Lung disease causes significant morbidity and mortality, and is exacerbated by environmental injury, for example through lipopolysaccharide (LPS) or ozone (O
). Toll-like receptors (TLRs) orchestrate ...immune responses to injury by recognizing pathogen- or danger-associated molecular patterns. TLR4, the prototypic receptor for LPS, also mediates inflammation after O
, triggered by endogenous hyaluronan. Regulation of TLR4 signaling is incompletely understood. TLR5, the flagellin receptor, is expressed in alveolar macrophages, and regulates immune responses to environmental injury. Using in vivo animal models of TLR4-mediated inflammations (LPS, O
, hyaluronan), we show that TLR5 impacts the in vivo response to LPS, hyaluronan and O
. We demonstrate that immune cells of human carriers of a dominant negative TLR5 allele have decreased inflammatory response to O
exposure ex vivo and LPS exposure in vitro. Using primary murine macrophages, we find that TLR5 physically associates with TLR4 and biases TLR4 signaling towards the MyD88 pathway. Our results suggest an updated paradigm for TLR4/TLR5 signaling.
Background The genetic determinants of the human innate immune response are poorly understood. Apolipoprotein (Apo) E, a lipid-trafficking protein that affects inflammation, has well-described ...wild-type (ε3) and disease-associated (ε2 and ε4) alleles, but its connection to human innate immunity is undefined. Objective We sought to define the relationship of APOε4 to the human innate immune response. Methods We evaluated APOε4 in several functional models of the human innate immune response, including intravenous LPS challenge in human subjects, and assessed APOε4 association to organ injury in patients with severe sepsis, a disease driven by dysregulated innate immunity. Results Whole blood from healthy APOε3 / APOε4 volunteers induced higher cytokine levels on ex vivo stimulation with Toll-like receptor (TLR) 2, TLR4, or TLR5 ligands than blood from APOε3 / APOε3 patients, whereas TLR7/8 responses were similar. This was associated with increased lipid rafts in APOε3/APOε4 monocytes. By contrast, APOε3 / APOε3 and APOε3 / APOε4 serum neutralized LPS equivalently and supported similar LPS responses in Apoe -deficient macrophages, arguing against a differential role for secretory APOE4 protein. After intravenous LPS, APOε3/APOε4 patients had higher hyperthermia and plasma TNF-α levels and earlier plasma IL-6 than APOε3/APOε3 patients. APOE4-targeted replacement mice displayed enhanced hypothermia, plasma cytokines, and hepatic injury and altered splenic lymphocyte apoptosis after systemic LPS compared with APOE3 counterparts. In a cohort of 828 patients with severe sepsis, APOε4 was associated with increased coagulation system failure among European American patients. Conclusions APOε4 is a determinant of the human innate immune response to multiple TLR ligands and associates with altered patterns of organ injury in human sepsis.
The TLR2 interactome was defined in cells upon exposure to the TLR2 agonist Pam3CSK4 and the HMG CoA reductase inhibitor simvastatin, singly and in combination, by applying ...coimmunoprecipitation-based cross-linking proteomics. Two different crosslinkers with different spacer chain lengths were used for fixing protein complexes. The results indicate that ACTR1A is a potential interactor of TLR2. Functional studies using RNA interference confirm that ACTR1A is required for TLR2 signaling to induction of pro-inflammatory cytokines.
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Highlights
•An innovative co-IP crosslinking proteomics study was designed for the TLR2 interactome.•Proteomic profiling revealed combinatorial effects of simvastatin and Pam3CSK4 on the TLR2 interactome.•ACTR1A and MARCKSL1 proteins were identified as potential interactors of TLR2 during the immune response.•ACTR1A has important modulatory actions on the TLR2 pro-inflammatory signaling cascade.
Toll-like receptor 2 (TLR2) is a pattern recognition receptor that, upon ligation by microbial molecules, interacts with other proteins to initiate pro-inflammatory responses by the cell. Statins (hydroxymethylglutaryl coenzyme A reductase inhibitors), drugs widely prescribed to reduce hypercholesterolemia, are reported to have both pro- and anti-inflammatory effects upon cells. Some of these responses are presumed to be driven by effects on signaling proteins at the plasma membrane, but the underlying mechanisms remain obscure. We reasoned that profiling the effect of statins on the repertoire of TLR2-interacting proteins might provide novel insights into the mechanisms by which statins impact inflammation. In order to study the TLR2 interactome, we designed a coimmunoprecipitation (IP)-based cross-linking proteomics study. A hemagglutinin (HA)-tagged-TLR2 transfected HEK293 cell line was used to precipitate the TLR2 interactome upon cell exposure to the TLR2 agonist Pam3CSK4 and simvastatin, singly and in combination. To stabilize protein interactors, we used two different chemical cross-linkers with different spacer chain lengths. Proteomic analysis revealed important combinatorial effects of simvastatin and Pam3CSK4 on the TLR2 interactome. After stringent data filtering, we identified alpha-centractin (ACTR1A), an actin-related protein and subunit of the dynactin complex, as a potential interactor of TLR2. The interaction was validated using biochemical methods. RNA interference studies revealed an important role for ACTR1A in induction of pro-inflammatory cytokines. Taken together, we report that statins remodel the TLR2 interactome, and we identify ACTR1A, a part of the dynactin complex, as a novel regulator of TLR2-mediated immune signaling pathways.
Arsenic exposure via drinking water impacts millions of people worldwide. Although arsenic has been associated epidemiologically with increased lung infections, the identity of the lung cell types ...targeted by peroral arsenic and the associated immune mechanisms remain poorly defined.
We aimed to determine the impact of peroral arsenic on pulmonary antibacterial host defense.
Female C57BL/6 mice were administered drinking water with 0, 250 ppb, or 25 ppm sodium arsenite for 5 wk and then challenged intratracheally with
, or lipopolysaccharide. Bacterial clearance and immune responses were profiled.
Arsenic had no effect on bacterial clearance in the lung or on the intrapulmonary innate immune response to bacteria or lipopolysaccharide, as assessed by neutrophil recruitment to, and cytokine induction in, the airspace. Alveolar macrophage TNFα production was unaltered. By contrast, arsenic-exposed mice had significantly reduced plasma TNFα in response to systemic lipopolysaccharide challenge, together suggesting that the local airway innate immune response may be relatively preserved from arsenic intoxication. Despite intact intrapulmonary bacterial clearance during pneumonia, arsenic-exposed mice suffered dramatically increased bacterial dissemination to the bloodstream. Mechanistically, this was linked to increased respiratory epithelial permeability, as revealed by intratracheal FITC-dextran tracking, serum Club Cell protein 16 measurement, and other approaches. Consistent with barrier disruption at the alveolar level, arsenic-exposed mice had evidence for alveolar epithelial type 1 cell injury.
Peroral arsenic has little effect on local airway immune responses to bacteria but compromises respiratory epithelial barrier integrity, increasing systemic translocation of inhaled pathogens and small molecules. https://doi.org/10.1289/EHP1878.
MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression by promoting degradation and/or repressing translation of specific target mRNAs. Several miRNAs have been identified that ...regulate the amplitude of the innate immune response by directly targeting Toll-like receptor (TLR) pathway members and/or cytokines. miR-33a and miR-33b (the latter present in primates but absent in rodents and lower species) are located in introns of the sterol regulatory element-binding protein (SREBP)-encoding genes and control cholesterol/lipid homeostasis in concert with their host gene products. These miRNAs regulate macrophage cholesterol by targeting the lipid efflux transporters ATP binding cassette (ABC)A1 and ABCG1. We and others have previously reported that Abca1−/− and Abcg1−/− macrophages have increased TLR proinflammatory responses due to augmented lipid raft cholesterol. Given this, we hypothesized that miR-33 would augment TLR signaling in macrophages via a raft cholesterol-dependent mechanism. Herein, we report that multiple TLR ligands down-regulate miR-33 in murine macrophages. In the case of lipopolysaccharide, this is a delayed, Toll/interleukin-1 receptor (TIR) domain-containing adapter-inducing interferon-β-dependent response that also down-regulates Srebf-2, the host gene for miR-33. miR-33 augments macrophage lipid rafts and enhances proinflammatory cytokine induction and NF-κB activation by LPS. This occurs through an ABCA1- and ABCG1-dependent mechanism and is reversible by interventions upon raft cholesterol and by ABC transporter-inducing liver X receptor agonists. Taken together, these findings extend the purview of miR-33, identifying it as an indirect regulator of innate immunity that mediates bidirectional cross-talk between lipid homeostasis and inflammation.
Background Sensitization to house dust mite allergens is strongly correlated with asthma. Der p 7 elicits strong IgE antibody and T-cell responses in patients with mite allergy. However, the ...structure and biological function of this important allergen are unknown. Allergen function might contribute to allergenicity, as shown for the protease activity of group 1 mite allergens and the interaction with the innate immune system by group 2 mite allergens. Objective We sought to determine the crystal structure of Der p 7 and to investigate its biological function. Methods X-ray crystallography was used to determine the Der p 7 structure. Nuclear magnetic resonance analysis and biochemical assays were used to examine the binding of Der p 7 to predicted ligands. Results Der p 7 has an elongated structure, with two 4-stranded antiparallel β-sheets that wrap around a long C-terminal helix. The fold of Der p 7 is similar to that of LPS-binding protein (LBP), which interacts with Toll-like receptors after binding LPS and other bacterially derived lipid ligands. Nuclear magnetic resonance and biochemical assays indicate that Der p 7 does not bind LPS but binds with weak affinity to the bacterial lipopeptide polymyxin B in the predicted binding site of Der p 7. Conclusions Der p 7 binds a bacterially derived lipid product, a common feature of some allergens. The finding that the group 7, as well as the group 2, mite allergens are structurally similar to different proteins in the Toll-like receptor pathway further strengthens the connections between dust mites, innate immunity, and allergy.
During viral infection, extracellular dsRNA is a potent signaling molecule that activates many innate immune cells, including macrophages. TLR3 is a well-known receptor for extracellular dsRNA, and ...internalization of extracellular dsRNA is required for endosomal TLR3 activation. Preserved inflammatory responses of TLR3-deficient macrophages to extracellular dsRNA strongly support a TLR3-independent mechanism in dsRNA-mediated immune responses. The present study demonstrated that CD11b/CD18 (Mac-1 macrophage-1 Ag), a surface integrin receptor, recognized extracellular dsRNA and induced macrophage immune responses. CD11b deficiency reduced inflammatory cytokine induction elicited by polyinosinic:polycytidylic acid (poly I:C; a synthetic dsRNA) in mouse sera and livers, as well as in cultured peritoneal macrophages. dsRNA-binding assay and confocal immunofluorescence showed that Mac-1, especially the CD11b subunit, interacted and colocalized with poly I:C on the surface of macrophages. Further mechanistic studies revealed two distinct signaling events following dsRNA recognition by Mac-1. First, Mac-1 facilitated poly I:C internalization through the activation of PI3K signaling and enhanced TLR3-dependent activation of IRF3 in macrophages. Second, poly I:C induced activation of phagocyte NADPH oxidase in a TLR3-independent, but Mac-1-dependent, manner. Subsequently, phagocyte NADPH oxidase-derived intracellular reactive oxygen species activated MAPK and NF-κB pathways. Our results indicate that extracellular dsRNA activates Mac-1 to enhance TLR3-dependent signaling and to trigger TLR3-independent, but Mac-1-dependent, inflammatory oxidative signaling, identifying a novel mechanistic basis for macrophages to recognize extracellular dsRNA to regulate innate immune responses. This study identifies Mac-1 as a novel surface receptor for extracellular dsRNA and implicates it as a potential therapeutic target for virus-related inflammatory diseases.
•Role of prohibitins (PHBs) in macrophage signaling was studied using PHB knockdown.•PHB knockdown decreases expression and signaling of lipid raft-dependent receptors.•Plasma membrane packing and ...raft formation are altered in PHB knockdown macrophages.•PHB controls the composition of cellular fatty acids.•PHBs impact macrophage signaling by regulating lipid rafts.
Prohibitins (PHB1 and PHB2) are ubiquitously expressed proteins which play critical roles in multiple biological processes, and together form the ring-like PHB complex found in phospholipid-rich cellular compartments including lipid rafts. Recent studies have implicated PHB1 as a mediator of fatty acid transport as well as a membrane scaffold mediating B lymphocyte and mast cell signal transduction. However, the specific role of PHBs in the macrophage have not been characterized, including their role in fatty acid uptake and lipid raft-mediated inflammatory signaling. We hypothesized that the PHB complex regulates macrophage inflammatory signaling through the formation of lipid rafts. To evaluate our hypothesis, RAW 264.7 macrophages were transduced with shRNA against PHB1, PHB2, or scrambled control (Scr), and then stimulated with lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-α), which activate lipid raft-dependent receptor signaling (CD14/TLR4 and TNFR1, respectively). PHB1 knockdown was lethal, whereas PHB2 knockdown (PHB2kd), which also resulted in decreased PHB1 expression, led to attenuated nuclear factor-kappa-B (NF-κB) activation and subsequent cytokine and chemokine production. PHB2kd macrophages also had decreased cell surface TNFR1, CD14, TLR4, and lipid raft marker ganglioside GM1 at baseline and post-stimuli. Post-LPS, PHB2kd macrophages did not increase the concentration of cellular saturated, monounsaturated, and polyunsaturated fatty acids. This was accompanied by decreased lipid raft formation and modified plasma membrane molecular packing, further supporting the PHB complex's importance in lipid raft formation. Taken together, these data suggest a critical role for PHBs in regulating macrophage inflammatory signaling via maintenance of fatty acid composition and lipid raft structure.
Prohibitins are proteins found in phospholipid-rich cellular compartments, including lipid rafts, that play important roles in signaling, transcription, and multiple other cell functions. Macrophages are key cells in the innate immune response and the presence of membrane lipid rafts is integral to signal transduction, but the role of prohibitins in macrophage lipid rafts and associated signaling is unknown. To address this question, prohibitin knockdown macrophages were generated and responses to lipopolysaccharide and tumor necrosis factor-alpha, which act through lipid raft-dependent receptors, were analyzed. Prohibitin knockdown macrophages had significantly decreased cytokine and chemokine production, transcription factor activation, receptor expression, lipid raft assembly and membrane packing, and altered fatty acid remodeling. These data indicate a novel role for prohibitins in macrophage inflammatory signaling through regulation of fatty acid composition and lipid raft formation.
Toll-like receptors (TLRs) are pathogen-recognition receptors that trigger the innate immune response. Recent reports have identified accessory proteins that provide essential support to TLR function ...through ligand delivery and receptor trafficking. Herein, we introduce leucine-rich repeats (LRRs) and calponin homology containing 4 (Lrch4) as a novel TLR accessory protein. Lrch4 is a membrane protein with nine LRRs in its predicted ectodomain. It is widely expressed across murine tissues and has two expression variants that are both regulated by lipopolysaccharide (LPS). Predictive modeling indicates that Lrch4 LRRs conform to the horseshoe-shaped structure typical of LRRs in pathogen-recognition receptors and that the best structural match in the protein database is to the variable lymphocyte receptor of the jawless vertebrate hagfish. Silencing Lrch4 attenuates cytokine induction by LPS and multiple other TLR ligands and dampens the in vivo innate immune response. Lrch4 promotes proper docking of LPS in lipid raft membrane microdomains. We provide evidence that this is through regulation of lipid rafts as Lrch4 silencing reduces cell surface gangliosides, a metric of raft abundance, as well as expression and surface display of CD14, a raft-resident LPS co-receptor. Taken together, we identify Lrch4 as a broad-spanning regulator of the innate immune response and a potential molecular target in inflammatory disease.
Lipid raft membrane microdomains organize signaling by many prototypical receptors, including the Toll-like receptors (TLRs) of the innate immune system. Raft-localization of proteins is widely ...thought to be regulated by raft cholesterol levels, but this is largely on the basis of studies that have manipulated cell cholesterol using crude and poorly specific chemical tools, such as β-cyclodextrins. To date, there has been no proteome-scale investigation of whether endogenous regulators of intracellular cholesterol trafficking, such as the ATP binding cassette (ABC)A1 lipid efflux transporter, regulate targeting of proteins to rafts. Abca1−/− macrophages have cholesterol-laden rafts that have been reported to contain increased levels of select proteins, including TLR4, the lipopolysaccharide receptor. Here, using quantitative proteomic profiling, we identified 383 proteins in raft isolates from Abca1+/+ and Abca1−/− macrophages. ABCA1 deletion induced wide-ranging changes to the raft proteome. Remarkably, many of these changes were similar to those seen in Abca1+/+ macrophages after lipopolysaccharide exposure. Stomatin-like protein (SLP)-2, a member of the stomatin-prohibitin-flotillin-HflK/C family of membrane scaffolding proteins, was robustly and specifically increased in Abca1−/− rafts. Pursuing SLP-2 function, we found that rafts of SLP-2-silenced macrophages had markedly abnormal composition. SLP-2 silencing did not compromise ABCA1-dependent cholesterol efflux but reduced macrophage responsiveness to multiple TLR ligands. This was associated with reduced raft levels of the TLR co-receptor, CD14, and defective lipopolysaccharide-induced recruitment of the common TLR adaptor, MyD88, to rafts. Taken together, we show that the lipid transporter ABCA1 regulates the protein repertoire of rafts and identify SLP-2 as an ABCA1-dependent regulator of raft composition and of the innate immune response.
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