Specific cytokines/chemokines induce GWB in THP-1 cells and human PBMCs
Since our previous work demonstrated that GWB can be used as biomarkers for miRNA activity (Pauley KM, unpublished data), we began to examine a variety of cytokines and chemokines for their ability to stimulate miRNA activity in human monocytic THP-1 cells. THP-1 cells were treated with 10 ng/ml TNF-α, IFN-α, IFN-β, IFN-γ, IL-12p70, M-CSF, IL-4, IL-10, or 25 ng/ml MCP-1 for 4 hours. Indirect immunofluorescence (IIF) was performed using a human anti-GWB serum to detect GWB in the cells. As shown in Figure , the proinflammatory cytokines/chemokines TNF-α, IFN-α, IFN-β, IFN-γ, and MCP-1 resulted in a significant increase in the number of GWB per cell compared with untreated cells cultured in parallel (P < 0.0001, as determined using one-way analysis of variance). However, IL-12p70, M-CSF, IL-4, and IL-10 had no significant effect on the number of GWB. TNF-α elicited the strongest response in THP-1 cells, with fourfold increase in the average number of GWB per cell (Figure ). These experiments were repeated at least three times, with reproducible results each time.
Figure 1 TNF-α treatment results in increased number of GWB in THP-1 and human PBMCs. (a) THP-1 cells were treated with 10 ng/ml TNF-α, IFN-α, IFN-β, IFN-γ, IL-12p70, M-CSF, IL-4, IL-10, or 25 ng/ml MCP-1 for 4 hours. IIF (more ...)
Next, we decided to examine the effect of TNF-α stimulation on human PBMC GWB. GWB staining using human PBMCs from a healthy donor, after 4 hours stimulation with TNF-α (1 ng/ml), is shown. Similar to THP-1 cells, the number of GWB per cell increased 3.5-fold after TNF-α stimulation of PBMCs (Figure ; P < 0.0001, as determined by Mann Whitney test). These data indicated that THP-1 cells may be suitable substitutes for human PBMCs in some of the subsequent experiments.
RA patient PBMCs exhibit increased expression of miR-146a, miR-155, miR-132, and miR-16
In Figure we showed that TNF-α is a potent inducer of GWB and therefore miRNA activity. Our preliminary studies and work from other investigators have confirmed that TNF-α stimulation induces the expression of certain miRNAs, including miR-146a and miR-155 [20
]. Based on these data and the important role played by TNF-α in RA pathogenesis and therapies, we began to investigate the expression levels of miRNA in RA patients as compared with those in healthy and disease control individuals. PBMCs were obtained from patients (n
= 17 RA patients and n
= 4 disease control individuals) and healthy donors (n
= 9) and isolated by Ficoll density-gradient centrifugation. Initially, RA PBMCs were monitored by IIF for GWB; however, we did not observe an increased number of GWB in RA compared with healthy control individuals (not shown). This discrepancy could be due to limited sensitivity in the quantitation of GWB.
As shown in Figure , the average relative expression levels of miR-146a, miR-155, miR-132, and miR-16 were 2.6-, 1.8-, 2.0-, and 1.9-fold, respectively, higher for RA patients than for healthy control individuals (P < 0.01 for miR-146a and P < 0.05 for miR-155, miR-132, and miR-16, as determined by one-way analysis of variance). The expression of miRNA let-7a was not significantly different between RA patients and healthy control individuals (Figure ). Disease control miRNA expression resembled that in healthy control individuals.
Figure 2 RA patients exhibit aberrant expression of miR-146a, miR-155, miR-132 and miR-16 versus healthy controls. (a) RNA was isolated from healthy control individuals (n = 9), disease control individuals (n = 4), and RA patient (n = 17). PBMCs and relative expression (more ...)
To examine the relationship between RA disease activity and miRNA expression levels, patients were classified into inactive/remission and active patients, based on C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) values. Three patients with normal CRP and ESR (Table ) were classified as inactive, whereas eight patients with elevated CRP and/or ESR (Table ) were classified as active (Figure ). Those patients with incomplete or no available data were omitted (Table ). miRNA expression levels were compared between the groups. Interestingly, high miR-146a and miR-16 expression levels appeared to correlate with active disease, whereas low expression level correlated with inactive disease (Figure ; P < 0.05, as determined by t-test). These data indicates that miR-146a and miR-16 expression levels may be a useful marker of RA disease activity. Further studies involving a larger patient cohort are needed to determine fully whether monitoring miRNA expression as a marker for disease activity can improve upon CRP or ESR measurements.
Figure shows the miRNA expression levels in two samples from a single RA patient collected over a 2-month interval, during which time this patient's CRP and ESR values increased despite methotrexate treatment. The miRNA levels of this patient were largely unchanged over the 2-month interval, remaining elevated compared with those in healthy control individuals. This indicates that the elevated miRNA expression in this patient may reflect the patient's lack of improvement, as indicated by the increased CRP and ESR values. In this patient, miR-146a, miR-155, and miR-132 expression levels were stable over this time period, whereas the expression levels of miR-16 and miRNA let-7a increased by approximately 3.5-fold and 2.4-fold, respectively. A larger patient population must be examined in order to determine whether miRNA expression levels may be indicative of treatment efficacy.
To further analyze the increased miRNA expression exhibited by these RA patients, we compared miR-146a, miR-155, and miR-132 expression levels with patient clinical and demographic data (Table ) and found no significant trends or correlations between high expression levels and age, race, or medications. Patients receiving no medications at the time of miRNA analysis exhibited the same trend toward elevated miRNA expression, indicating that treatment with medications is not responsible for the increased miRNA expression in RA patients.
Recently, two reports [26
] showed increased miR-146 and miR-155 expression levels in RA synovial tissue and fibroblasts. Stanczyk and coworkers [27
] reported a fourfold increase in miR-146a expression and a twofold increase in miR-155 expression in RA synovial fibroblasts compared with osteoarthritis synovial fibroblasts. They also demonstrated that miR-155 expression can repress the induction of matrix metalloproteinases 3 and 1, indicating that miR-155 may be involved in modulating the destructive properties of RA synovial fibroblasts. However, in that report, miR-155 expression from RA PBMCs was not significantly different from that in control PBMCs. This discrepancy could be due to differences in experimental techniques or patient populations. Nakasa and colleagues [26
] also reported an approximately fourfold increase in miR-146a expression in RA synovial tissue. Our data demonstrate that RA patient PBMCs exhibit elevated miRNA expression in a similar manner to RA synovial tissue, with a 2.6-fold increase in miR-146a expression and a 1.8-fold increase in miR-155 expression. Because of the invasiveness involved in collecting samples, monitoring miRNA expression in RA synovial tissue is, in most cases, limited to extremely severe disease in patients undergoing joint surgery or replacement. Because blood collection is not invasive, this allows for easy sample collection over time, which is a distinct advantage when monitoring disease activity and treatment efficacy.
Monocyte/macrophage population of RA PBMCs exhibits increased miRNA expression
Because PBMCs are composed of a mixed cell population, the two main components of which are monocytes/macrophages and lymphocytes, we wished to determine which cell population in RA patients exhibits increased miRNA expression. PBMCs were isolated from RA patients (n = 2) and incubated in tissue culture dishes at 37°C for 1 hour. The monocyte/macrophage population adhered to the dish, whereas lymphocytes remained in suspension. The adherent cells were washed five times with sterile PBS, and the nonadherent cells were collected and washed with sterile PBS. The purity of the adherent population was approximately 80%, as determined by microscopy. RNA was isolated from the cells, miRNA expression was analyzed by qRT-PCR, and the data were normalized within the total group of patient and control samples.
The expression levels of miR-146a, miR-155, miR-132, and miR-16 were 2.8-, 1.6-, 4.2-, and 3.4-fold higher, respectively, in monocytes than in lymphocytes (Figure ). Let-7a expression was similar between monocytes and lymphocytes (not shown). Figure shows the average expression (miR-146a, miR-155, miR-132, and miR-16 combined) for the monocyte and lymphocyte populations of two RA patients (P < 0.02, as determined by Mann-Whitney test). These findings suggest that monocytes/macrophages contribute to the increased miRNA expression observed in RA patients more than lymphocytes, but further studies must be performed to confirm this observation.
Figure 3 Monocyte/macrophage fraction of PBMCs exhibit increased miRNA expression compared with lymphocyte fraction. PBMCs were collected from RA patients and separated into monocyte/macrophage and lymphocyte populations by allowing the monocytes/macrophages to (more ...)
TRAF6 and IRAK-1 expression is similar between RA patients and control individuals
Because most of the RA patients exhibited increased expression of miR-146a compared with healthy and disease control individuals, we decided to examine the expression of two confirmed targets of miR-146a, namely TRAF6 and IRAK-1 [21
]. TRAF6 and IRAK-1 mRNA expression levels were analyzed by qRT-PCR (Figure ). RA patients exhibited increased miR-146a production compared with control individuals, and we therefore expected to observe decreased TRAF6 and/or IRAK-1 expression in RA patients as compared with control individuals. However, TRAF6 and IRAK-1 mRNA expression levels were very similar between RA patients and control individuals, and overall the mRNA levels of TRAF6 and IRAK-1 did not exhibit the same degree of variability between patients that we observed with miRNA expression. This may indicate that TRAF6 and IRAK-1 transcripts are under other levels of control. To confirm this discrepancy, we analyzed TRAF6 and IRAK-1 protein levels by IIF in one healthy control individual and one RA patient whose miR-146a level was increased (Figure ). PBMCs were processed for IIF as previously described and were stained for TRAF6 and IRAK-1. Image J software was used to quantify the relative level of fluorescence for at least 20 cells. As shown in Figure , there was no significant difference in TRAF6 or IRAK-1 protein levels between the RA patient and healthy control individual, which is consistent with the mRNA analysis.
Figure 4 TRAF6 and IRAK-1 expression levels are similar between RA patients, healthy controls, and disease controls. RNA was isolated from PBMCs from healthy control individuals (n = 9), disease control individuals (n = 4) and RA patients (n = 14), and mRNA expression (more ...)
It is interesting to speculate that this lack of regulation of TRAF6/IRAK-1 by miR-146a could play a role in RA pathogenesis, especially because it has been reported that inhibition of IRAK-1 using antisense oligonucleotides results in decreased LPS-induced cytokine production [28
], and our preliminary data have shown that transfection of miR-146a into THP-1 monocytes results in knockdown of TRAF6 and IRAK-1 expression and inflammatory cytokine production (Pauley KM and coworkers, unpublished data). To investigate this possibility further, we transfected siRNA targeting TRAF6 and/or IRAK-1 into THP-1 cells. The knockdown efficiency was determined by analyzing TRAF6 and IRAK-1 mRNA levels by qRT-PCR, and at least 80% and 60% knockdown was achieved for TRAF6 and IRAK-1, respectively (Figure ). Two days after transfection, knockdown and control cells were treated with 1 μg/ml LPS for 24 hours. Culture supernatants were collected and cytokines/chemokines were quantitatively detected using a human cytokine multiplex assay. TNF-α production was drastically reduced in the TRAF6 and/or IRAK-1 deficient cells compared with mock transfected cells (Figure ), whereas MCP-1 production was not affected by TRAF6 or IRAK-1 knockdown (Figure ).
Figure 5 Knockdown of TRAF6 and/or IRAK-1 results in decreased TNF-α production in THP-1 cells. THP-1 cells were transfected with siRNA targeting TRAF6 and/or IRAK-1. (a) 48 hours after transfection, mRNA levels of TRAF6 and IRAK-1 were analyzed by qRT-PCR (more ...)
These data demonstrate that TRAF6 and IRAK-1 are required for the production of TNF-α in THP-1 cells. Taken together, it is reasonable to hypothesize that the absence of TRAF6/IRAK-1 regulation by miR-146a in RA patients could contribute to the prolonged production of TNF-α that many of these patients exhibit. Furthermore, it would be interesting to investigate the expression patterns of miR-146a, TRAF6, and IRAK-1 in RA patients who are responsive to anti-TNF-α therapy versus those who are not responsive. Clearly, further studies are needed to elucidate the role played by miR-146a regulation in RA pathogenesis and the mechanism by which TRAF6/IRAK-1 escape miR-146a regulation.