Genetic redirection of T cells for cancer therapy

Abstract

Ft is a facultative intracellular pathogen that infects many cell types, including neutrophils. In previous work, we demonstrated that the type B Ft strain LVS disrupts NADPH oxidase activity throughout human neutrophils, but how this is achieved is incompletely defined. Here, we used several type A and type B strains to demonstrate that Ft-mediated NADPH oxidase inhibition is more complex than appreciated previously. We confirm that phagosomes containing Ft opsonized with AS exclude flavocytochrome b(558) and extend previous results to show that soluble phox proteins were also affected, as indicated by diminished phosphorylation of p47(phox) and other PKC substrates. However, a different mechanism accounts for the ability of Ft to inhibit neutrophil activation by formyl peptides, Staphylococcus aureus, OpZ, and phorbol esters. In this case, enzyme targeting and assembly were normal, and impaired superoxide production was characterized by sustained membrane accumulation of dysfunctional NADPH oxidase complexes. A similar post-assembly inhibition mechanism also diminished the ability of anti-Ft IS to confer neutrophil activation and bacterial killing, consistent with the limited role for antibodies in host defense during tularemia. Studies of mutants that we generated in the type A Ft strain Schu S4 demonstrate that the regulatory factor fevR is essential for NADPH oxidase inhibition, whereas iglI and iglJ, candidate secretion system effectors, and the acid phosphatase acpA are not. As Ft uses multiple mechanisms to block neutrophil NADPH oxidase activity, our data strongly suggest that this is a central aspect of virulence.

Subversion of Host Recognition and Defense Systems by Francisella spp

“…In contrast, another study using F. novicida determined that AcpA did not play a role in replication in murine macrophages (22). In F. tularensis, deletion of acpA, or even of acpA, acpB, and acpC together, did not influence virulence in murine macrophages or human monocytes (48,146). These conflicting data indicate that AcpA is not required for Francisella replication under all conditions tested.…”

“…Its requirement may depend on the species or host cells used and the specific infection conditions. In addition to blocking assembly, F. tularensis can also inhibit the generation of ROS when NADPH oxidase assembly is induced by exogenous stimuli (146). In spite of the formation of this complex, F. tularensis can inhibit the production of ROS.…”

Characterization of Filifactor alocis and its immune evasion strategies employed against human neutrophils.

“…Neither virulent nor avirulent strains of F. tularensis were effectively killed by neutrophils and ~70% of internalized bacteria remained viable 2-4-8 hrs after neutrophil challenge (128). The live vaccine strain of F. tularensis (LVS) induces a minimal respiratory burst response compared to formalin or periodate-killed LVS as assessed by NBT staining (347).…”

“…Using these two antimicrobial mechanisms, neutrophils create an inhospitable environment for most pathogens, which results in bacterial cell death (72). However, some pathogens have evolved mechanisms that allow for interference with the engulfment process or ensure their survival inside neutrophils through modulation of phagosomal maturation, and creation of a more hospitable phagosomal environment (72,(126)(127)(128)(129)(130).…”

“…In order to more closely determine the role of NADPH oxidase assembly upon neutrophil challenge with F. alocis, we used confocal immunostaining to detect oxidase components and their localization to the bacterial-containing phagosome. Several studies in human and mice neutrophils show that different pathogenic microbes can prevent or delay ROS production when 40% or less of the oxidase components were recruited to the phagosomal structure (127,128,132,139,140,142,285,(308)(309)(310)(311)(312)(313)(314)(315)(316)(317)(318)(319)(320)(321)(322)(323)(324).…”