Induction of Cytoplasmic Rods and Rings Structures by Inhibition of the CTP and GTP Synthetic Pathway in Mammalian Cells

Background: Cytoplasmic filamentous rods and rings (RR) structures were identified using human autoantibodies as probes. In the present study, the formation of these conserved structures in mammalian cells and functions linked to these structures were examined. Methodology/Principal Findings: Distinct cytoplasmic rods (,3–10 mm in length) and rings (,2–5 mm in diameter) in HEp-2 cells were initially observed in immunofluorescence using human autoantibodies. Co-localization studies revealed that, although RR had filament-like features, they were not enriched in actin, tubulin, or vimentin, and not associated with centrosomes or other known cytoplasmic structures. Further independent studies revealed that two key enzymes in the nucleotide synthetic pathway cytidine triphosphate synthase 1 (CTPS1) and inosine monophosphate dehydrogenase 2 (IMPDH2) were highly enriched in RR. CTPS1 enzyme inhibitors 6-diazo-5-oxo-L-norleucine and Acivicin as well as the IMPDH2 inhibitor Ribavirin exhibited dose-dependent induction of RR in.95 % of cells in all cancer cell lines tested as well as mouse primary cells. RR formation by lower concentration of Ribavirin was enhanced in IMPDH2-knockdown HeLa cells whereas it was inhibited in GFP-IMPDH2 overexpressed HeLa cells. Interestingly, RR were detected readily in untreated mouse embryonic stem cells (.95%); upon retinoic acid differentiation, RR disassembled in these cells but reformed when treated with Acivicin

Autoimmune Hepatitis During Ledipasvir/Sofosbuvir Treatment of Hepatitis C: A Case Report

Abstract We report the case of a woman with chronic hepatitis C and idiopathic thrombocytopenic purpura (ITP) who developed autoimmune hepatitis (AIH) during antiviral therapy with ledipasvir (LDV)/sofosbuvir (SOF). The onset of acute hepatitis rose two weeks after starting treatment with LDV/SOF when HCV‐RNA tested negative, suggesting a link between rapid HCV clearance and de novo autoimmune diseases. Conclusion: This case report proposes new immunologic scenarios in patients with hepatitis C virus (HCV) with laboratory or clinical signs of autoimmunity during direct‐acting antiviral (DAA) therapy

The expression of rods versus rings was not correlated with the cell cycle.

<p>HEp-2 cells were co-stained with rabbit anti-CENP-F/Alexa 568 goat anti-rabbit IgG (A) and human anti-RR serum 604/Alexa 488 goat anti-human IgG (B). G1 cells had little or no CENP-F staining, whereas late S/G2 and mitotic (M) cells showed strong staining for CENP-F. Nuclei counterstained with DAPI (blue). Bar, 10 µm.</p

Inhibition of CTPS1-induced formation of RR in mouse primary cardiomyocytes, fibroblasts, and endothelial cells.

<p>Mouse primary cardiomyocytes prepared together with fibroblasts and endothelial cells, were treated with 2 mM DON and cultured for 24 h. Cells were co-stained with human anti-RR serum IT2006 (green) and mouse anti-actinin monoclonal antibody (red). Nuclei counterstained with DAPI (blue). Actinin-positive cardiomyocytes (A, B, red) as well as actinin-negative fibroblast or endothelial cells (A, C) all show distinct rods. The percentage of cells with RR displayed is shown in the lower right corner with the total number of cells counted indicated in parentheses (A, all cells; B, actinin-positive cardiomyocytes only; C; actinin-negative fibroblast and endothelial cells). Bar, 10 µm.</p

The distribution of cytoplasmic rods and rings was independent of the Golgi complex and centrosomes, and these structures were not enriched in tubulin or vimentin.

<p>(A) Merged image of HEp-2 co-stained with human anti-RR prototype serum 604/Alexa 488 goat anti-human Ig (green) and rabbit anti-giantin (Golgi marker)/Alexa 568 goat anti-rabbit Ig (red). Rods are often presented adjacent (short arrows) or perpendicular (long arrows) to the nucleus while rings (arrowheads) are found either 1 or 2 to a cell. M, mitotic cell. HEp-2 cells were also co-stained with serum 604/Alexa 488 goat anti-human Ig (green, B,E,H) and different cytoplasmic markers using mouse anti-tubulin (C), anti-vimentin (F), anti-pericentrin (I), followed by Alexa 568 goat anti-mouse Ig (red). Nuclei were counterstained with DAPI (blue). Bar, 10 µm.</p

Inhibition of CTPS1-induced formation of RR in several human cancer cell lines.

<p>Induction of RR observed in human cancer cell lines HeLa (A), CAL 27 (B), THP-1 (C), and HCT116 (D) when treated with 2 mM DON in culture for 24 h, fixed, and co-stained with human anti-RR serum It2006 (green) and rabbit anti-giantin (red). Untreated controls showed few or no RR. The percentage of cells with RR displayed for each condition is shown in the upper right corner with the total number of cells counted indicated in parentheses. Bar, 5 µm.</p

Inhibition of CTPS1- or IMPDH2-induced formation of rods and rings.

<p>Untreated HEp-2 cells (A) and cells treated with inhibitors 2 mM DON (CTPS inhibitor, B), 2 mM Acivicin (CTPS inhibitor, C), or 2 mM Ribavirin (IMPDH2 inhibitor, D) for 24 h were co-stained with human anti-RR serum It2006 (green) and rabbit anti-giantin (red). Nuclei were counterstained with DAPI (blue). The percentage of cells with RR displayed for each experiment is shown in the lower right corner with the total number of cells counted indicated in parentheses. Bar, 10 µm.</p

CTPS1 and IMPDH2 were highly enriched in RR and human anti-RR prototype serum It2006 recognized IMPDH2.

<p>(A) Enrichment of CTPS1, detected by rabbit anti-CTPS1 (green), to RR (arrows) identified by It2006 (red). Nuclei were counterstained with DAPI (blue). (B) IMPDH2 stained by rabbit anti-IMPDH2 (red) localized to RR (arrows) detected by It2006 (green). Bar, 10 µm. (C) Immunoprecipitation (IP) analysis using an extract of [³⁵S]-methionine-labeled K562 cells and rabbit anti-IMPDH2, It2006, and a second human anti-RR serum 609. It2006 recognized a 55 kDa protein band that co-migrated with IMPDH2 immunoprecipitated by rabbit anti-IMPDH2. Serum 609 did not immunoprecipitate the 55 kDa protein. (D) IP-Western blot demonstrated that serum It2006 immunoprecipitated IMPDH2, which was recognized by both mouse monoclonal and rabbit anti-IMPDH2 antibodies. NHS, control normal human serum.</p

Differential time requirements for RR induction in HEp-2 cells treated with different CTPS1 and IMPDH2 inhibitors at 2 mM concentration.

1<p>Ribavirin induced RR as early as 30 min and between 1 to 24 h, nearly all cells had RR (p<0.0001, 0 min vs all time points and 30 min vs 1 to 24 h). There is no statistical significance between 1 to 24 h after Bonferroni's adjustment;</p>2<p>Induction of RR by DON was slower than Ribavirin (p<0.0001, 1, 2, and 3 h; ns, 30 min and 24 h) and the percentage of RR positive cells gradually increased after 1–3 h and was nearly all cells at 24 h.</p>3<p>Acivicin had similar pattern to DON in which RR positive cells gradually increased after 1 h and nearly all cells at 24 h.</p>4<p>Ribavirin vs DON and DON vs Acivicin, ns; Ribavirin vs Acivicin, p = 0.03;</p>5<p>Ribavirin vs DON and Ribavirin vs Acivicin, p<0.0001; DON vs Acivicin, ns;</p>6<p>Ribavirin vs DON, DON vs Acivicin, Ribavirin vs Acivicin, p<0.0001;</p>7<p>ns between Ribavirin, DON, and Acivicin.</p

Uninduced mouse embryonic stem cells expressed predominantly cytoplasmic rings that were disassembled during retinoic acid-induced differentiation and reassembled when treated with Acivicin.

<p>(A) Mouse 3T3 cells for comparison shown with both rods (80–90%, arrowhead) and rings (10–20%, arrows). (B) Undifferentiated mouse ESCs shown with ∼90% rings (arrows) and few rods. (C) ESCs treated with RA for 4 days showed no RR. (D) RR were induced in these RA-differentiated cells by treatment with 2 mM Acivicin for 24 h. Cells were stained with human anti-RR serum It2006 (green, A–D) and co-stained with rabbit anti-giantin (red, A, B only). Nuclei counterstained with DAPI (blue). The percentage of ESCs with RR displayed for each condition is shown in the lower right corner with the total number of cells counted indicated in parentheses. Bar, 5 µm.</p