Background/Purpose: Axial spondyloarthritis (AxSpA) is a chronic inflammatory arthritis characterized by monocyte activation and Th17 cell expansion. While HLA-B*27 is the strongest genetic risk factor for AxSpA, its mechanism especially in human remains unclear.

Methods: Human peripheral blood mononuclear cells (PBMCs) from 97 AS patients were used in this study. Single-cell RNA sequencing (scRNA-seq) were used to compare monocyte populations between HLA-B*27⁺ and HLA-B*27⁻ healthy donors, as well as between AxSpA patients and healthy controls. Flow cytometry and enzyme-linked immunosorbent assay (ELISA) were used to investigate the outcome of T-cell and monocyte co-cultures. Small interfering RNAs or inhibitors were used to study the function of genes. The knockdown efficiency was determined by quantitative PCR and Western blotting.

Results: We identified a distinct CCL20⁺ monocyte subset that is expanded in HLA-B*27⁺ individuals and increased in AxSpA patients. Silencing HLA-B in AxSpA monocytes reduced CCL20+ signature genes, suggesting HLA-B*27 drives their expansion. CCL20⁺ monocytes are hyper-responsive to both microbial and T cell-derived stimuli. Moreover, T cell-activated monocytes promoted Th17 responses, suggesting a bidirectional inflammatory loop central to AxSpA pathology. Finally, an siRNA screen targeting AxSpA-associated risk genes enriched in CCL20⁺ monocytes identified ZC3H12C, ERN1, and IL1R1 as key regulators of T cell-induced monocyte activation.

Conclusion: Together, these findings uncover a novel HLA-B*27–driven inflammatory pathway in AxSpA involving CCL20⁺ monocyte expansion and monocyte–T cell crosstalk, and point to new candidate targets for therapeutic intervention.

Figure 1. HLA-B*27 is associated with an increased proportion of CCL20+ cells in peripheral blood monocytes of healthy controls and Axial SpA patients. (A) UMAP visualization of the CD14+ classical monocytes from 982 healthy donors (sourced from Yazar S. et al. 2022, PMID: 35389779). (B) Heatmap of normalized and scaled expression of key marker genes in different subsets of CD14+ monocytes. (C) Frequency of CCL20+ cells in classical monocytes in healthy donors carrying different HLA-B alleles. (D) UMAP visualization of the CD14+ classical monocyte subsets from 29 healthy controls, 10 AxSpA patients, 28 PsA patients, and 24 Psoriasis patients (sourced from Alber S. et al. 2022, PMID: 35634297). (E) ) Color coding for CD14+ monocytes from different groups of donors. (F) Comparing the frequency of monocyte subsets in CD14+ classical monocytes between groups. (G) Volcano plots showing genes differentiated expressed between control and HLA-B-siRNA-transfected CD14+ monocytes from 4 HLA-B*27+ AxSpA patients (FDR < 0.05). (H) HLA-B knockdown gene signature score (calculated using the top 50 genes downregulated by HLA-B siRNA) across monocyte subsets. Wilcox test was carried out for statistics. P < 0.05 was considered significant.

Figure 2. CCL20+ monocytes are hyper-responsive to LPS stimulation. (A) Pathway analysis for top 100 genes that are enriched in CCL20+ monocyte using MSigDB 2000 database. (B) Experimental schematic for treatment of PBMCs from AxSpA patients before single cell RNA sequencing. (C) UMAP visualization of transcriptionally distinct populations of CD14+ monocytes extracted from unstimulated and LPS-stimulated PBMCs. (D) Heatmap of normalized and scaled expression of key marker genes in different subsets of CD14+ monocytes. (E, F) Expression of TNF, IL6, IL1B, and IL23A in different CD14+ monocyte subsets. The Wilcoxon test was performed for the statistical analysis in panels (E) and (F). A p-value of < 0.05 was considered significant (*: p < 0.05, **: p < 0.01, ***: p < 0.001).

Figure 3. Activated T-cells induce cytokine production by Axial SpA monocytes. (A) Representative flow cytometry plots and summary graphs showing cytokine production by CD14+ monocytes from PBMCs simulated with T cell activation beads (TAB) or LPS. (AxSpA patients, n=13). (B) Cytokines secreted to the supernatant of PBMCs from AxSpA patients after stimulation with TAB or LPS for 16 hours (n=11). (C) PBMCs from sex and age-matched healthy controls or AxSpA patients were treated with TAB for 16h. IL-1β, IL-23 and TNF-α in the culture supernatant were measured using ELISA (n=10 for each group). (D) Whole PBMCs or monocyte-depleted PBMCs from AxSpA patients stimulated with TAB for 16h. IL-1β, IL-23, TNF-α and IL-6 in the culture supernatant were measured using ELISA (n=4). (E) Isolated CD3+ T-cells from PBMCs from AxSpA patients (n=9) were cultured alone or co-cultured with autologous monocytes in the absence/presence of TAB for 16 hours. The levels of cytokines IL-1β, IL-23, TNF-α and IL-6 in the culture supernatant were measured using ELISA. Data are represented as mean and SEM of independent donors in independent experiments. The one-way ANOVA were performed for the statistical analysis in panels (A) and (B). The paired and unpaired two-tailed Student’s t-test was calculated for (A),(B),(D) and (E). The unpaired two-tailed Student’s t-test was calculated for (C). A p-value of < 0.05 was considered significant (*: p < 0.05, **: p < 0.01, ***: p < 0.001).

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/ccl20-monocytes-expanded-by-hla-b27-fuel-th17-generation-in-axial-spondyloarthritis/