Dendritic cells in host response to biologic scaffolds.

Tissue regeneration and repair require a highly complex and orchestrated series of events that require inflammation, but can be compromised when inflammation is excessive or becomes chronic. Macrophages are one of the first cells to contact and respond to implanted materials, and mediate the inflammatory response. The series of events following macrophage association with biomaterials has been well-studied. Dendritic cells (DCs) also directly interact with biomaterials, are critical for specific immune responses, and can be activated in response to interactions with biomaterials. Yet, much less is known about the responses by DCs. This review discusses what we know about DC response to biomaterials, the underlying mechanisms involved, and how DCs can be influenced by the macrophage response to biomaterials. Lastly, I will discuss how biomaterials can be manipulated to enhance or suppress DC function to promote a specific desirable immune response - a major goal for implantable biologically active therapeutics.

Mitochondrial DNA in the tumour microenvironment activates neutrophils and is associated with worse outcomes in patients with advanced epithelial ovarian cancer.

BACKGROUND: Advanced cancer causes necrosis and releases damage-associated molecular patterns (DAMPs). Mitochondrial DAMPs activate neutrophils, including generation of neutrophil extracellular traps (NETs), which are injurious, thrombogenic, and implicated in metastasis. We hypothesised that extracellular mitochondrial DNA (mtDNA) in ascites from patients with epithelial ovarian cancer (EOC) would correlate with worse outcomes. METHODS: Banked ascites supernatants from patients with newly diagnosed advanced EOC were analysed for mtDNA, neutrophil elastase, and activation of healthy donor neutrophils and platelets. TCGA was mined for expression of SELP and ELANE. RESULTS: The highest quartile of ascites mtDNA correlated with reduced progression-free survival (PFS) and a higher likelihood of disease progression within 12-months following primary surgery (n = 68, log-rank, p = 0.0178). NETs were detected in resected tumours. Ascites supernatants chemoattracted neutrophils, induced NETs, and activated platelets. Ascites exposure rendered neutrophils suppressive, based on abrogation of ex vivo stimulated T cell proliferation. Increased SELP mRNA expression correlated with worse overall survival (n = 302, Cox model, p = 0.02). CONCLUSION: In this single-centre retrospective analysis, ascites mtDNA correlated with worse PFS in advanced EOC. Mitochondrial and other DAMPs in ascites may activate neutrophil and platelet responses that facilitate metastasis and obstruct anti-tumour immunity. These pathways are potential prognostic markers and therapeutic targets.

Toll-like receptor signaling in macrophages is regulated by extracellular substrate stiffness and Rho-associated coiled-coil kinase (ROCK1/2).

Macrophages participate in immunity, tissue repair and tissue homeostasis. Activation of Toll-like receptors (TLRs) by conserved exogenous or endogenous structures initiates signaling cascades that result in the release of cytokines such as tumor necrosis factor α (TNFα). Extracellular substrate stiffness is known to regulate functions of non-immune cells through a process called mechanotransduction, yet less is known about how physical cues affect macrophage function or TLR signaling. To investigate this question, we cultured murine primary bone marrow-derived macrophages (BMMs) and RAW264.7 cells on fibronectin-coated polyacrylamide (PA) gels of defined stiffnesses (1, 20 and 150 kPa) that approximate the physical properties of physiologic tissues. BMMs on all gels were smaller and more circular than those on rigid glass. Macrophages on intermediate stiffness 20 kPa PA gels were slightly larger and less circular than those on either 1 or 150 kPa. Secretion of the pro-inflammatory cytokine, TNFα, in response to stimulation of TLR4 and TLR9 was increased in macrophages grown on soft gels versus more rigid gels, particularly for BMMs. Inhibition of the rho-associated coiled-coil kinase 1/2 (ROCK1/2), key mediators in cell contractility and mechanotransduction, enhanced release of TNFα in response to stimulation of TLR4. ROCK1/2 inhibition enhanced phosphorylation of the TLR downstream signaling molecules, p38, ERK1/2 and NFκB. Our data indicate that physical cues from the extracellular environment regulate macrophage morphology and TLR signaling. These findings have important implications in the regulation of macrophage function in diseased tissues and offer a novel pharmacological target for the manipulation of macrophage function in vivo.

Complex Negative Regulation of TLR9 by Multiple Proteolytic Cleavage Events.

TLR9 is an innate immune receptor important for recognizing DNA of host and foreign origin. A mechanism proposed to prevent excessive response to host DNA is the requirement for proteolytic cleavage of TLR9 in endosomes to generate a mature form of the receptor (TLR9(471-1032)). We previously described another cleavage event in the juxtamembrane region of the ectodomain that generated a dominant-negative form of TLR9. Thus, there are at least two independent cleavage events that regulate TLR9. In this study, we investigated whether an N-terminal fragment of TLR9 could be responsible for regulation of the mature or negative-regulatory form. We show that TLR9(471-1032), corresponding to the proteolytically cleaved form, does not function on its own. Furthermore, activity is not rescued by coexpression of the N-terminal fragment (TLR9(1-440)), inclusion of the hinge region (TLR9(441-1032)), or overexpression of UNC93B1, the last of which is critical for trafficking and cleavage of TLR9. TLR9(1-440) coimmunoprecipitates with full-length TLR9 and TLR9(471-1032) but does not rescue the native glycosylation pattern; thus, inappropriate trafficking likely explains why TLR9(471-1032) is nonfunctional. Lastly, we show that TLR9(471-1032) is also a dominant-negative regulator of TLR9 signaling. Together, these data provide a new perspective on the complexity of TLR9 regulation by proteolytic cleavage and offer potential ways to inhibit activity through this receptor, which may dampen autoimmune inflammation.

Safe Recombinant Outer Membrane Vesicles that Display M2e Elicit Heterologous Influenza Protection.

Recombinant, Escherichia coli-derived outer membrane vesicles (rOMVs), which display heterologous protein subunits, have potential as a vaccine adjuvant platform. One drawback to rOMVs is their lipopolysaccharide (LPS) content, limiting their translatability to the clinic due to potential adverse effects. Here, we explore a unique rOMV construct with structurally remodeled lipids containing only the lipid IVa portion of LPS, which does not stimulate human TLR4. The rOMVs are derived from a genetically engineered B strain of E. coli, ClearColi, which produces lipid IVa, and which was further engineered in our laboratory to hypervesiculate and make rOMVs. We report that rOMVs derived from this lipid IVa strain have substantially attenuated pyrogenicity yet retain high levels of immunogenicity, promote dendritic cell maturation, and generate a balanced Th1/Th2 humoral response. Additionally, an influenza A virus matrix 2 protein-based antigen displayed on these rOMVs resulted in 100% survival against a lethal challenge with two influenza A virus strains (H1N1 and H3N2) in mice with different genetic backgrounds (BALB/c, C57BL/6, and DBA/2J). Additionally, a two-log reduction of lung viral titer was achieved in a ferret model of influenza infection with human pandemic H1N1. The rOMVs reported herein represent a potentially safe and simple subunit vaccine delivery platform.

Electrostatically self-assembled biodegradable microparticles from pseudoproteins and polysaccharide: fabrication, characterization, and biological properties.

Electrostatically self-assembling hybrid microparticles derived from novel cationic unsaturated arginine-based poly(ester amide) polymers (UArg-PEA) and anionic hyaluronic acid (HA) were fabricated into sub-micron-sized particles in aqueous medium with subsequent UV crosslinking treatment to stabilize the structure. These hybrid microparticles were characterized for size, charge, viscosity, chemical structure, morphology, and biological properties. Depending on the feed ratio of cationic UArg-PEA to anionic HA, the crosslinked microparticles formed spherical structures of 0.772-22.08 µm in diameter, whereas the uncrosslinked microparticles formed a core with an outer petal-like structure of 2.49-15 µm in diameter. It was discovered that the morphological structure of the self-assembled microparticles had a profound influence on their biological properties. At a 1:1 feed ratio of UArg-PEA to HA, the uncrosslinked microparticles showed no cytotoxicity toward NIH 3T3 fibroblasts at concentrations up to 20 µg/mL, and the crosslinked particles exhibited no cytotoxicity at concentrations up to 10 µg/mL. The UArg-PEA/HA hybrid microparticles exhibited a significantly lower macrophage-induced proinflammatory response (via TNF-α) than that from a pure hyaluronic acid control while retaining the beneficial anti-inflammatory IL-10 production by HA. The UArg-PEA/HA microparticles also stimulated size-dependent induction of arginase activity. Therefore, self-assembling these two types of biomaterials in a favorable nontoxic aqueous environment, having complementary biological properties like those of the currently reported UArg-PEA/HA hybrid microparticles, may provide a new class of biomaterials to improve the overall tissue microenvironment for promoting wound healing.

Cutting edge: a TLR9 cytoplasmic tyrosine motif is selectively required for proinflammatory cytokine production.

Compartmentalization of nucleic acid sensing TLR9 has been implicated as a mechanism to prevent recognition of self nucleic acid structures. Furthermore, recognition of CpG DNA in different endosomal compartments leads to the production of the proinflammatory cytokine TNF-α, or type I IFN. We previously characterized a tyrosine-based motif at aa 888-891 in the cytoplasmic tail of TLR9 important for appropriate intracellular localization. In this article, we show that this motif is selectively required for the production of TNF, but not IFN. In response to CpG DNA stimulation, the proteolytically processed 80-kDa fragment is tyrosine phosphorylated. Although Y888 is not itself phosphorylated, the structure of this motif is necessary for both TLR9 phosphorylation and TNF-α production in response to CpG DNA. We conclude that bifurcation in TLR9 signaling is regulated by a critical tyrosine motif in the cytoplasmic tail.

Heterozygous carriage of a dysfunctional Toll-like receptor 9 allele affects CpG oligonucleotide responses in B cells.

Toll-like receptors (TLR) are employed by the innate immune system to detect microbial pathogens based on conserved microbial pathogen molecules. For example, TLR9 is a receptor for CpG-containing microbial DNA, and its activation results in the production of cytokines and type I interferons from human B cells and plasmacytoid dendritic cells, respectively. Both are required for mounting an efficient antibacterial or antiviral immune response. These effects are mimicked by synthetic CpG oligodeoxynucleotides (ODN). Although several hyporesponsive TLR9 variants have been reported, their functional relevance in human primary cells has not been addressed. Here we report a novel TLR9 allele, R892W, which is hyporesponsive to CpG ODN and acts as a dominant-negative in a cellular model system. The R892W variant is characterized by increased MyD88 binding and defective co-localization with CpG ODN. Whereas primary plasmacytoid dendritic cells isolated from a heterozygous R892W carrier responded normally to CpG by interferon-α production, carrier B cells showed impaired IL-6 and IL-10 production. This suggests that heterozygous carriage of a hyporesponsive TLR9 allele is not associated with complete loss of TLR9 function but that TLR9 signals elicited in different cell types are regulated differently in human primary cells.

Participation of MyD88 and interleukin-33 as innate drivers of Th2 immunity to Trichinella spiralis.

Trichinella spiralis is a highly destructive parasitic nematode that invades and destroys intestinal epithelial cells, injures many different tissues during its migratory phase, and occupies and transforms myotubes during the final phase of its life cycle. We set out to investigate the role in immunity of innate receptors for potential pathogen- or danger-associated molecular patterns (PAMPs or DAMPs). Focusing on the MyD88-dependent receptors, which include Toll-like receptors (TLRs) and interleukin-1 (IL-1) family members, we found that MyD88-deficient mice expelled worms normally, while TLR2/4-deficient mice showed accelerated worm expulsion, suggesting that MyD88 was active in signaling pathways for more than one receptor during intestinal immunity. A direct role for PAMPs in TLR activation was not supported in a transactivation assay involving a panel of murine and human TLRs. Mice deficient in the IL-1 family receptor for the DAMP, IL-33 (called ST2), displayed reduced intestinal Th2 responses and impaired mast cell activation. IL-33 was constitutively expressed in intestinal epithelial cells, where it became concentrated in nuclei within 2 days of infection. Nuclear localization was an innate response to infection that occurred in intestinal regions where worms were actively migrating. Th2 responses were also compromised in the lymph nodes draining the skeletal muscles of ST2-deficient mice, and this correlated with increased larval burdens in muscle. Our results support a mechanism in which the immune system recognizes and responds to tissue injury in a way that promotes Th2 responses.

Antimicrobial peptides inhibit polyinosinic-polycytidylic acid-induced immune responses.

Viral proteins and nucleic acids stimulate TLRs to elicit production of cytokines, chemokines, and IFNs. Because of their immunostimulatory activity, several TLR agonists are being developed as vaccine adjuvants and cancer immunotherapeutics. However, TLR signaling is modified by disease state, which could enhance or impair therapeutic efficacy. For example, in the skin of psoriasis patients, the human cationic antimicrobial peptide LL37 is highly expressed and binds to host DNA. Association with LL37 enhances DNA uptake into intracellular compartments, where it stimulates TLR9-dependent overproduction of IFNs. Polyinosinic-polycytidylic acid (poly(I:C)), an analog of viral dsRNA, is recognized by TLR3 and is currently in preclinical trials as an inducer of type I IFN. If LL37 similarly enhanced IFN production, use of poly(I:C) might be contraindicated in certain conditions where LL37 is elevated. In this study, we show that TLR3 signaling was not enhanced, but was dramatically inhibited, by LL37 or mouse cathelicidin-related antimicrobial peptide in macrophages, microglial cells, and dendritic cells. Inhibition correlated with formation of a strong complex between antimicrobial peptides and poly(I:C), which partially inhibited poly(I:C) binding to TLR3. Therefore, after injury or during existing acute or chronic inflammation, when LL37 levels are elevated, the therapeutic activity of poly(I:C) will be compromised. Our findings highlight the importance of using caution when therapeutically delivering nucleic acids as immunomodulators.

Traditional biochemical assays for studying toll-like receptor 9.

Understanding the mechanistic basis of receptor activation and regulation can offer therapeutic targets for disease treatment. Evidence is emerging for a role of the normally foreign responsive Toll-like receptors (TLRs) in the development of autoimmunity through response to self-patterns. Regulatory mechanisms governing this class of receptors are poorly understood, and failures within this system likely contribute to development of autoimmunity. In this article, we review biochemical assays used to study one of the self-pattern responsive TLRs, TLR9, and suggest that these studies are critical for development of new targets for autoimmune therapies.

Racing CARs to veterinary immuno-oncology.

Chimeric antigen receptors (CARs) have demonstrated remarkable promise in human oncology over the past two decades, yet similar strategies in veterinary medicine are still in development. CARs are synthetically engineered proteins comprised of a specific antigen-binding single chain variable fragment (ScFv) fused to the signaling domain of a T cell receptor and co-receptors. Patient T cells engineered to express a CAR are directed to recognize and kill target cells, most commonly hematological malignancies. The U.S Food and Drug Administration (FDA) has approved multiple human CAR T therapies, but translation of these therapies into veterinary medicine faces many challenges. In this review, we discuss considerations for veterinary use including CAR design and cell carrier choice, and discuss the future promise of translating CAR therapy into veterinary oncology.

Multifunctional role of dextran sulfate sodium for in vivo modeling of intestinal diseases.

BACKGROUND: Inflammatory bowel diseases (IBDs) are chronic, relapsing disorders that affect the gastrointestinal tract of millions of people and continue to increase in incidence each year. While several factors have been associated with development of IBDs, the exact etiology is unknown. Research using animal models of IBDs is beginning to provide insights into how the different factors contribute to disease development. Oral administration of dextran sulfate sodium (DSS) to mice induces a reproducible experimental colitis that models several intestinal lesions associated with IBDs. The murine DSS colitis model can also be adapted to quantify intestinal repair following injury. Understanding the mechanistic basis behind intestinal repair is critical to development of new therapeutics for IBDs because of their chronic relapsing nature. RESULTS: The murine DSS colitis model was adapted to provide a system enabling the quantification of severe intestinal injury with impaired wound healing or mild intestinal injury with rapid restoration of mucosal integrity, by altering DSS concentrations and including a recovery phase. We showed that through a novel format for presentation of the clinical disease data, the temporal progression of intestinal lesions can be quantified on an individual mouse basis. Additionally, parameters for quantification of DSS-induced alterations in epithelial cell populations are included to provide insights into mechanisms underlying the development of these lesions. For example, the use of the two different model systems showed that toll-like receptor 9, a nucleic acid-sensing pattern recognition receptor, is important for protection only following mild intestinal damage and suggests that this model is superior for identifying proteins necessary for intestinal repair. CONCLUSIONS: We showed that using a murine DSS-induced experimental colitis model system, and presenting data in a longitudinal manner on a per mouse basis, enhanced the usefulness of this model, and provided novel insights into the role of an innate immune receptor in intestinal repair. By elucidating the mechanistic basis of intestinal injury and repair, we can begin to understand the etiology of IBDs, enabling development of novel therapeutics or prophylactics.

Negative regulation of signaling by a soluble form of toll-like receptor 9.

Nucleic acid structures are highly conserved through evolution and when self nucleic acids are aberrantly detected by toll-like receptors (TLRs) they contribute to autoimmune disease. For this reason, multiple regulatory mechanisms exist to prevent immune responses to self nucleic acids. TLR9 is a nucleic acid-sensing TLR that is regulated at multiple levels including association with accessory proteins, intracellular localization and proteolytic processing. In the endolysosomal compartment TLR9 is proteolytically processed to an 80 kDa form (p80) and this processing is a prerequisite for activation. Here, we identified a soluble form of TLR9 (sTLR9) generated by a novel proteolytic event that cleaved TLR9 between amino acids 724-735. Similar to p80, sTLR9 was generated in endosomes. However, generation of sTLR9 was independent of the cysteine protease cathepsin B, active at acidic pH, but partially dependent on cathepsin S, a protease active at neutral pH. Most importantly, sTLR9 inhibited TLR9-dependent signaling. Altogether, these data support a model where an intrinsic proteolytic processing mechanism negatively regulates TLR9 signaling. A proper balance between the independent proteolytic events probabably contributes to regulation of TLR9-mediated innate immunity and prevention of autoimmune disease.

Heat shock protein gp96 regulates Toll-like receptor 9 proteolytic processing and conformational stability.

Nucleic acid-sensing Toll-like receptors (TLRs) initiate innate immune responses to foreign RNA and DNA, yet can detect and respond to host DNA. To avoid autoimmune pathologies, nucleic acid sensing TLRs are tightly regulated. TLR9 primarily resides in the endoplasmic reticulum, traffics to endosomes, is proteolytically processed and responds to DNA. The heat shock protein gp96 is one of several accessory proteins that regulate intracellular trafficking of TLR9. In the absence of gp96, TLR9 fails to exit the endoplasmic reticulum, and therefore gp96-deficient macrophages fail to respond to CpG DNA. However, absence of gp96 precludes studies on potential chaperoning functions of gp96 for TLR9. Here we demonstrate that pharmacologic interference with gp96 function inhibits TLR9 signaling. TLR9 remains associated with gp96 during intracellular trafficking, and gp96-specific inhibitors increase TLR9 sensitivity to proteolytic degradation. We propose that gp96 is critical for both TLR9 egress from the ER, and for protein conformational stability in the endosomal compartment. These studies highlight the importance of examining gp96-specific inhibitors for modulating TLR9 activation, and the treatment autoimmune diseases.

TLR9 regulates Th1 responses and cooperates with TLR2 in mediating optimal resistance to Mycobacterium tuberculosis.

To investigate the role of Toll-like receptor (TLR)9 in the immune response to mycobacteria as well as its cooperation with TLR2, a receptor known to be triggered by several major mycobacterial ligands, we analyzed the resistance of TLR9(-/-) as well as TLR2/9 double knockout mice to aerosol infection with Mycobacterium tuberculosis. Infected TLR9(-/-) but not TLR2(-/-) mice displayed defective mycobacteria-induced interleukin (IL)-12p40 and interferon (IFN)-gamma responses in vivo, but in common with TLR2(-/-) animals, the TLR9(-/-) mice exhibited only minor reductions in acute resistance to low dose pathogen challenge. When compared with either of the single TLR-deficient animals, TLR2/9(-/-) mice displayed markedly enhanced susceptibility to infection in association with combined defects in proinflammatory cytokine production in vitro, IFN-gamma recall responses ex vivo, and altered pulmonary pathology. Cooperation between TLR9 and TLR2 was also evident at the level of the in vitro response to live M. tuberculosis, where dendritic cells and macrophages from TLR2/9(-/-) mice exhibited a greater defect in IL-12 response than the equivalent cell populations from single TLR9-deficient animals. These findings reveal a previously unappreciated role for TLR9 in the host response to M. tuberculosis and illustrate TLR collaboration in host resistance to a major human pathogen.

MARCO, TLR2, and CD14 are required for macrophage cytokine responses to mycobacterial trehalose dimycolate and Mycobacterium tuberculosis.

Virtually all of the elements of Mycobacterium tuberculosis (Mtb) pathogenesis, including pro-inflammatory cytokine production, granuloma formation, cachexia, and mortality, can be induced by its predominant cell wall glycolipid, trehalose 6,6'-dimycolate (TDM/cord factor). TDM mediates these potent inflammatory responses via interactions with macrophages both in vitro and in vivo in a myeloid differentiation factor 88 (MyD88)-dependent manner via phosphorylation of the mitogen activated protein kinases (MAPKs), implying involvement of toll-like receptors (TLRs). However, specific TLRs or binding receptors for TDM have yet to be identified. Herein, we demonstrate that the macrophage receptor with collagenous structure (MARCO), a class A scavenger receptor, is utilized preferentially to "tether" TDM to the macrophage and to activate the TLR2 signaling pathway. TDM-induced signaling, as measured by a nuclear factor-kappa B (NF-kappaB)-luciferase reporter assay, required MARCO in addition to TLR2 and CD14. MARCO was used preferentially over the highly homologous scavenger receptor class A (SRA), which required TLR2 and TLR4, as well as their respective accessory molecules, in order for a slight increase in NF-kappaB signaling to occur. Consistent with these observations, macrophages from MARCO(-/-) or MARCO(-/-)SRA(-/-) mice are defective in activation of extracellular signal-related kinase 1/2 (ERK1/2) and subsequent pro-inflammatory cytokine production in response to TDM. These results show that MARCO-expressing macrophages secrete pro-inflammatory cytokines in response to TDM by cooperation between MARCO and TLR2/CD14, whereas other macrophage subtypes (e.g. bone marrow-derived) may rely somewhat less effectively on SRA, TLR2/CD14, and TLR4/MD2. Macrophages from MARCO(-/-) mice also produce markedly lower levels of pro-inflammatory cytokines in response to infection with virulent Mtb. These observations identify the scavenger receptors as essential binding receptors for TDM, explain the differential response to TDM of various macrophage populations, which differ in their expression of the scavenger receptors, and identify MARCO as a novel component required for TLR signaling.

Early response of mucosal epithelial cells during Toxoplasma gondii infection.

The innate immune response of mucosal epithelial cells during pathogen invasion plays a central role in immune regulation in the gut. Toxoplasma gondii is a protozoan intracellular parasite that is usually transmitted through oral infection. Although much of the information on immunity to T. gondii has come from i.p. infection models, more recent studies have revealed the importance of studying immunity following infection through the natural peroral route. Oral infection studies have identified many of the key players in the intestinal response; however, they have relied on responses detected days to weeks following infection. Much less is known about how the gut epithelial layer senses and reacts during initial contact with the pathogen. Given the importance of epithelial cells during pathogen invasion, this study uses an in vitro approach to isolate the key players and examine the early response of intestinal epithelial cells during infection by T. gondii. We show that human intestinal epithelial cells infected with T. gondii elicit rapid MAPK phosphorylation, NF-kappaB nuclear translocation, and secretion of IL-8. Both ERK1/2 activation and IL-8 secretion responses were shown to be MyD88 dependent and TLR2 was identified to be involved in the recognition of the parasite regardless of the parasite genotype. Furthermore, we were able to identify additional T. gondii-regulated genes in the infected cells using a pathway-focused array. Together, our findings suggest that intestinal epithelial cells were able to recognize T. gondii during infection, and the outcome is important for modulating intestinal immune responses.

Macrophage uptake of oxidized and acetylated low-density lipoproteins and generation of reactive oxygen species are regulated by linear stiffness of the growth surface.

Macrophages are key players in the development of atherosclerosis: they scavenge lipid, transform into foam cells, and produce proinflammatory mediators. At the same time, the arterial wall undergoes profound changes in its mechanical properties. We recently showed that macrophage morphology and proinflammatory potential are regulated by the linear stiffness of the growth surface. Here we asked whether linear stiffness also regulates lipid uptake by macrophages. We cultured murine bone marrow-derived macrophages (BMMs) on polyacrylamide gels modeling stiffness of healthy (1kPa) and diseased (10-150kPa) blood vessels. In unprimed BMMs, increased linear stiffness increased uptake of oxidized (oxLDL) and acetylated (acLDL) low density lipoproteins and generation of reactive oxygen species, but did not alter phagocytosis of bacteria or silica particles. Macrophages adapted to stiff growth surfaces had increased mRNA and protein expression of two key lipoprotein receptors: CD36 and scavenger receptor b1. Regulation of the lipoprotein receptor, lectin-like receptor for ox-LDL, was more complex: mRNA expression decreased but surface protein expression increased with increased stiffness. Focal adhesion kinase was required for maximal uptake of oxLDL, but not of acLDL. Uptake of oxLDL and acLDL was independent of rho-associated coiled coil kinase. Through pharmacologic inhibition and genetic deletion, we found that transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive ion channel, plays an inhibitory role in the uptake of acLDL, but not oxLDL. Together, these results implicate mechanical signaling in the uptake of acLDL and oxLDL, opening up the possibility of new pharmacologic targets to modulate lipid uptake by macrophages in vivo.

Inhibition of TLR3 and TLR4 function and expression in human dendritic cells by helminth parasites.

Patent lymphatic filariasis is characterized by antigen-specific T-cell unresponsiveness with diminished IFN-gamma and IL-2 production and defects in dendritic cell (DC) function. Because Toll-like receptors (TLRs) play an important role in pathogen recognition and TLR expression is diminished on B and T cells of filaria-infected individuals, we examined the effect of live microfilariae (mf) on expression and function of TLRs in human DCs. We show that mf-exposed monocyte-derived human DCs (mhDCs) demonstrate marked diminution of TLR3 and TLR4 mRNA expression compared with mf-unexposed mhDCs that translated into loss of function in response to appropriate TLR ligands. Exposure to mf significantly down-regulated production of IFN-alpha, MIP-1alpha, IL-12p70, and IL-1alpha following activation with poly I:C, and of IL-12p40 following activation with poly I:C or LPS. mRNA expression of MyD88, the adaptor molecule involved in TLR4 signaling, was significantly diminished in mhDCs after exposure to mf. Moreover, mf interfered with NF-kappaB activation (particularly p65 and p50) following stimulation with poly I:C or LPS. These data suggest that mf interfere with mhDC function by altering TLR expression and interfering with both MyD88-dependent signaling and a pathway that ultimately diminishes NF-kappaB activity. This down-regulated NF-kappaB activity impairs mhDC-produced cytokines needed for full T-cell activation.