6 research outputs found
CYLD Proteolysis Protects Macrophages from TNF-Mediated Auto-necroptosis Induced by LPS and Licensed by Type I IFN
SummaryTumor necrosis factor (TNF) induces necroptosis, a RIPK3/MLKL-dependent form of inflammatory cell death. In response to infection by Gram-negative bacteria, multiple receptors on macrophages, including TLR4, TNF, and type I IFN receptors, are concurrently activated, but it is unclear how they crosstalk to regulate necroptosis. We report that TLR4 activates CASPASE-8 to cleave and remove the deubiquitinase cylindromatosis (CYLD) in a TRIF- and RIPK1-dependent manner to disable necroptosis in macrophages. Inhibiting CASPASE-8 leads to CYLD-dependent necroptosis caused by the TNF produced in response to TLR4 ligation. While lipopolysaccharides (LPS)-induced necroptosis was abrogated in Tnf−/− macrophages, a soluble TNF antagonist was not able to do so in Tnf+/+ macrophages, indicating that necroptosis occurs in a cell-autonomous manner. Surprisingly, TNF-mediated auto-necroptosis of macrophages requires type I IFN, which primes the expression of key necroptosis-signaling molecules, including TNFR2 and MLKL. Thus, the TNF necroptosis pathway is regulated by both negative and positive crosstalk
Opsonic monoclonal antibodies enhance phagocytic killing activity and clearance of Mycobacterium tuberculosis from blood in a quantitative qPCR mouse model
BACKGROUND : Patients with impaired immunity often have rapid progression of tuberculosis (TB) which can lead to
highly lethal Mycobacterium tuberculosis (MTB) sepsis. Opsonic monoclonal antibodies (MABs) directed against
MTB that enhance phagocytic killing activity and clearance of MTB from blood may be useful to enhance TB
immunity.
METHODS : BALB/c mice were immunized with ethanol-killed MTB (EK-MTB) and MABs were produced and
screened by ELISA for binding to killed and live Mycobacterium smegmatis (SMEG) and MTB. MAB opsonophagocytic
killing activity (OPKA) was examined using SMEG with HL60 and U-937 cells and MTB with U-937 cells.
Clearance of MTB from blood was evaluated in Institute of Cancer Research (ICR) mice given opsonic anti-MTB
MABs or saline (control) 24 h prior to intravenous infusion with 108 CFUs gamma-irradiated MTB (HN878). MTB
levels in murine blood collected 0.25, 4 and 24 h post-challenge were assessed by qPCR. MAB binding to
peptidoglycan (PGN) was examined by ELISA using PGN cell wall mixture and ultra-pure PGN.
RESULTS : Two MABs (GG9 and JG7) bound to killed and live SMEG and MTB (susceptible and resistant), and
promoted OPKA with live MTB. MAB JG7 significantly enhanced OPKA of MTB. Both MABs significantly
enhanced clearance of killed MTB from murine blood at 4 and 24 h as measured by qPCR. These opsonic MABs
bound to PGN, a major cell wall constituent.
CONCLUSIONS : Anti-MTB MABs that promote bactericidal phagocytic activity of MTB and enhance clearance of killed
MTB from the blood, may offer an immunotherapeutic approach for treatment of MTB bacteremia or sepsis, and
augment treatment of multi-drug resistant (MDR) or extensively drug resistant (XDR) TB.This work was supported by Longhorn Vaccines and Diagnostics, LLC.
Live MTB work was additionally supported from post-graduate student
bursaries to Bong-Akee Shey from the University of Pretoria and from
grant 105830 to PBF by the National Research Foundation of South Africa.Post-graduate student
bursaries to Bong-Akee Shey from the University of Pretoria and from
grant 105830 to PBF by the National Research Foundation of South Africa.https://www.heliyon.comam2020Medical Microbiolog
CYLD Proteolysis Protects Macrophages from TNF-Mediated Auto-necroptosis Induced by LPS and Licensed by Type I IFN
Tumor necrosis factor (TNF) induces necroptosis, a RIPK3/MLKL-dependent form of inflammatory cell death. In response to infection by Gram-negative bacteria, multiple receptors on macrophages, including TLR4, TNF, and type I IFN receptors, are concurrently activated, but it is unclear how they crosstalk to regulate necroptosis. We report that TLR4 activates CASPASE-8 to cleave and remove the deubiquitinase cylindromatosis (CYLD) in a TRIF- and RIPK1-dependent manner to disable necroptosis in macrophages. Inhibiting CASPASE-8 leads to CYLD-dependent necroptosis caused by the TNF produced in response to TLR4 ligation. While lipopolysaccharides (LPS)-induced necroptosis was abrogated in Tnf−/− macrophages, a soluble TNF antagonist was not able to do so in Tnf+/+ macrophages, indicating that necroptosis occurs in a cell-autonomous manner. Surprisingly, TNF-mediated auto-necroptosis of macrophages requires type I IFN, which primes the expression of key necroptosis-signaling molecules, including TNFR2 and MLKL. Thus, the TNF necroptosis pathway is regulated by both negative and positive crosstalk
Conserved Influenza Hemagglutinin, Neuraminidase and Matrix Peptides Adjuvanted with ALFQ Induce Broadly Neutralizing Antibodies
A universal influenza candidate vaccine that targets multiple conserved influenza virus epitopes from hemagglutinin (HA), neuraminidase (NA) and matrix (M2e) proteins was combined with the potent Army liposomal adjuvant (ALFQ) to promote induction of broad immunity to seasonal and pandemic influenza strains. The unconjugated and CRM-conjugated composite peptides formulated with ALFQ were highly immunogenic and induced both humoral and cellular immune responses in mice. Broadly reactive serum antibodies were induced across various IgG isotypes. Mice immunized with the unconjugated composite peptide developed antibody responses earlier than mice immunized with conjugated peptides, and the IgG antibodies were broadly reactive and neutralizing across Groups 1 and 2 influenza viruses. Multi-epitope unconjugated influenza composite peptides formulated with ALFQ provide a novel strategy for the development of a universal influenza vaccine. These synthetic peptide vaccines avoid the pitfalls of egg-produced influenza vaccines and production can be scaled up rapidly and economically
Unconjugated Multi-Epitope Peptides Adjuvanted with ALFQ Induce Durable and Broadly Reactive Antibodies to Human and Avian Influenza Viruses
An unconjugated composite peptide vaccine targeting multiple conserved influenza epitopes from hemagglutinin, neuraminidase, and matrix protein and formulated with a safe and highly potent adjuvant, Army Liposome formulation (ALFQ), generated broad and durable immune responses in outbred mice. The antibodies recognized specific epitopes in influenza peptides and several human, avian, and swine influenza viruses. Comparable antibody responses to influenza viruses were observed with intramuscular and intradermal routes of vaccine administration. The peptide vaccine induced cross-reactive antibodies that recognized influenza virus subtypes A/H1N1, A/H3N2, A/H5N1, B/Victoria, and B/Yamagata. In addition, immune sera neutralized seasonal and pandemic influenza strains (Group 1 and Group 2). This composite multi-epitope peptide vaccine, formulated with ALFQ and administered via intramuscular and intradermal routes, provides a high-performance supra-seasonal vaccine that would be cost-effective and easily scalable, thus moving us closer to a viable strategy for a universal influenza vaccine and pandemic preparedness