untreated – – – 7,4 – –
   P17 16 M ERA celecoxib – – – – 1,6 –
   P18 11 F oligoJIA ANA+ methotrexate + adalimumab – – – – 17,0 0,0
   P18’ 11 F oligoJIA ANA+ methotrexate + adalimumab – – – – 17,9 1,5
   P18’’ 11 F oligoJIA ANA+ Methotrexate + Adalimumab – – – – 25,0 2,4
   P19 18 F oligoJIA untreated – – – – 24,0 2,4
   P20 16 F oligoJIA untreated – – – – 1,6 0,5
   P21 12 F oligoJIA untreated 8,8 – – – 1,0 34,1
   P22 14 M ERA Adalimumab 21,8 1,8 0,2 – – –
   P23 17 F oligoJIA Infliximab + Methotrexate + prednisone 23,7 0,3 1,1 –
   – –
   P24 13 M polyJIA Cortancyl + Arava 7201,1 1620,7 – – –
   P24’ 13 M polyJIA Cortancyl + Arava 8,5 1,6 0,0 – – –
   P25 18 F sJIA Ruxolitinib + Prednisone 3,2 0,4 0,3 – – –
   P26 18 F oligoJIA Methotrexate 13,2 – – –
   P27 18 M polyJIA RF- ANA+ Methotrexate + Cortancyl 0,0 1,0 0,1 – – –
   P28 3 F oligoJIA untreated 3,3 1,7 0,1 – – –
   P29 17 F oligoJIA untreated 10,5 – – – – –
   P30 13 M oligoJIA untreated 4,0 – – – – –
   P31 5 F oligoJIA ANA+ untreated 3,3 – – – – –
   P31’ 5 F oligoJIA ANA+ untreated 3,5 – – – – –
   P31’’ 5 F oligoJIA ANA+ untreated 3,3 – – – – –
   P32 5 F sJIA anti-IL-1 – 2,2 6,3 – – –
   P33 12 F sJIA Tocilizumab + Cortancyl + Methotrexate – 27,9 980,3 – – –
   P34 5 F polyJIA RF- Tocilizumab – 13,5 1,1 – – –
   P35 18 F sJIA Tocilizumab – 0,0 1,3 –
   P36 16 M sJIA Tocilizumab + Cortancyl – 12,1 486,9 – – –
   [136]Open in a new tab

   SF, synovial fluid; oligoJIA, oligoarticular juvenile idiopathic
   arthritis; ERA, enthesitis-related arthritis; polyJIA, polyarticular
   juvenile idiopathic arthritis; sJIA, systemic juvenile idiopathic
   arthritis; RF, rheumatoid factor; ANA, antinuclear factor. The Null and
   CB columns correspond to TNF-α assay data by Simoa performed on
   supernatants of isolated monocytes from blood or synovial fluid of JIA
   patients. Null: untreated monocytes, CB: monocytes treated with 20 μM
   CB.

Cell stimulation

   PBMCs were initially seeded at a concentration of 2.106 cells per
   milliliter (mL), while monocytes isolated from either PBMCs or SFMCs
   were seeded at a concentration of 1.10^6 cells per mL. Prior to a
   16-hour stimulation with the TLR-7/8 agonist Resiquimod – R848 at a
   concentration of 5 µg/mL or as specified, the cells were pre-treated
   for 1 hour with AMD3100 (Sigma-Aldrich) and/or clobenpropit (CB) (Sigma
   Aldrich) at a concentration of 20 µM (or other specified
   concentration). Subsequently, flow cytometry or mass cytometry
   techniques were employed to collect the cells, and supernatants were
   collected to detect cytokine levels. For intracellular staining,
   Brefeldin A (BFA) was added to the cells 30 minutes after stimulation
   and incubated for 5 hours.

   THP1-dual cells (Invivogen) were seeded at a concentration of 1.106
   cells per mL in RPMI 1640 (Invitrogen, Gaithersburg, MD) (R10) medium
   supplemented with 10% heat-inactivated fetal bovine serum, 2mM
   glutamine (Hyclone, Logan, UT), 25mM HEPES, and 1% Pen-Strep. Before a
   24-hour stimulation with the TLR-7/8 agonist Resiquimod – R848 at a
   concentration of 5 µg/mL, the cells were pre-treated for 1 hour with
   clobenpropit (CB) (Sigma Aldrich) or Histamine at concentrations
   ranging from 1 to 100 µM. The activity of the NF-κB reporter was
   measured using QUANTI-Blue, a SEAP detection reagent, following the
   instructions provided by the supplier.

Mass cytometry

   Peripheral blood mononuclear cells (PBMCs) obtained from healthy donors
   were stimulated with R848 in the presence of CB. Brefeldin A was added
   overnight to the culture. Following stimulation, PBMCs were treated
   with a mixture of surface antibodies and Rh isotopes in PBS for 20
   minutes at room temperature. Both commercially available and
   custom-conjugated antibodies were used to create a panel for phenotypic
   and functional analysis. Two DNA interchelators, Rhodium isotope mass
   103 and Iridium isotopes mass 191 & 193, were employed to determine
   cell viability and gate the singlet cells during analysis. Prior to
   analysis, each antibody was titrated using PBMCs. Subsequently, the
   cells were fixed with Fix-I solution (Maxpar Fix-I buffer; Fluidigm)
   for 15 minutes at room temperature. After fixation, the cells were
   incubated with anti-cytokine antibodies and Ir isotopes in Perm-S
   solution for 30 minutes at room temperature (Maxpar Perm-S buffer;
   Fluidigm). Before CyTOF acquisition, the cells underwent three washes
   with highly pure water and were resuspended in water at a maximum
   concentration of 500,000 cells/mL (Maxpar Water; Fluidigm). Data
   acquisition was performed using a CyTOF2 instrument (Fluidigm) and the
   analysis included data cleaning using FlowJo software and the
   application of the viSNE algorithm in Cytobank for the primary
   analysis. The table ([137] Table 2 ) provides the details of antibodies
   coupled with the respective metals.

Table 2.

   Antibodies for mass cytometry.
   Targets       Metals Clones    Suppliers         References