Murine Toll-Like Receptor 2 Activation Induces Type I Interferon Responses from Endolysosomal Compartments

Background: Toll-like receptors (TLRs) are among the first-line sentinels for immune detection and responsiveness to pathogens. The TLR2 subfamily of TLRs (TLR1, TLR2, TLR6) form heterodimers with each other and are thus able to recognize a broad range of components from several microbes such as yeast, Gram-positive bacteria and protozoa. Until now, TLR2 activation by bacterial ligands has long been associated with pro-inflammatory cytokines but not type I interferon responses. Methodology/Principal Findings: Using a variety of transgenic mice, here we provide in vivo and in vitro data showing that TLR2 activation does in fact induce interferon-beta and that this occurs via MyD88-IRF1 and-IRF7 pathways. Interestingly, by microscopy we demonstrate that although a cell surface receptor, TLR2 dependent induction of type I interferons occurs in endolysosomal compartments where it is translocated to upon ligand engagement. Furthermore, we could show that blocking receptor internalization or endolysosomal acidification inhibits the ability of TLR2 to trigger the induction type I interferon but not pro-inflammatory responses. Conclusion/Significance: The results indicate that TLR2 activation induces pro-inflammatory and type I interferon responses from distinct subcellular sites: the plasma membrane and endolysosomal compartments respectively. Apart from identifying and characterizing a novel pathway for induction of type I interferons, the present study offers new insights into how TLR signaling discriminates and regulates the nature of responses to be elicited against extracellular and endocytosed microbes

Mast cells initiate early anti-Listeria host defences.

The Gram-positive bacterium Listeria monocytogenes (L. m.) is the aetiological agent of listeriosis. The early phase listeriosis is characterized by strong innate host responses that play a major role in bacterial clearance. This is emphasized by the fact that mice deficient in T and B cells have a remarkable ability to control infection. Mast cells, among the principal effectors of innate immunity, have largely been studied in the context of hyper-reactive conditions such as allergy and autoimmune diseases. In the present study, we evaluated the significance of mast cells during the early phase of listeriosis. Compared with controls, mice depleted of mast cells showed hundred-fold higher bacterial burden in spleen and liver and were significantly impaired in neutrophil mobilization. Although L. m. interacts with and triggers mast cell degranulation, bacteria were hardly found within such cells. Mainly neutrophils and macrophages phagozytosed L. m. Thus, mast cells control infection not via direct bacterial uptake, but by initiating neutrophils influx to the site of infection. We show that this is initiated by pre-synthesized TNF-alpha, rapidly secreted by mast cell upon activation by L. m. We also show that upon recruitment, neutrophils also become activated and additionally secrete TNF-alpha thus amplifying the anti-L. m. inflammatory response

The deubiquitinase MYSM1 dampens NOD2-mediated inflammation and tissue damage by inactivating the RIP2 complex

NOD2 is essential for antimicrobial innate immunity and tissue homeostasis, but require tight regulation to avert pathology. A focal point of NOD2 signaling is RIP2, which upon polyubiquitination nucleates the NOD2:RIP2 complex, enabling signaling events leading to inflammation, yet the precise nature and the regulation of the polyubiquitins coordinating this process remain unclear. Here we show that NOD2 signaling involves conjugation of RIP2 with lysine 63 (K63), K48 and M1 polyubiquitin chains, as well as with non-canonical K27 chains. In addition, we identify MYSM1 as a proximal deubiquitinase that attenuates NOD2:RIP2 complex assembly by selectively removing the K63, K27 and M1 chains, but sparing the K48 chains. Consequently, MYSM1 deficient mice have unrestrained NOD2-mediated peritonitis, systemic inflammation and liver injury. This study provides a complete overview of the polyubiquitins in NOD2:RIP2 signaling and reveal MYSM1 as a central negative regulator restricting these polyubiquitins to prevent excessive inflammation

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

Listeria monocytogenes Desensitizes Immune Cells to Subsequent Ca2+ Signaling via Listeriolysin O-Induced Depletion of Intracellular Ca2+ Stores▿

Listeriolysin O (LLO), the pore-forming toxin of Listeria monocytogenes, is a prototype of the cholesterol-dependent cytolysins (CDCs) secreted by several pathogenic and nonpathogenic gram-positive bacteria. In addition to mediating the escape of the bacterium into the cytosol, this toxin is generally believed to be a central player in host-pathogen interactions during L. monocytogenes infection. LLO triggers the influx of Ca2+ into host cells as well as the release of Ca2+ from intracellular stores. Thus, many of the cellular responses induced by LLO are related to calcium signaling. Interestingly, in this study, we report that prolonged exposure to LLO desensitizes cells to Ca2+ mobilization upon subsequent stimulations with LLO. Cells preexposed to LLO-positive L. monocytogenes but not to the LLO-deficient Δhly mutant were found to be highly refractory to Ca2+ induction in response to receptor-mediated stimulation. Such cells also exhibited diminished Ca2+ signals in response to stimulation with LLO and thapsigargin. The presented results suggest that this phenomenon is due to the depletion of intracellular Ca2+ stores. The ability of LLO to desensitize immune cells provides a significant hint about the possible role played by CDCs in the evasion of the immune system by bacterial pathogens

Listeria monocytogenes induces T cell receptor unresponsiveness through pore-forming toxin listeriolysin O.

The success of many pathogens relies on their ability to circumvent the innate and adaptive immune defenses. How bacterial pathogens subvert adaptive immune defenses is not clear. Cholesterol-dependent cytolysins (CDCs) represent an expansive family of homologous pore-forming toxins that are produced by more than 20 gram-positive bacterial species. Listeriolysin O (LLO), a prototype CDC, is the main virulence factor of Listeria monocytogenes

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

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