Ankylosing spondylitis macrophage production of higher levels of interleukin-23 in response to lipopolysaccharide without induction of a significant unfolded protein response

Abstract

Objective: Previous studies of the HLA-B27-transgenic rat model of ankylosing spondylitis (AS) suggested that macrophages develop an intracellular stress response called the unfolded protein response (UPR) and, as a result, secrete increased amounts of cytokines in response to Toll-like receptor agonists such as lipopolysaccharide (LPS). Our objective was to determine whether macrophages from AS patients also undergo a UPR and secrete increased cytokines/chemokines in response to LPS.

Methods: Peripheral blood monocytes isolated from 10 AS patients and 10 healthy controls were differentiated in vitro with macrophage colony-stimulating factor. Select samples were treated with interferon-γ (IFNγ) to up-regulate class I major histocompatibility complex (HLA-B) expression prior to stimulation with LPS for either 3 hours (for RNA) or 8-24 hours (for supernatant). UPR induction was assessed by measuring the expression of messenger RNA for ERdj4, BiP, and CCAAT/enhancer binding protein homologous protein 10 (CHOP).

Results: Although IFNγ treatment up-regulated HLA-B expression (2-fold; P < 0.0001), neither IFNγ nor LPS substantially enhanced BiP or CHOP expression (<1.3-fold). ERdj4 expression increased weakly, but not significantly, in AS samples treated with IFNγ plus LPS (2.2-fold; P = 0.31). In response to LPS, AS macrophages secreted more CXCL9, interleukin-10 (IL-10), IL-12p70, IL-23, and tumor necrosis factor α than did control macrophages (P ≤ 0.025). The most striking difference was observed for IL-23 (median 265 pg/ml in AS patients versus 9 pg/ml in controls; P = 0.0007). We did not detect significant differences in IL-6, IL-8, or IFNβ production.

Conclusion: The greater production of IL-23 by AS patient macrophages in response to LPS provides further support for the development of Th17/IL-23-directed therapy. Since significant UPR induction was not detected in AS patient macrophages, the relationship between UPR and inflammatory cytokine production remains unclear.

Figure 1. HLA-B and UPR-regulated gene expression

A) Human macrophages were treated with various doses of tunicamycin (Tu μg/mL) for the times indicated. Gene expression was normalized to 4h untreated controls to yield fold change. Data was combined from 4 independent experiments with bars representing geometric means and vertical bars showing +/− standard errors of the mean. The geometric mean best represents the typical fold change but downweights large fold changes. B) Analysis of AS patients (AS) and controls (C): individual data points with lines connecting unstimulated and stimulated samples are on the left, and box plots showing median, 25^th percentile, 75^th percentile (boxes), means (black squares) and 10^th and 90^th percentiles (bars), are on the right. Subject macrophages were unstimulated (−), or treated with IFN-γ for 24h followed by 100 ng/mL LPS (+) for 3h. Relative gene expression was determined by quantitative PCR with normalization to 18S rRNA. HLA-B expression represents data from 8 patients and 8 controls. P=0.0006 for upregulation of HLA-B in combined patient and control samples. CHOP and BiP expression represent data from 9 patients and 10 controls. ERdj4 expression shows data from 9 patients and 7 controls.

Figure 2. Increased IL-23, CXCL9, IL-12(p70) and TNF-α production in AS patient macrophages

Individual sample data points are on the left and box plots showing median, 25^th and 75^th percentiles (boxes) and 10^th and 90^th percentiles (error bars) are on the right. A) IL-23: AS patient (AS, open triangles) or control samples (C, open circles) were treated with 10 or 100 ng/mL LPS for 24h. See Table 3 for associated p-values and numbers per group. B) CXCL-9: AS patient and control samples were left untreated (−) or treated with IFN-γ (+) for 24h, and then stimulated with 10 ng/mL LPS for another 24h. C) IL-12 (p70): All samples were pre-treated with IFN-γ for 24h and then stimulated with either 10 or 100 ng/mL LPS for another 8h. The outlier points for IL-12 production in response to both doses of LPS were from the same control subject. D) TNF-α: Macrophages were left untreated (−) or treated with IFN-γ (+) for 24h and then stimulated with 100 ng/mL LPS for 8h.