Serum Neopterin Levels among Hepatitis C-Positive Living-Donor Renal Transplant Recipients

Background The role of neopterin as a marker of cell-mediated immunity for immunological monitoring after transplantation is of great potential interest. Neopterin levels among hepatitis C virus (HCV)-positive recipients of living-donor renal transplantation (LDRT) have not been previously described. Methods Twenty-two HCV-positive (group I) and 10 HCV-negative (group II) recipients of LDRT were serially monitored for serum neopterin levels by enzyme-linked immunosorbent assay (ELISA). Group I patients were monitored thrice, ie, before transplantation, day 10, and 6 months post transplantation, while group II patients were monitored twice (day 10 and 6 months post transplantation). Peripheral blood T-lymphocyte subsets (CD3, CD4, CD8, CD4 + CD25 + , CD 16+56 ) and Thl/Th2 cytokines were monitored concomitantly by flow cytometry. Results Ten days post transplantation, there was a significant increase in neopterin and neopterin/creatnine levels among group I patients. There was a positive correlation between activated T-lymphocyte (CD4 + CD25 + ) and neopterin early post transplantation (day 10). Th2 cytokines IL-10 and IL-5 showed a positive correlation with neopterin levels on day 10 and 6 months post transplantation, respectively. Neopterin levels did not show association with either HCV viral load or allograft rejection among our study cohort. Conclusion Increased monocyte/macrophage activation with elevated serum neopterin was detected among group I patients on day 10 post transplantation, but it could not predict rejection. It appears that IL-10 either from a regulatory or nonregulatory source helps in the maintenance of stable graft early post transplantation. Further, it would be of interest to assess the role of neopterin in chronic allograft nephropathy and long-term graft outcome

Endogenous IL-22 Plays a Dual Role in Arthritis: Regulation of Established Arthritis via IFN-γ Responses

<div><p>Objective</p><p>IL-22 is elevated in patients with inflammatory arthritis and correlates with disease activity. IL-22 deficient mice have reduced incidence of arthritis. Recombinant IL-22 restrains progression of arthritis via increase in IL-10 responses when administered prior to onset of arthritis. These findings imply a possible dual role of IL-22 in inflammatory arthritis depending on the phase of arthritis. Experiments outlined here were designed to elucidate the contribution of endogenous IL-22 before and after the onset of arthritis.</p><p>Methods</p><p>Collagen induced arthritis (CIA) was induced in DBA1 or IFN-γ deficient mice following immunization with collagen and complete Freund's adjuvant. Anti-IL-22 antibody or isotype control were administered prior to or after onset of arthritis and disease progression assessed by clinical scoring and histopathology. IL-22, IL-17 and IFN-γ responses were measured by ELISA and flowcytometry. Anti-collagen antibody responses were analyzed by ELISA. Expression of IL-22R1 in CD4+ cells was elucidated by flowcytometry and real time PCR.</p><p>Results</p><p>Collagen specific IL-22 responses were expanded during arthritis and IL-22 producing cells were discrete from IL-17 or IFN-γ producing cells. Neutralization of IL-22 after onset of arthritis resulted in significant increase in Th1 responses and significantly reduced severity of arthritis. CD4+ cells from arthritic mice showed increased surface expression of IL-22R1. In vitro, CD4+T cells cultured with antigen presenting cells in the presence or absence of IL-22 suppressed or induced IFN-γ, respectively. The protective effect of anti-IL-22 was reversed in IFN-γ deficient mice. Moreover, administration of anti-IL-22 prior to onset of arthritis augmented arthritis severity.</p><p>Conclusion</p><p>We show for the first time that IL-22 plays a dual role: protective prior to the onset of arthritis and pathogenic after onset of arthritis. The pathogenic effect of IL-22 is dependent on suppression of IFN-γ responses. IL-17 responses remained unchanged with the administration of anti-IL22 antibody. IL-22R1 is upregulated on CD4+T cells during arthritis and regulates IFN-γ in T cells.</p></div

Administration of anti-IL-22 antibody is associated with altered B and T cell responses.

<p>Experimental design was the same as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093279#pone-0093279-g002" target="_blank">figure 2</a>. <b>A.</b> Sera from mice receiving either anti-IL-22 antibody or isotype control were analyzed for anti-collagen IgG1, IgG2a and IgG2b antibodies by ELISA. <b>B, C & D:</b> Splenocytes from mice receiving anti-IL-22 or isotype control were stimulated with anti-CD3 (5 ug/ml for 3 days) or collagen (100 ug/ml for 7 days) and IL-17A, IFN-γ and IL-10 were measured in culture supernatants by ELISA. For some experiments IL-10 was measured by real-time PCR from splenocytes of mice receiving anti-IL-22 antibody or isotype control and expressed as fold change over GAPDH. Data is representative of 2 independent experiments (8 mice per group) with each dot representing an individual mouse. <b>E:</b> Single cell suspensions of the paws from mice receiving anti-IL22 or isotype control were stimulated with anti-CD3 (5 ug/ml for 3 days) and IL-17A was measured in culture supernatants by ELISA. Each dot represents an individual mouse.</p

Increased expression of IL-22R1 on CD4 cells from arthritic mice and regulation of IFN-γ responses in T cells by IL-22.

<p><b>A:</b> Expression of IL-22R1 and IL-10R2 were analyzed by realtime PCR in CD4+, CD19+, and CD11c+ cells enriched from splenocytes from various phases of arthritis. GAPDH was used as internal control. Data represented as fold change over GAPDH expression. Data is representative of 3 independent experiments. <b>B:</b> Splenocytes from various phases of arthritis were stained for CD4, and IL-22R1 and analyzed by flowcytometry. RatIgG was used as isotype control. Data shown is gated on mononuclear cells based on forward and side scatter followed by gating on CD4+ cells. Data is representative of 3 independent experiments. <b>C& D:</b> CD4 T, CD11c, and B cells were enriched from splenocytes of arthritic mice and co-cultured with recombinant IL-22 (100 ng/ml, Insight Genomics, USA), or anti-IL-22 antibody (10 ug/ml) or mouse isotype control (10 ug/ml, Biolegend) and/or collagen for 7 days. Supernatants were analyzed for IFN-γ (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093279#pone-0093279-g005" target="_blank">Fig. 5C</a>) or IL-17A (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093279#pone-0093279-g005" target="_blank">Fig. 5D</a>) by ELISA. Data is representative of two independent experiments.</p

The protective effect of anti-IL-22 is reversed in IFN-γ deficient mice.

<p><b>A:</b> 16 female B6.129S7-ifng^tm1Ts (B6.IFNγKO) mice were immunized with collagen and CFA. Mice with total score ≥1 and arthritis duration of ≥4 days were randomized to receive anti-IL-22 antibody (7–8 mice) or isotype control (7–8 mice) intraperitoneally at 100 ug/mouse/day for 8–9 days. Mice were scored for clinical arthritis every other day. Data shows cumulative arthritis score in 2 groups over time. <b>B:</b> Histologic scoring of paws for various aspects of joint damage and inflammation assessed after H&E staining. Data is from 4 mice per group. <b>C:</b> Hematoxylin and eosin staining of representative paws from the two groups of mice, magnification of 10×.</p

Induction of IL-22 response during arthritis.

<p><b>A:</b> DBA mice were immunized with collagen and CFA. Splenocytes (5×10⁶/ml) from naïve mice, mice during initiation phase (2 weeks following immunization with collagen and CFA) or arthritic mice were stimulated with collagen (100 ug/ml) for 7 days or unstimulated. Supernatants were analyzed for IL-22 by ELISA. Data shown is representative of 3 independent experiments with 3 mice per experiment. <b>B:</b> Splenocytes (5×10⁶/ml) from arthritic mice were stimulated with varying concentrations of collagen (0, 10, 50 or 100 ug/ml) for 7 days and IL-22 was measured in supernatant by ELISA. Data shown is representative of 3 independent experiments with 3 mice per experiment. <b>C:</b> Splenocytes from arthritic mice were stimulated with PMA/ionomycin and Brefeldin A for 6 hours, stained intra-cellularly for IL-17, IFN-γ or IL-22 and analyzed by flow-cytometry. Data shown is gated on mononuclear cells based on forward and side scatter. Data is representative of 3 independent experiments with 3 mice per experiment.</p

Neutralization of IL-22 after onset of arthritis reduces severity of arthritis.

<p><b>A.</b> 16 DBA mice were immunized with collagen and CFA. Mice with total score ≥1 and arthritis duration of ≥4 days were randomized to receive anti-IL-22 antibody (7–8 mice) or isotype control (7–8 mice) intraperitoneally at 100 ug/mouse/day for 10–12 days. Mice were scored for clinical arthritis every other day by a person blinded to interventions. Data shows cumulative arthritis score in 2 groups over time. Data shown is representative of 2 independent experiments. <b>B.</b> Histologic scoring of paws for various aspects of joint damage and inflammation assessed after H&E staining. Data is from 4 mice per group. <b>C.</b> Data shows the histology of hematoxylin and eosin staining of representative paws from the two groups of mice, magnification of 10×.</p