Russell D. Salter

Regulation of inflammation is necessary to balance sufficient pathogen clearance with excessive tissue damage. Central to regulating inflammation is the switch from a pro-inflammatory pathway to an anti-inflammatory pathway. Macrophages are well-positioned to initiate this switch, and as such are the target of multiple therapeutics. One such potential therapeutic is methylthioadenosine (MTA), which inhibits TNFα production following LPS stimulation. We found that MTA could block TNFα production by multiple TLR ligands. Further, it prevented surface expression of CD69 and CD86 and reduced NF-KB signaling. We then determined that the mechanism of this action by MTA is signaling through adenosine A2 receptors. A2 receptors and TLR receptors synergized to promote an anti-inflammatory phenotype, as MTA enhanced LPS tolerance. In contrast, IL-1β production and processing was not affected by MTA exposure. Taken together, these data demonstrate that MTA reprograms TLR activation pathways via adenosine receptors to promote resolution of inflammation.

The nucleotide-binding oligomerization domain–like receptor family, pyrin domain–containing 3 (NLRP3) inflammasome drives many inflammatory processes and mediates IL-1 family cytokine release. Inflammasome activators typically damage cells and may release lysosomal and mitochondrial products into the cytosol. Macrophages triggered by the NLRP3 inflammasome activator nigericin show reduced mitochondrial function and decreased cellular ATP. Release of mitochondrial reactive oxygen species (ROS) leads to subsequent lysosomal membrane permeabilization (LMP). NLRP3-deficient macrophages show comparable reduced mitochondrial function and ATP loss, but maintain lysosomal acidity, demonstrating that LMP is NLRP3 dependent. A subset of wild-type macrophages undergo subsequent mitochondrial membrane permeabilization and die. Both LMP and mitochondrial membrane permeabilization are inhibited by potassium, scavenging mitochondrial ROS, or NLRP3 deficiency, but are unaffected by cathepsin B or caspase-1 inhibitors. In contrast, IL-1β secretion is ablated by potassium, scavenging mitochondrial ROS, and both cathepsin B and caspase-1 inhibition. These results demonstrate interplay between lysosomes and mitochondria that sustain NLRP3 activation and distinguish cell death from IL-1β release.

Pore-forming toxins are utilized by bacterial and mammalian cells to exert pathogenic effects and induce cell lysis. In addition to rapid plasma membrane repair, macrophages respond to pore-forming toxins through activation of the NLRP3 inflammasome, leading to IL-1β secretion and pyroptosis. The structural determinants of pore-forming toxins required for NLRP3 activation remain unknown. Here, we demonstrate using streptolysin O (SLO) that pore-formation controls IL-1β secretion and direct toxicity. An SLO mutant incapable of pore-formation did not promote direct killing, pyroptosis or IL-1β production. This indicated that pore formation is necessary for inflammasome activation. However, a partially active mutant (SLO N402C) that was less toxic to macrophages than wild-type SLO, even at concentrations that directly lysed an equivalent number of red blood cells, enhanced IL-1β production but did not alter pyroptosis. This suggests that direct lysis may attenuate immune responses by preventing macrophages from successfully repairing their plasma membrane and elaborating more robust cytokine production. We suggest that mutagenesis of pore-forming toxins represents a strategy to enhance adjuvant activity.

Aberrant activation of macrophages in arterial walls by oxidized lipoproteins can lead to atherosclerosis. Oxidized lipoproteins convert macrophages to foam cells through lipid uptake and TLR signaling. To investigate the relative contributions of lipid uptake and TLR signaling in foam cell formation, we established an in vitro assay using liposomes of defined lipid compositions. We found that TLRs signaling through Toll/IL-1R domain-containing adapter inducing IFN-β promoted foam cell formation by inducing both NF-κB signaling and type I IFN production, whereas TLRs that do not induce IFN, like TLR2, did not enhance foam cell formation. Addition of IFN-α to TLR2 activator promoted robust foam cell formation. TLR signaling further required peroxisome proliferator-activated receptor α, as inhibition of peroxisome proliferator-activated receptor α blocked foam cell formation. We then investigated the ability of endogenous microparticles (MP) to contribute to foam cell formation. We found that lipid-containing MP promoted foam cell formation, which was enhanced by TLR stimulation or IFN-α. These MP also stimulated foam cell formation in a human skin model. However, these MP suppressed TNF-α production and T cell activation, showing that foam cell formation can occur by immunosuppressive MP. Taken together, the data reveal novel signaling requirements for foam cell formation and suggest that uptake of distinct types of MP in the context of activation of multiple distinct TLR can induce foam cell formation.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding ...

Immunization against beta-amyloid (Aβ) is a promising approach for the treatment of Alzheimer’s disease, but the optimal timing for the vaccination remains to be determined. Preventive immunization approaches may be more efficacious and associated with fewer side-effects; however, there is only limited information available from primate models about the effects of preclinical vaccination on brain amyloid composition and the neuroinflammatory milieu. Ten non-human primates (NHP) of advanced age (18–26 years) and eight 2-year-old juvenile NHPs were immunized at 0, 2, 6, 10 and 14 weeks with aggregated Aβ₄₂ admixed with monophosphoryl lipid A as adjuvant, and monitored for up to 6 months. Anti-Aβ antibody levels and immune activation markers were assessed in plasma and cerebrospinal fluid samples before and at several time-points after immunization. Microglial activity was determined by [¹¹C]PK11195 PET scans acquired before and after immunization, and by post-mortem immunohistochemical and real-time PCR evaluation. Aβ oligomer composition was assessed by immunoblot analysis in the frontal cortex of aged immunized and non-immunized control animals. All juvenile animals developed a strong and sustained serum anti-Aβ IgG antibody response, whereas only 80 % of aged animals developed detectable antibodies. The immune response in aged monkeys was more delayed and significantly weaker, and was also more variable between animals. Pre- and post-immunization [¹¹C]PK11195 PET scans showed no evidence of vaccine-related microglial activation. Post-mortem brain tissue analysis indicated a low overall amyloid burden, but revealed a significant shift in oligomer size with an increase in the dimer:pentamer ratio in aged immunized animals compared with non-immunized controls (P < 0.01). No differences were seen in microglial density or expression of classical and alternative microglial activation markers between immunized and control animals. Our results indicate that preventive Aβ immunization is a safe therapeutic approach lacking adverse CNS immune system activation or other serious side-effects in both aged and juvenile NHP cohorts. A significant shift in the composition of soluble oligomers towards smaller species might facilitate removal of toxic Aβ species from the brain.

ATP-mediated activation of the purinergic receptor P2X₇ elicits morphological changes and proinflammatory responses in macrophages. These changes include rapid shedding of microvesicles (MV) and the nonconventional secretion of cytokines, such as IL-1β and IL-18 following priming. In this study, we demonstrate the activation potential of P2X₇-induced MV isolated from nonprimed murine macrophages. Cotreatment of nonprimed macrophages with ATP and calcium ionophore induced a rapid release of MV that were predominantly 0.5–1 μm in size. Exposure of primary murine bone marrow-derived macrophages to these MV resulted in costimulatory receptor upregulation and TNF-α secretion. Cell homogenates or supernatants cleared of MV did not activate macrophages. MV-mediated activation was p38 MAPK and NF-κB dependent, and partially dependent on TLR4 activity, but was high-mobility group box 1 independent. Biochemical fractionation of the MV demonstrated that the phospholipid fraction, not the protein fraction, mediated macrophage activation through a TLR4-dependent process. P2X₇ activation is known to induce calcium-independent phospholipase A₂, calcium-dependent phospholipase A₂, and phospholipase D activities, but inhibition of these enzymes did not inhibit MV generation or shedding. However, blocking phospholipase D activity resulted in release of MV incapable of activating recipient macrophages. These data demonstrate a novel mechanism of macrophage activation resulting from exposure to MV from nonprimed macrophages, and identifies phospholipids in these MV as the biologically active component. We suggest that phospholipids delivered by MV may be mediators of sterile inflammation in a number of diseases.

The P2X₇ receptor exhibits significant allelic polymorphism in humans, with both loss and gain of function variants potentially impacting on a variety of infectious and inflammatory disorders. At least five loss-of-function polymorphisms (G150R, R307Q, T357S, E496A, and I568N) and two gain-of-function polymorphisms (H155Y and Q460R) have been identified and characterized to date. In this study, we used RT-PCR cloning to isolate and characterize P2X₇ cDNA clones from human PBMCs and THP-1 cells. A previously unreported variant with substitutions of V80M and A166G was identified. When expressed in HEK293 cells, this variant exhibited heightened sensitivity to the P2X₇ agonist (BzATP) relative to the most frequent allele, as shown by pore formation measured by fluorescent dye uptake into cells. Mutational analyses showed that A166G alteration was critical for the gain-of-function change, while V80M was not. Full-length variants with multiple previously identified nonsynonymous SNPs (H155Y, H270R, A348T, and E496A) were also identified. Distinct functional phenotypes of the P2X₇ variants or mutants constructed with multiple polymorphisms were observed. Gain-of-function variations (A166G or H155Y) could not rescue the loss-of-function E496A polymorphism. Synergistic effects of the gain-of-function variations were also observed. We also identified the A348T alteration as a weak gain-of-function variant. Thus, these results identify the new gain-of-function variant A166G and demonstrate that multiple-gene polymorphisms contribute to functional phenotypes of the human P2X₇ receptor. Furthermore, the results demonstrate that the C-terminal of the cysteine-rich domain 1 of P2X₇ is critical for regulation of P2X₇-mediated pore formation.

Summary:  Ca²⁺‐driven responses in dendritic cells (DCs) are less well characterized than in lymphocytes. When DCs undergo a sequence of activation/maturation events, typically beginning with exposure to pathogens in the periphery, Ca²⁺ entry into the cytosol from stores in the endoplasmic reticulum or from outside the cell can occur at various steps and participate in intracellular signaling. However, not all cellular processes identified in these cells are Ca²⁺ dependent. While immigration of precursor DCs into the peripheral tissues as well as emigration to secondary lymphoid sites following microbial challenge depend on processes that involve Ca²⁺, other processes such as DC maturation in response to Toll‐like receptor agonist stimulation appear not to. Certain microbial stimuli and host‐derived chemokines induce Ca²⁺ entry that is important for the induced responses. In this article, we review the current state of our understanding of the role of Ca²⁺ in DC biology and argue that homeostatic control of Ca²⁺ levels in these cells is critical for maintaining their proper function. We also consider evidence for intercellular transmission of Ca²⁺ signals between DCs that are physically linked by thin membranous extensions termed tunneling nanotubules.