The impact of C-reactive protein levels on the effectiveness of upadacitinib in patients with rheumatoid arthritis: a 12-month prospective, non-interventional German study.

OBJECTIVES: We investigated whether the effectiveness of upadacitinib in rheumatoid arthritis (RA) treatment is affected by baseline CRP levels in a real-world setting. METHODS: UPwArds was a prospective, non-interventional study. Patients had moderate-to-severe RA and an inadequate response or intolerance to ≥1 disease-modifying anti-rheumatic drug (DMARD). The primary endpoint was clinical remission (Clinical Disease Activity Index [CDAI] ≤2.8) at 6 months. Secondary endpoints at 12 months included clinical remission and low disease activity assessed by CDAI and Simple Disease Activity Index criteria, DAS28-CRP <2.6/≤3.2, and patient-reported outcomes. The impact of baseline CRP levels (normal vs. above the upper limit of normal [ULN]) on primary and secondary endpoints was evaluated. The effect of concomitant MTX and prior inadequate response to biologic or targeted synthetic DMARDs (b/tsDMARD-IR) on the effectiveness of upadacitinib was also assessed. Safety was evaluated through 12 months. RESULTS: 518 patients were included in the effectiveness analyses. At 6 months, 24.4% of patients achieved the primary endpoint (CDAI ≤2.8). At 12 months, similar proportions of patients with normal CRP and CRP above the ULN at baseline achieved CDAI ≤2.8 (27.3% and 29.1%) and other key secondary endpoints. The effectiveness of upadacitinib was comparable with and without concomitant MTX and in b/tsDMARD-naive and b/tsDMARD-IR patients. The safety results were consistent with the known safety profile of upadacitinib; no new safety signals were identified. CONCLUSIONS: Upadacitinib therapy was effective for RA in a real-world setting. Baseline CRP levels had no significant impact on the effectiveness of upadacitinib.

Quantitative phosphoproteomics reveals link between Helicobacter pylori infection and RNA splicing modulation in host cells.

The Gram-negative, spiral-shaped bacterium Helicobacter pylori is a common human pathogen that causes chronic inflammation of the human gastric mucosa, leading to peptic ulceration and/or gastric cancer. Here, we analyzed changes in the phosphoproteome of gastric epithelial cells (AGS) upon infection with H. pylori using a combination of SILAC, phosphoprotein enrichment, 2-DE, and MALDI TOF/TOF-MS. From a total of 526 spots we identified 391 protein species (143 proteins) and quantified 332 (127 proteins). Nearly, one-third of the identified proteins (40/143) were associated with the spliceosome or RNA splicing. The abundance of 20 proteins was altered by H. pylori infection, in particular, a number of serine arginine-rich (SR) proteins involved in the regulation and control of alternative splicing. Importantly, the combined methodologies enabled the detection of infection-dependent protein species-specific regulation, suggesting functional modulation of individual protein species. These findings reveal unexpected new insights into the mechanisms of host cell manipulation by H. pylori, which are likely associated with gastric pathologies, including gastric cancer.

Proteomic identification of secreted proteins of Propionibacterium acnes.

BACKGROUND: The anaerobic Gram-positive bacterium Propionibacterium acnes is a human skin commensal that resides preferentially within sebaceous follicles; however, it also exhibits many traits of an opportunistic pathogen, playing roles in a variety of inflammatory diseases such as acne vulgaris. To date, the underlying disease-causing mechanisms remain ill-defined and knowledge of P. acnes virulence factors remains scarce. Here, we identified proteins secreted during anaerobic cultivation of a range of skin and clinical P. acnes isolates, spanning the four known phylogenetic groups. RESULTS: Culture supernatant proteins of P. acnes were separated by two-dimensional electrophoresis (2-DE) and all Coomassie-stained spots were subsequently identified by MALDI mass spectrometry (MALDI-MS). A set of 20 proteins was secreted in the mid-exponential growth phase by the majority of strains tested. Functional annotation revealed that many of these common proteins possess degrading activities, including glycoside hydrolases with similarities to endoglycoceramidase, ß-N-acetylglucosaminidase and muramidase; esterases such as lysophospholipase and triacylglycerol lipase; and several proteases. Other secreted factors included Christie-Atkins-Munch-Petersen (CAMP) factors, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and several hypothetical proteins, a few of which are unique to P. acnes. Strain-specific differences were apparent, mostly in the secretion of putative adhesins, whose genes exhibit variable phase variation-like sequence signatures. CONCLUSIONS: Our proteomic investigations have revealed that the P. acnes secretome harbors several proteins likely to play a role in host-tissue degradation and inflammation. Despite a large overlap between the secretomes of all four P. acnes phylotypes, distinct differences between predicted host-tissue interacting proteins were identified, providing potential insight into the differential virulence properties of P. acnes isolates. Thus, our data presents a rich resource for guiding much-needed investigations on P. acnes virulence factors and host interacting properties.

Analysis of T4SS-induced signaling by H. pylori using quantitative phosphoproteomics.

Helicobacter pylori is a Gram-negative bacterial pathogen colonizing the human stomach. Infection with H. pylori causes chronic inflammation of the gastric mucosa and may lead to peptic ulceration and/or gastric cancer. A major virulence determinant of H. pylori is the type IV secretion system (T4SS), which is used to inject the virulence factor CagA into the host cell, triggering a wide range of cellular signaling events. Here, we used a phosphoproteomic approach to investigate tyrosine signaling in response to host-pathogen interaction, using stable isotope labeling in cell culture (SILAC) of AGS cells to obtain a differential picture between multiple infection conditions. Cells were infected with wild type H. pylori P12, a P12Δ CagA deletion mutant, and a P12Δ PAI deletion mutant to compare signaling changes over time and in the absence of CagA or the T4SS. Tryptic peptides were enriched for tyrosine (Tyr) phosphopeptides and analyzed by nano-LC-Orbitrap MS. In total, 85 different phosphosites were found to be regulated following infection. The majority of phosphosites identified were kinases of the MAPK family. CagA and the T4SS were found to be key regulators of Tyr phosphosites. Our findings indicate that CagA primarily induces activation of ERK1 and integrin-linked factors, whereas the T4SS primarily modulates JNK and p38 activation.

The Helicobacter pylori virulence effector CagA abrogates human ß-defensin 3 expression via inactivation of EGFR signaling.

Antimicrobial peptides are constituents of the first-line innate mucosal defense system that acts as a barrier to establishment of infection. The highly successful human gastric pathogen, Helicobacter pylori, is able to persistently colonize its host despite inducing expression of several antimicrobial peptides, including human ß-defensin 3 (hBD3). We find that hBD3 is highly active against H. pylori in vitro and is rapidly induced during early infection via EGFR-dependent activation of MAP kinase and JAK/STAT signaling. However, during prolonged infection, hBD3 was subsequently downregulated by the H. pylori virulence determinant CagA. Upon translocation into host cells, CagA activated the cellular tyrosine phosphatase, SHP-2, terminating EGFR activation and downstream signaling and increasing bacterial viability. Chemical inhibition and knockdown of SHP-2 expression rescued hBD3 synthesis and bactericidal activity. Thus, we reveal how cagPAI-positive H. pylori strains use CagA to evade a key innate mucosal defense pathway to support the establishment of persistent infection.

Helicobacter pylori-induced modification of the histone H3 phosphorylation status in gastric epithelial cells reflects its impact on cell cycle regulation.

Post-translational modifications of core histones are important components of the epigenetic landscape. Recent investigations of bacterial or toxin-induced effects on histone phosphorylation and acetylation in host cells have linked the changes to transcriptional alterations of key cellular response pathways. However, these changes may have other reasons and functional consequences. Here, we show that infection of gastric epithelial cell lines with the carcinogenic bacterium Helicobacter pylori leads to changes in histone H3 phosphorylation: type IV secretion system (T4SS)-dependent decreases of H3 phosphorylation levels at serine 10 (pH3Ser10) and threonine 3 (pH3Thr3) were observed. Immunofluorescence experiments with pH3Ser10 and cyclin B1 revealed that a H. pylori-induced transient pre-mitotic arrest was responsible for the observed reduction. This causal link was substantiated further by showing that H. pylori causes a strong decrease of the cell division cycle 25 (CDC25C) phosphatase. As a consequence, mitotic histone H3 kinases such as vaccinia-related kinase 1 (VRK1) and Aurora B were not fully activated in infected cells. We show that VRK1 activity, measured using a kinase activity assay, was reduced after H. pylori infection by approximately 40%. Moreover, overexpression of VRK1, but not Aurora B, compensated for the H. pylori-induced decrease of pH3Ser10. Rephosphorylation of H3Ser10 was IkappaB kinase alpha (IKKalpha)-dependent and occurred at later time points of infection. Taken together, our work highlights the impact of bacterial pathogens on host cell chromatin; this modulation reflects the subversion of key cellular processes such as cell cycle progression.