Original Contribution 2 PGD 2 and PGE 2 regulate gene expression of Prx 6 in primary macrophages via Nrf2

Prostaglandin 26 Free radicals 27 Peroxiredoxin 6 (Prx 6) is a bifunctional enzyme with both glutathione peroxidase and acidic Ca 2+ -28 independent phospholipase A 2 activities. We have recently shown that exposure of murine bone marrow-29 derived macrophages to LPS and IFN-γ leads to induction of COX-2 expression and secretion of PGE 2 , up-30 regulating Prx 6 mRNA levels. This study was designed to investigate various prostaglandins (PGs) for their 31 ability to induce gene expression of Prx's, in particular Prx 6, and to determine the underlying regulatory 32 mechanisms. We provide evidence that both conventional and cyclopentenone PGs enhance Prx 6 mRNA 33 expression. Treatment with either activators or inhibitors of adenylate cyclase as well as cAMP analogs 34 indicated that Prx 6 gene expression is regulated by adenylate cyclase in response to PGD 2 or PGE 2 . 35 Furthermore, our study revealed that JAK2, PI3K, PKC, and p38 MAPK contribute to the PGD 2 -or PGE 2 -36 dependent Prx 6 induction. Using stimulated macrophages from Nrf2-deficient mice or activators of Nrf2 and 37 PPARγ, we found that Nrf2, but not PPARγ, is involved in the PG-dependent increase in Prx 6 mRNA 38 expression. In summary, our data suggest multiple signaling pathways of Prx 6 regulation by PGs and 39 identified Nrf2 as a critical player mediating transcriptional induction. 40 © 2011 Elsevier Inc. All rights reserved

Protocol for isolation of microbiota-derived membrane vesicles from mouse blood and colon

Summary: Bacterial membrane vesicles have emerged as gadgets allowing remote communication between the microbiota and distal host organs. Here we describe a protocol for enriching vesicles from serum and colon that could widely be adapted for other tissues. We detail pre-clearing of serum or colon fluids using 0.2-μm syringe filters and their concentration by centrifugal filter devices. We also describe vesicle isolation with qEV size exclusion columns and finally the concentration of isolated vesicle fractions for downstream analyses.For complete details on the use and execution of this protocol, please refer to Erttmann et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Infection with <i>Y. pseudotuberculosis</i> prevents SuperFasLigand-induced caspase 3 activation.

<p>PMNs were left untreated or infected for 30∶1 followed by addition of 2 µM STS, 1 mM sulfasalazine, 50 ng/ml SFL or 10 ng/ml TNFα and 5 µg/ml CHX and incubation for 1, 3, 6 and 12 h. Caspase 3 activity in rate of FU is indicated. Data are presented as mean with SEM (N = 4).</p

Hypothetical model of signal transduction regulating bacteria-induced survival of PMNs.

<p>TLR ligation by bacteria or bacterial products leads to activation of PKCε. Hypothetically PKCε becomes phosphorylated and binds 14-3-3β in a MyD88-dependent manner. Activation of PKCε can occur via PI3K or PC-PLC that provides the PKCε-required DAG. PC-PLC and PKCε transfer signal through a so far unidentified pathway potentially independent of TRAF-6, but dependent on <i>de novo</i>-protein synthesis. Simultaneously, p44/42 MAPK and JNK are activated via TRAF6 and provoke translocation of activator protein 1 (AP-1) to the nucleus. TRAF6 also activates NFκB by IκB degradation. The resulting AP-1−/NFκB-induced transcription and production of anti-apoptotic and pro-inflammatory proteins, and the inactivation of proapoptotic proteins, together contribute to suppression of caspase 3 activity. Arrows mark activation, while marks inactivation of target proteins. Black labels are well documented events while red labels are hypothetical. IKK: IκB kinase; IκBα: inhibitor of NFκB; MAL: MyD88 adaptor-like; MEK: mitogen-activated protein kinase kinase; MKK: mitogen-activated protein kinase kinase; TAK1: transforming growth factor β-activated kinase 1; TIRAP: toll-interleukin 1 receptor (TIR) domain-containing adaptor protein; TLR: Toll-like receptor; TRAF-6: TNF receptor associated factor 6; TREM-1: triggering receptor expressed on myeloid cells 1.</p

TLR-mediated induction of IL-8 and TNFα gene expression requires PC-PLC and PKC.

<p>(A) PMNs were treated with 10 ng/ml LPS for 3–12 h or with (B) 2 µM Ro 318220 or (C) 50 µM D609 for 1 h followed by LPS stimulation for 3 h. IL-8 and TNFα gene expression normalized to RPLP0 and relative to untreated/vehicle-treated PMNs is indicated (N = 3); ***<i>p</i><0.001, **<i>p</i><0.01, *<i>p</i><0.05.</p

Bacteria-induced PMN survival requires PC-PLC and PKC.

<p>PMNs were treated with (A) 2 µM Ro 318220, (B) 4 µM U-73122, 10 µM Et-18-OCH₃, 50 µM D609 or 5 µM MAFP for 1 h followed by 30 min infection with YPIIIpc or pIB102 at MOI 10∶1 and incubation for indicated time periods. Caspase 3 activity in rate of FU is indicated. Data are presented as mean with SEM (N = 4); *<i>p</i><0.05, ***<i>p</i><0.001 compared to 1 h control unless indicated differentially; <i>+++p</i><0.001 compared to 12 h control.</p

PI3K, but not tyrosine kinases contribute to bacteria-induced PMN survival.

<p>(A) PMNs were treated with 25 µM genistein or 25 µM LY294002 for 1 h before infection with YPIIIpc or pIB102 at MOI 10∶1 for 30 min followed by incubation for 1 and 12 h. Caspase 3 activity in rate of FU is indicated. Data are presented as mean with SEM (N = 3). (B) PMNs were infected with YPIIIpc or pIB102 at MOI 10∶1 for indicated periods of time. Protein extracts were subjected to Western blot analysis and probed with antibodies against phospho-JNK/SAPK, phospho-p38 MAPK, phospho-p44/42 MAPK and β-actin. One experiment representative of three performed is shown. (C) PMNs were preincubated with 5 µM SB202190, 20 µM PD98059 or 20 µM SP600125 for 1 h before infection with YPIIIpc or pIB102 at MOI 10∶1 for 30 min and further incubation for 1 and 12 h. Caspase 3 activity in rate of FU is indicated. Data are presented as mean with SEM (N = 3); (A, C) <i>**p</i><0.01, ***<i>p</i><0.001 compared to 1 h control unless indicated differentially, <i>++p</i><0.01, <i>+++p</i><0.001 compared to 12 h control.</p

Different types of bacterial species induce PMN survival that is mediated by TLR2 and TLR4.

<p>(A) PMNs were exposed to heat-killed or live YPIIIpc or pIB102, <i>E. coli</i> MC4100 or <i>S. aureus</i> Newman at MOI 1∶1, 10∶1, 25∶1 for 30 min and further incubated for 1, 3, 6 and 12 h in gentamicin-containing medium. Untreated PMNs were used as control. Caspase 3 activity in rate of FU is indicated. Data are presented as mean with SEM (N≥3). (B) PMNs were infected with YPIIIpc or pIB102 at MOI 10∶1 or stimulated with 1, 10 and 100 ng/ml ultrapure LPS or Pam₂CSK₄ for 30 min followed by an incubation for 1, 3, 6 and 12 h. Caspase 3 activity in rate of FU is indicated. Data are presented as mean with SEM (N≥3).</p

Bacterial components stimulate release of IL-8 and TNFα by PMNs triggering further recruitment of PMNs.

<p>(A, B) PMNs were infected with YPIIIpc, pIB102, <i>E. coli</i> MC4100 or <i>S. aureus</i> Newman at MOI 10∶1 for 30 min followed by incubation for additional 12 h. Concentration of IL-8 (A) and TNFα (B) in cell culture supernatants were determined by immunoassays. Data are presented as mean with SEM (N = 4); *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001. (C, D) In transwell plates supernatant of PMNs that were stimulated with 10 ng/ml LPS (C) or 100 ng/ml Pam₃CSK₄ (D) for 3, 6 or 12 h or that remained untreated, were used as attractants of calcein-AM-stained PMNs. Medium containing 10 nM fMLP was used as positive chemoattractant control while LPS- or Pam₃CSK₄-containing medium was used to exclude side effects. (C) Migration rate in fluorescence intensity over time (0–120 min) and (D) after 120 min is indicated. Data are presented as mean with SEM, **<i>p</i><0.01. One experiment representative of three is shown.</p

<i>Y. pseudotuberculosis</i> infection blocks caspase 3 and caspase 8 activity in human PMNs.

<p>(A) PMNs were left untreated or infected for 30 min with <i>Y. pseudotuberculosis</i> strain YPIIIpc or pIB102 at indicated MOIs followed by 1, 3, 6 and 12 h incubation in gentamicin-containing medium. Caspase 3 and 8 activities in lysates were determined using fluorometric caspase assays. The caspase activity in rate of fluorescence units (FU) is indicated. (B) PMNs were incubated with DMSO or with a specific caspase 3 or caspase 8 inhibitor for 1, 3, 6 and 12 h. Caspase 3 and 8 activities in PMN lysates were determined. The caspase activity in rate of fluorescence units (FU) is indicated. (A, B) Data are presented as mean with SEM (N = 3); ***<i>p</i><0,001 compared to 1 h control; +<i>p</i><0,05, ++<i>p</i><0,01, +++<i>p</i><0,001 compared to correlating time point of control panel. (C) PMNs were left untreated or infected with <i>Y. pseudotuberculosis</i> strain YPIIIpc or pIB102 at MOI 10∶1 for 30 min followed by 1, 3, 6 and 12 h incubation in gentamicin-containing medium. Cell lysates were used for Western blot analysis and probed with antibodies against caspase-3, PARP, and β-actin respectively. One experiment representative of at least three performed is shown.</p