Targeting the chemokine receptor CXCR4 with histamine analog to reduce inflammation in juvenile arthritis

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: arthritis; cytokines; inflammation; monocytes; treatment.

Figure 1

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.

Figure 2

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.

Figure 3

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.

Figure 4

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.

Figure 5

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.