ABSTRACT
Humans that are heterozygous for the common S180L polymorphism in the Toll-like receptor (TLR) adaptor Mal (encoded by TIRAP) are protected from a number of infectious diseases, including tuberculosis (TB), whereas those homozygous for the allele are at increased risk. The reason for this difference in susceptibility is not clear. We report that Mal has a TLR-independent role in interferon-gamma (IFN-γ) receptor signaling. Mal-dependent IFN-γ receptor (IFNGR) signaling led to mitogen-activated protein kinase (MAPK) p38 phosphorylation and autophagy. IFN-γ signaling via Mal was required for phagosome maturation and killing of intracellular Mycobacterium tuberculosis (Mtb). The S180L polymorphism, and its murine equivalent S200L, reduced the affinity of Mal for the IFNGR, thereby compromising IFNGR signaling in macrophages and impairing responses to TB. Our findings highlight a role for Mal outside the TLR system and imply that genetic variation in TIRAP may be linked to other IFN-γ-related diseases including autoimmunity and cancer.
Subject(s)
Interferon-gamma/metabolism , Macrophages/physiology , Membrane Glycoproteins/metabolism , Mycobacterium tuberculosis/immunology , Receptors, Interleukin-1/metabolism , Tuberculosis, Pulmonary/immunology , Animals , Autophagy/genetics , Genetic Association Studies , Genetic Predisposition to Disease , Genotype , HEK293 Cells , Humans , Immunity, Innate/genetics , MAP Kinase Signaling System/genetics , Macrophages/microbiology , Membrane Glycoproteins/genetics , Mice , Mice, Knockout , Polymorphism, Genetic , Protein Binding/genetics , RNA, Small Interfering/genetics , Receptors, Interferon/metabolism , Receptors, Interleukin-1/genetics , Tuberculosis, Pulmonary/genetics , Interferon gamma ReceptorABSTRACT
The ability of particulate materials to provoke inflammatory immune responses has been well documented. In the case of endogenous and environmental particulates, these effects can often lead to pathological disorders. In contrast, particulate adjuvants incorporated into vaccines promote immune responses, which in turn provide efficient protection against infectious diseases. In recent years, studies have revealed that the NLRP3 inflammasome plays a key role in particulate-driven inflammation and its associated cytotoxicity. Hence, this chapter covers protocols useful to (1) assess NLRP3 inflammasome activation triggered by particulate adjuvants or materials in mouse bone marrow-derived dendritic cell (BMDCs) differentiated cultures, and (2) measure particle-induced cytotoxicity. More specifically, protocols are described for the preparation and differentiation of BMDCs, their priming and stimulation using particulate NLRP3 agonists such as monosodium urate monohydrate (MSU) and the vaccine adjuvant alum. We then detail protocols to assess particulate-driven cytotoxicity via flow cytometry using annexin V-propidium iodide (PI) and novel dye LIVE/DEAD(®) aqua stain. General considerations are provided that warn against the use of endotoxin-contaminated particles and emphasize the use of experimental controls. Suggestions are also outlined for further assessment of the immunomodulatory effects of particulate materials in vivo using the mouse peritonitis model.
Subject(s)
Adjuvants, Immunologic/toxicity , Carrier Proteins/metabolism , Cytotoxicity, Immunologic/drug effects , Inflammasomes/metabolism , Particulate Matter/toxicity , Animals , Apoptosis Regulatory Proteins , Blotting, Western , CARD Signaling Adaptor Proteins , Cell Differentiation/drug effects , Cell Differentiation/immunology , Cytoskeletal Proteins/metabolism , Dendritic Cells/cytology , Dendritic Cells/drug effects , Dendritic Cells/immunology , Dendritic Cells/metabolism , Mice , Microscopy, Confocal , NLR Family, Pyrin Domain-Containing 3 Protein , Particulate Matter/immunologyABSTRACT
The inflammatory effects of carbon nanoparticles (NPs) are highly disputed. Here it is demonstrated that endotoxin-free preparations of raw carbon nanotubes (CNTs) are very limited in their capacity to promote inflammatory responses in vitro, as well as in vivo. Upon purification and selective oxidation of raw CNTs, a higher dispersibility is achieved in physiological solutions, but this process also enhances their inflammatory activity. In synergy with toll-like receptor (TLR) ligands, CNTs promote NLRP3 inflammasome activation and it is shown for the first time that this property extends to spherical carbon nano-onions (CNOs) of 6 nm in size. In contrast, the benzoic acid functionalization of purified CNTs and CNOs leads to significantly attenuated inflammatory properties. This is evidenced by a reduced secretion of the inflammatory cytokine IL-1ß, and a pronounced decrease in the recruitment of neutrophils and monocytes following injection into mice. Collectively, these results reveal that the inflammatory properties of carbon NPs are highly dependent on their physicochemical characteristics and crucially, that chemical surface functionalization allows significant moderation of these properties.
