Internalization and phagosome escape required forFrancisellato induce human monocyte IL-1β processing and release

Abstract

Macrophage responses to Francisella infection have been characterized previously by subdued proinflammatory responses; however, these studies have generally focused on macrophage cell lines or monocyte-derived macrophages. Therefore, we studied the ability of fresh human blood monocytes to engulf and respond to Francisella by using the live vaccine strain variant and Francisella novicida. Because Francisella organisms have been reported to escape from the phagolysosome into the cytosol, we hypothesized that this escape may trigger the activation of caspase-1. Francisella tularensis variants were readily taken up by fresh human CD14 ؉ monocytes, inducing the release of IL-1␤, as well as IL-8, in a timeand dose-dependent fashion. Importantly, whereas live and dead Escherichia coli, F. novicida, and live vaccine strain, as well as the LPS of E. coli, were able to induce abundant IL-1␤ mRNA synthesis and intracellular pro-IL-1␤ production, only live Francisella induced enhanced IL-1␤ processing and release (51 ؎ 10 vs. 7.1 ؎ 2.1 ng͞ml, for F. novicida vs. E. coli LPS; P ‫؍‬ 0.0032). Cytochalasin D blocked the Francisella internalization and the Francisella-induced monocyte IL-1␤ processing and release but not that induced by the exogenous stimulus E. coli LPS. Also, killing bacteria did not block uptake but significantly diminished the IL-1␤ processing and release that was induced by Francisella. Blocking bacterial escape from the phagosome into the cytosol also decreased IL-1␤ but not IL-8 release. These findings demonstrate that Francisella organisms efficiently induce IL-1␤ processing and release in fresh monocytes by means of a sensing system that requires the uptake of live bacteria capable of phagosome escape.bacteria ͉ caspase-1 ͉ cytokine ͉ phagocytosis M onocytes and macrophages, which are the first-line guards of the innate host-defense system, recognize and phagocytose pathogens. The innate immune system senses the invasion of pathogenic microorganisms through the recognition of pathogen-associated molecular patterns by Toll-like receptors (TLRs), of which there are 10 that are known in humans, as well as non-TLRs (1-3). Pathogen recognition induces the release of many proinflammatory and antiinflammatory cytokines, which provide a balanced inflammatory response. At the same time, prototypical intracellular pathogens like Francisella tularensis depend on the intracellular milieu of mononuclear cells for growth and replication (4, 5). In this context, a class of intracellular innate host-defense molecules, which recognize and respond to internalized pathogens, has been identified (3, 6). These molecules [historically called CATERPILLERs (3) and nucleotide-binding oligomerization domain-leucine-rich repeats (NOD-LRRs) (7), and, most recently, NACHT leucine-rich repeats (NLRs) (8)] are homologues of plant-disease-resistance genes and contain leucine-rich repeats, much like TLRs. The human family of NLR consists of 22 (8) or 23 (7) proteins. These NLRs, similar to TLRs, function to regulate pathogen recogniti...

Depletion of the Ubiquitin-binding Adaptor Molecule SQSTM1/p62 from Macrophages Harboring cftr ΔF508 Mutation Improves the Delivery of Burkholderia cenocepacia to the Autophagic Machinery

“…18412) ACAGAUGCCAGAAUCGGAA, CUGCU-CAGGAGGAGACGAU, GAACAGAUGGAGUCGGGAA, and CCAUGGGUUUCUCGGAUGA; siRNA against NDP52 (Dharmacon, catalog no. 76815) CAACACAGAGGGUCAAkit and Amaxa equipment, as described previously (30,31). Successful knockdown was confirmed by immunoblot analysis for each experiment.…”

“…15596-026) and then converted to cDNA. Gene expression was calculated as relative copy numbers, as described previously (30,33). Briefly, C t values of the p62 gene were subtracted from the average Ct of two housekeeping genes (GADPH and CAP1), and the resulting ⌬C t was used in the following equation: RCN ϭ (2 Ϫ⌬Ct ) 100.…”

“…Briefly, C t values of the p62 gene were subtracted from the average Ct of two housekeeping genes (GADPH and CAP1), and the resulting ⌬C t was used in the following equation: RCN ϭ (2 Ϫ⌬Ct ) 100. The relative copy number of a gene is represented as the number of copies relative to the 100 copies of average housekeeping genes (30,33).…”

In vivo negative selection screen identifies genes required for Francisella virulence

“…In activated macrophages, however, the ability of Francisella to escape the phagosome and replicate in the cytosol correlates with the activation of the inflammasome, which contains the host proteins ASC and caspase-1. Inflammasome activation is critical for host defense and leads to the induction of cell death in infected cells and the concomitant release of the proinflammatory cytokines IL-1␤ and IL-18 (8,9). These responses may function to limit Francisella's intracellular growth niche and activate the powerful host innate immune system.…”

Interfering with Immunity: Detrimental Role of Type I IFNs during Infection

“…Although the PRR responsible for type I IFN induction during F. tularensis infection remains to be identified, IFN-b expression is greatly reduced in STING-deficient cells (104), suggesting a possible involvement of DNA-sensing receptors, such as cGAS and IFI16, in the process (105). Furthermore, cytosolic bacterial nucleic acids from F. tularensis (103) were shown to induce absent in melanoma 2 inflammasome complex formation and secretion of the proinflammatory cytokines IL-1b and IL-18 (102,104,106,107), providing further evidence that bacterial DNA is able to access cytoplasmic PRRs.…”