In this study we examined gene expression patterns in macrophages derived over one week in culture, rather than complex mixtures of cells examined immediately after removal from the individual. We found that macrophages derived from subjects with AS exhibit a ‘reverse’ IFN signature where genes typically upregulated by IFN are underexpressed, and genes downregulated by IFN are overexpressed, relative to healthy controls. This reverse IFN signature correlates with reduced expression of the IFN-γ gene, suggesting that an autocrine effect may be involved. These results provide insight into gene expression differences that may be inherent to individuals with AS, a disease that is largely genetically determined (1
), and thus may be informative for upstream mechanisms contributing to pathogenesis.
We derived macrophages over several days ex vivo
in order to eliminate gene expression differences that might be the result of in vivo
effects of serum and tissue factors. Thus, finding differences in the relative expression of IFN-regulated genes was initially surprising. However, autocrine effects of type I (IFN-α/β) and type II IFNs (IFN-γ) produced by macrophages have been well documented (27
), suggesting two plausible explanations for this result. The first is that macrophages from patients produce less IFN (type I or type II) than cells from healthy controls, resulting in lower expression of genes positively regulated by IFN, and higher expression of IFN-repressed genes. A second possibility is that IFN production is similar, but that AS patient macrophages are less responsive. The normalization of expression of IFN-responsive genes across patients and controls when cells were treated with exogenous IFN-γ indicates that there is no inherent defect in responsiveness to this cytokine. However, since cells were maximally stimulated (100 U/ml for 24 h), we cannot rule out subtle differences that might occur with lower concentrations for brief periods. In support of the first mechanism, we found significantly lower expression of the IFN-γ gene in macrophages from AS patients. This was specific for IFN-γ, as IFN-β mRNA levels were no different between patients and controls. Further support for a defect in expression of IFN-γ comes from the LPS stimulation experiment, where there is little to no induction of IFN-γ in AS patients. Our results are consistent with a mechanism whereby low levels of IFN-γ produced by cultured macrophages maintain the expression levels of IFN-responsive genes. Lower expression of IFN-γ by macrophages from AS patients would lead to down regulation of IFN-induced genes and upregulation of IFN repressed genes, relative to healthy controls. We were unable to measure immunoreactive IFN-γ protein in culture supernatants from healthy controls or AS patients, consistent with the notion that these are autocrine effects of low levels of the cytokine (unpublished observations). Since there is a large overlap in genes responsive to IFN-γ and IFN-β (29
), we cannot rule out autocrine effects of IFN-β. However, this seems unlikely since there is no difference in expression of the IFN-β gene or its responsiveness to LPS.
We considered other possible sources of IFN-γ in the macrophage cultures. NK cells and T cells are quantitatively important producers of IFN-γ during innate and adaptive immune responses, respectively. Indeed, it has been shown that even small numbers of these cells persisting in cultures of bone marrow-derived macrophages or dendritic cells (DCs), can be important producers of IFN-γ (particularly when cells were stimulated with IL-12), that had previously been attributed to the macrophages or DCs (30
). This is unlikely to account for our results for several reasons. First, to the extent that this occurs with peripheral blood derived macrophages as it does with bone marrow preparations, we would expect patient and healthy control cultures to be affected similarly. Second, T cell carryover is more likely to be a problem with IL-12 stimulation, since it is a potent inducer of IFN-γ production by NK and T cells. Finally, the LPS stimulation results, where we used low concentrations (10 ng/ml) for a brief period (3 h), are most consistent with an abnormality in macrophage IFN-γ expression, since T cells and most NK cells are not readily responsive to this Toll-like receptor agonist.
Previous studies of cytokine expression in spondyloarthritis and AS support the idea that IFN-γ expression may be dysregulated. Gut biopsy samples show a reduced Th1 profile in lymphocytes from spondyloarthritis patients (31
). IFN-γ was also lower in synovial tissue from spondyloarthritis patients compared to individuals with rheumatoid arthritis (32
), and lower in synovial fluid from HLA-B27 positive patients with Chlamydia-induced reactive arthritis compared to HLA-B27 negative patients (33
). A lower frequency of IFN-γ positive T cells has also been reported in AS patients (34
), consistent with previous observations showing that in vitro
stimulation of whole blood with Mycoplasma arthritidis superantigen, or mitogens (PHA and ConA) resulted in less IFN-γ production (35
). The nature of these differences, and whether they are primary or secondary, remains to be determined. However, our results raise the possibility that T cell and/or NK cell expression of IFN-γ may be altered similar to macrophages, particularly if the defect is genetic in origin.
Abnormal cytokine responses to arthritogenic bacteria have been implicated in pathogen persistence (36
). Along these lines it is worth noting that macrophage-derived IFN-γ is important for controlling infection with certain intracellular gram-negative bacteria (38
). In these studies, infection of macrophages with Chlamydia pneumonia
led to an increase in IFN-γ production. In IFN-γ receptor-deficient macrophages, bacterial load was significantly higher, implicating an autocrine/paracrine mechanism whereby IFN-γ signaling helps to limit intracellular bacterial growth. In addition, there is evidence to suggest that autocrine macrophage-derived IFN-γ plays a role in the pathogenesis of glomerulonephritis in a mouse model (27
). Thus, decreased IFN-γ production by macrophages could impair the host's ability to clear certain organisms, which in turn could contribute to the pathogenesis of spondyloarthritis. This has experimental support from recent studies of Chlamydia
-induced arthritis in rats, in which susceptibility and resistance between strains corresponds to the synovial decreased or enhanced IFN-γ expression, respectively (39
In addition to IFN-γ dysregulation hindering pathogen clearance, reduced expression can contribute to activation of the IL-23/IL-17 axis. For example, induction of arthritis with collagen immunization in IFN-γ-deficient mice results in greater expansion of IL-17-expressing CD4 T cells and increased IL-17 production (40
). There is increasing evidence for the involvement of this axis in AS and spondyloarthritis pathogenesis. We have recently found overexpression of IL-23 and IL-17, along with expansion of IL-17-expressing CD4 T cells, in the colon of HLA-B27 transgenic rats with spondyloarthritis-like disease (41
) (manuscript in preparation). In humans, IL-17 is overexpressed in synovial fluid of patients with undifferentiated spondyloarthritis (42
), and IL23R
(IL-23 receptor gene) polymorphisms have been associated with AS susceptibility (3
). The IL-23 receptor is expressed on developing T cells that become Th17 committed. This enables them to respond to IL-23, which in turn drives T cell survival and expression of IL-17 (43
). Thus, our results reported here raise the possibility that low IFN-γ expression could contribute to Th17 development in patients with AS.
Several studies have demonstrated that the HLA-B27 heavy chain has a tendency to misfold during synthesis and assembly into complexes with β2
m) and peptide in the endoplasmic reticulum (ER) (44
). Protein misfolding in the ER can lead to a stress response known as the unfolded protein response (UPR) resulting in increased transcription of many UPR target genes (45
). We have found that bone marrow derived macrophages from HLA-B27/human β2
m transgenic rats exhibit UPR activation when transgene expression is upregulated (46
), particularly when cells are exposed to combinations of IFN (type II or type I) and agents that activate NF-κB (e.g. TNF-α) (47
). Importantly, the UPR was not seen in bone marrow macrophages from HLA-B7/human β2
m transgenic rats despite comparable expression of heavy chain and human β2
), consistent with biochemical evidence that HLA-B7 does not misfold (48
). We did not observe evidence for UPR activation in the human peripheral blood derived macrophages studied here. However, despite 24 h of exposure to IFN-γ we also did not see significant upregulation of HLA-B mRNA transcripts (data not shown). Further studies on protein expression and/or qPCR quantitation were not possible due to limitations in material. Increased expression of the UPR target gene, BiP/Hspa5, has been reported in synovial fluid mononuclear cells from spondyloarthritis patients compared to rheumatoid arthritis controls (50
). Since these cells had been exposed to a highly inflammatory milieu, which contains numerous cytokines, it may be necessary to stimulate peripheral blood-derived human macrophages from AS/spondyloarthritis patients to additional stimuli that provide robust HLA class I upregulation in order to definitively determine whether UPR activation occurs.
It will be important to confirm and extend these results by comparing additional AS patients with other disease controls, examining T and NK cells for their capacity to produce IFN-γ, and determining whether this low IFN-γ production contributes to Th17 development and IL-17 overexpression. Such studies will help to determine the specificity and consequences of IFN-γ gene dysregulation for the pathogenesis of spondyloarthritis.