Each condition was tested in three replicate wells

Each condition was tested in three replicate wells. measured by real-time RT-PCR were significantly higher in temporal arteries from 50 patients compared to 20 controls (4.35??4.06 vs 0.51??0.75; as described (10, 11, 28, 29) with or without neutralizing anti-human IL-12p40 mouse monoclonal Ab (10?g/ml R&D Systems), or dexamethasone (DXM) (0.5?g/ml, Sigma-Aldrich). Each condition was tested in three replicate wells. Biopsies were frozen in TRIzol reagent for RNA extraction. Statistical Analysis MannCWhitney test, Spearman correlation, and KaplanCMeier survival curves analyzed with log-rank test were used for statistical analysis Rabbit Polyclonal to SH2D2A using SPSS software, version PASW 18.0. Results IL-12/23p40 and IL-23p19 Expression is Increased in Temporal Arteries From Patients With GCA As shown in Figures ?Figures1A,B,1A,B, IL-12/23p40 mRNA and IL-23p19 mRNA concentrations were significantly increased in temporal arteries from untreated patients compared to control arteries (4.35??4.06 vs 0.51??0.75 relative units; cultured temporal artery biopsies. Several molecules related to Th1 and Th17 differentiation were investigated (9, 11, 30). Neutralization of IL-12/IL-23p40 tended to decrease IFN ARS-1323 mRNA and, slightly, IFN-induced chemokine CXCL11 and CXCL10, but not CXCL9, mRNAs in cultured arteries. IL17 mRNA also tended to decrease and no apparent effect on TNF expression was observed. Conversely IL-6 and IL-1 mRNA involved in Th17 differentiation tended to increase, possibly as a compensatory mechanism (Figure ?(Figure6).6). As shown in the same figure, DXM significantly reduced IFN, CXCL10, IL-17, IL-6, and IL-1 and tended to reduce CXCL9 and TNF. Open in a separate window Figure 6 Changes in gene expression by blocking IL-12/23p40 on cultured GCA biopsies. mRNA concentrations of IFN, CXCL11, CXCL10, CXCL9, IL-17, IL-6, IL-1, and tumor necrosis ARS-1323 factor in 10 cultured control arteries (negative biopsies, Neg Bx) vs 10 cultured GCA-involved arteries untreated or exposed to anti-human IL-12p40 mouse monoclonal Ab (anti-p40) (10?g/ml), or dexamethasone (DXM) (0.5?g/ml). Statistical comparisons were performed between histologically negative and GCA-involved arteries and between GCA-involved arteries and anti-p40 treated and DXM treated. Bars represent mean??SEM, *gene (encoding for IL12/23p40) is associated with increased genetic risk for both diseases, although the putative functional impact of this variant on expression remains unknown (41). A recent open-label trial with ustekinumab, a monoclonal antibody neutralizing IL-12/23p40, suggests benefit in a small series of patients with refractory/relapsing GCA (42). Analysis of the peripheral blood compartment revealed reduction in both Th1 and Th17 polarization in a patient with GCA upon ustekinumab treatment (43). However, when we analyzed the effects of blocking IL-12/23 p40 on involved tissue from GCA ARS-1323 patients, only a trend, consistent with its known biology, was observed. IL-12/23p40 inhibition tended to reduce IFN expression as well as expression of ARS-1323 IFN induced chemokines CXCL10 and 11 but not CXCL9. IL-12/23p40 blockade slightly reduced IL-17 expression although, interestingly, cytokines involved in Th17 differentiation such as IL-1 and IL-6 increased, possibly as a compensatory mechanism. Effects in tissue may be more complex than those observed in the peripheral compartment (43) and stimuli and interactions with other elements in the microenvironment may configure a protective niche. Although the inhibitory effect of the anti-IL-12/23p40 antibody used in our study may not be equivalent to that of ustekinumab, which has been generated for therapeutic purposes, recent studies suggest that neutralizing IL-12/23p19 may have more potent effects than blocking IL-12/23p40 in suppressing inflammatory activity in other diseases (44). Our functional model has some limitations such as isolation from a functional immune system or induction of changes by the culture itself in the expression of some inflammatory molecules (10). Moreover, lesions are often segmental in GCA arteries and this may have increased variability in responses. However, in spite of these limitations this model has been useful to evidence functional modifications after therapeutic intervention with various agents, including biologic agents (10, 11). The increasingly recognized diversity of subunits configuring the IL-12/IL-6 superfamily of cytokines to which IL-12 and IL-23 belong, as well as the multiple potential partnership of subunits and ARS-1323 receptor chains providing pro-inflammatory and anti-inflammatory stimuli depending on the specific combinations, has added an unexpected complexity to this system (34). Our results clearly indicate that additional combinations to the classical heterodimers IL-12 and IL-23 may occur in GCA and may lead to incomplete or compensated responses to the blockade of single subunits. Ethics Statement The study was approved by the Ethics Committee of Hospital Clnic (Barcelona). All subjects gave written informed consent in accordance with the Declaration of Helsinki. Author Contributions GE-F, EP-R, and MC conceived experiments and interpreted data, GE-F, EP-R, and EL carried out experiments. GE-F and AG-M collected patient data. GE-F, JH-R, SP-G, and MC provided patient care. GE-F and EP-R generated figures. GE-F and MC wrote the manuscript. All.