Search

Pseudomonas aeruginosais an opportunistic pathogen that can establish acute and chronic infections in individuals that lack fully functional innate immunity. In particular, phagocytosis by neutrophils and macrophages is a key mechanism that modulates host control and clearance ofP. aeruginosa. Individuals with neutropenia or cystic fibrosis are highly susceptible toP. aeruginosainfection thus underscoring the importance of the host innate immune response. Cell-to-cell contact between host innate immune cells and the pathogen, a first step in phagocytic uptake, is facilitated by simple and complex glycan structures present at the host cell surface. We have previously shown that endogenous polyanionic N-linked glycans localized to the cell surface of phagocytes mediate binding and subsequent phagocytosis ofP. aeruginosa. However, the suite of glycans thatP. aeruginosabinds to on host phagocytic cells remains poorly characterized. Here we demonstrate, with the use of exogenous N-linked glycans and a glycan array, thatP. aeruginosaPAO1 preferentially attaches to a subset of glycans, including a bias towards monosaccharide versus more complex glycan structures. Consistent with these findings, we were able to competitively inhibit bacterial adherence and uptake by the addition of exogenous N-linked mono- and di-saccharide glycans. We discuss of findings in the context of previous reports ofP. aeruginosaglycan binding.IMPORTANCEP. aeruginosabinds to a variety of glycans as part of its interaction with host cells, and a number ofP. aeruginosa-encoded receptors and target ligands have been described that allow this microbe to bind to such glycans. Here we extend this work by studying the glycans used byP. aeruginosaPAO1 to bind to phagocytic cells and by using a glycan array to characterize the suite of such molecules that could facilitate host cell-binding by this microbe. This study provides an increased understanding of the glycans bound byP. aeruginosa, and furthermore, provides a useful dataset for future studies ofP. aeruginosa-glycan interactions.

Phagocytic cells are critical to host defense against Pseudomonas aeruginosa, a Gram-negative bacterium that is an opportunistic pathogen. Accordingly, susceptible individuals frequently have impaired innate immune responses, including those with cystic fibrosis or neutropenia. Previous studies identified that the downregulation, or loss, of bacterial flagellar motility enables bacteria to evade interactions with phagocytic cells that result in phagocytic uptake of the bacteria. However, the mechanistic bases for motility-dependent interactions between P. aeruginosa and host cell surfaces that lead to phagocytic uptake of the bacteria are poorly understood. A recent insight is that exogenous addition of a negatively charged phospholipid, phosphatidylinositol-(3,4,5)-triphosphate (PIP(3)), promotes the engagement of non-motile strains of P. aeruginosa with phagocytes leading to uptake of the bacteria. Thus, we hypothesized that the engagement of P. aeruginosa by phagocytic cells is mediated by motility-dependent interactions with cell-surface polyanions. Here we report that endogenous polyanionic N-linked glycans and heparan sulfate mediate bacterial binding of P. aeruginosa by human monocytic cells. These specific interactions resulted in P. aeruginosa phagocytosis, bacterial type 3 secretion system (T3SS)-mediated cellular intoxication and the IL-1β response of host innate immune cells. Importantly, the bacterial interactions with the glycans were motility-dependent and could be recapitulated with purified, immobilized glycans. Therefore, this work describes novel interactions of P. aeruginosa with specific phagocyte cell-surface glycans that modulate relevant host innate immune responses to the bacteria, including phagocytosis, inflammation and cytotoxicity.

Highlights • CIS-platin chemotherapy leads to an increase in the percentage of monocytes within the ascites in a murine model of epithelial ovarian cancer. • Ascites derived CD11b+ cells elicit an increased response from T-cells after treatment with CIS-platin. • CIS-platin chemotherapy increases tumor-antigen -specific IFN-ɣ responses from T-cells., Epithelial ovarian cancer (EOC) is an immunologically active malignancy, but thus far immune therapy has had limited success in clinical trials. One barrier to implementation of efficacious immune therapies is a lack of knowledge of the effect of chemotherapy on the monocyte-derived component of the immune infiltrate within the tumor. We utilized the ID8 murine EOC model to investigate alterations within tumor ascites that occur following administration of platinum chemotherapy. Cisplatin treatment resulted in a significant increase in monocytes within the ascites of tumor bearing mice. We identified that CD11b+ cells from the ascites of mice that have been treated with cisplatin elicits an increase in IFN-ɣ expression from CD8+T-cells compared to CD11b+ cells from a mouse treated with vehicle control (604.0 pg/mL v. 4328.0 pg/mL; p .05). These results indicate that treatment with cisplatin leads to an increase of anti-tumor activity within the ascites related to alterations in the ascites monocytes. Further investigation of these findings in humans is necessary to identify how these cells behave in different patient subgroups and if there is a role for monocyte directed therapy in conjunction with T-cell directed therapy and/or chemotherapy.

We are developing magnetic nanoparticle (NP) methods to characterize inflammation and infection in vivo. Peritoneal infection in C57BL/6 mice was used as a biological model. An intraperitoneal NP injection was followed by measurement of magnetic nanoparticle spectroscopy of Brownian rotation (MSB) spectra taken over time. MSB measures the magnetization of NPs in a low frequency alternating magnetic field. Two groups of three mice were studied; each group had two infected mice and one control with no infection. The raw MSB signal was compared with two derived metrics: the NP relaxation time and number of NPs present in the sensitive volume of the receive coil. A four compartment dynamic model was used to relate those physical properties to the relevant biological processes including phagocytic activity and migration. The relaxation time increased over time for all of the mice as the NPs were absorbed. The NP number decreased over time as the NPs were cleared from the sensitive volume of the receive coil. The composite p-values for all three rate constants were significant: raw signal, 0.0002, relaxation

Phagocytosis is the key mechanism for host control of Pseudomonas aeruginosa, a motile Gram-negative, opportunistic bacterial pathogen which frequently undergoes adaptation and selection for traits that are advantageous for survival. One such clinically relevant adaptation is the loss of bacterial motility, observed within chronic infections, that is associated with increased antibiotic tolerance and phagocytic resistance. Previous studies using phagocytes from a leukocyte adhesion deficiency type 1 (LAD-I) patient identified CD18 as a putative cell surface receptor for uptake of live P. aeruginosa. However, how bacterial motility alters direct engagement with CD18-containing integrins remains unknown. Here we demonstrate, with the use of motile and isogenic nonmotile deletion mutants of two independent strains of P. aeruginosa and with CRISPR-generated CD18-deficient cell lines in human monocytes and murine neutrophils, that CD18 expression facilitates the uptake of both motile and nonmotile P. aeruginosa. However, unexpectedly, mechanistic studies revealed that CD18 expression was dispensable for the initial attachment of the bacteria to the host cells, which was validated with ectopic expression of complement receptor 3 (CR3) by CHO cells. Our data support that surface N-linked glycan chains (N-glycans) likely facilitate the initial interaction of bacteria with monocytes and cooperate with CD18 integrins in trans to promote internalization of bacteria. Moreover, talin-1 and kindlin-3 proteins promote uptake, but not binding, of P. aeruginosa by murine neutrophils, which supports a role for CD18 integrin signaling in this process. These findings provide novel insights into the cellular determinants for phagocytic recognition and uptake of P. aeruginosa.

Evidence indicates that complex gene-environment interactions underlie the incidence and progression of Parkinson’s disease (PD). Neuroinflammation is a well-characterized feature of PD widely believed to exacerbate the neurodegenerative process. Environmental toxicants associated with PD, such as pesticides and heavy metals, can cause cellular damage and stress potentially triggering an inflammatory response. Toxicant exposure can cause stress and damage to cells by impairing mitochondrial function, deregulating lysosomal function, and enhancing the spread of misfolded proteins. These stress-associated mechanisms produce sterile triggers such as reactive oxygen species (ROS) along with a variety of proteinaceous insults that are well documented in PD. These associations provide a compelling rationale for analysis of sterile inflammatory mechanisms that may link environmental exposure to neuroinflammation and PD progression. Intracellular inflammasomes are cytosolic assemblies of proteins that contain pattern recognition receptors, and a growing body of evidence implicates the association between inflammasome activation and neurodegenerative disease. Characterization of how inflammasomes may function in PD is a high priority because the majority of PD cases are sporadic, supporting the widely held belief that environmental exposure is a major factor in disease initiation and progression. Inflammasomes may represent a common mechanism that helps to explain the strong association between exposure and PD by mechanistically linking environmental toxicant-driven cellular stress with neuroinflammation and ultimately cell death.

Cystic fibrosis (CF), the most common lethal genetic disease in Caucasians, is characterized by chronic bacterial lung infection and excessive inflammation, which lead to progressive loss of lung function and premature death. Although ivacaftor (VX-770) alone and ivacaftor in combination with lumacaftor (VX-809) improve lung function in CF patients with the Gly551Asp and del508Phe mutations, respectively, the effects of these drugs on the function of human CF macrophages are unknown. Thus studies were conducted to examine the effects of lumacaftor alone and lumacaftor in combination with ivacaftor (i.e., ORKAMBI) on the ability of human CF ( del508Phe/ del508Phe) monocyte-derived macrophages (MDMs) to phagocytose and kill Pseudomonas aeruginosa. Lumacaftor alone restored the ability of CF MDMs to phagocytose and kill P. aeruginosa to levels observed in MDMs obtained from non-CF (WT-CFTR) donors. This effect contrasts with the partial (~15%) correction of del508Phe Cl− secretion of airway epithelial cells by lumacaftor. Ivacaftor reduced the ability of lumacaftor to stimulate phagocytosis and killing of P. aeruginosa. Lumacaftor had no effect on P. aeruginosa-stimulated cytokine secretion by CF MDMs. Ivacaftor (5 µM) alone and ivacaftor in combination with lumacaftor reduced secretion of several proinflammatory cytokines. The clinical efficacy of ORKAMBI may be related in part to the ability of lumacaftor to stimulate phagocytosis and killing of P. aeruginosa by macrophages.

Acidic microenvironments commonly occur at sites of inflammation and bacterial infections. In the context of a Pseudomonas aeruginosa infection, we previously demonstrated that acidosis enhances the cellular proinflammatory interleukin (IL)-1β response in vitro. However, how pH alterations affect in vivo IL-1β responses and subsequent IL-1-driven inflammation during infection with P. aeruginosa is unclear. Here, we report that acidosis enhances in vivo IL-1β production and downstream IL-1 receptor-dependent responses during infection with P. aeruginosa in models of acute pneumonia and peritonitis. Importantly, we demonstrate that infection with P. aeruginosa within an acidic environment leads to enhanced production of a subset of proinflammatory cytokines, including chemokine (C-X-C) motif ligand 1, IL-6, and chemokine (C-C motif) ligand 2, and increased neutrophil recruitment. Furthermore, with the use of IL-1 receptor type 1-deficient mice, we identify the contribution of the IL-1 signaling pathway to the acidosis-enhanced inflammatory response and pathology. These data provide insights into the potential benefit of pH regulation during bacterial infections to control disease progression and immunopathology.

Complex interactions between genetic and environmental factors are widely believed to underlie the incidence and progression of Parkinson's disease (PD). Rotenone is a naturally occurring metabolic toxin employed as an insecticide and piscicide identified as a risk factor for the development of PD in agricultural workers. The Nlrp3 inflammasome is an intracellular mediator that can initiate an inflammatory cascade in response to cellular stress. Reports by others indicating that NLRP3 expression was detectable in tissues obtained from Alzheimer's disease patients and that the PD-associated protein α-synuclein could activate inflammasomes in cultured glial cells, prompted us to test the prediction that Nlrp3 was required for the development of Parkinson's-like changes resulting from rotenone exposure in mice. We exposed wild type and Nlrp3-/- mice to chronic low doses of intragastric rotenone and conducted longitudinal behavioral and serum cytokine analysis followed by evaluation of neuroinflammatory and neurodegenerative endpoints in brain tissues. We observed progressive rotenone-dependent changes in serum cytokine levels and circulating leukocytes in wild type mice not observed in Nlrp3-/- mice. Analysis of brain tissues revealed Nlrp3-dependent neuroinflammation and nigral cell loss in mice exposed to rotenone as compared with mice exposed to vehicle alone. Together, our findings provide compelling evidence of a role for Nlrp3 in nigral degeneration and neuroinflammation resulting from systemic rotenone exposure and suggest that the suppression of NLRP3 activity may be a rational neuroprotective strategy for toxin-associated PD.

Pseudomonas aeruginosa is a bacterial pathogen that causes severe chronic infections in immunocompromised individuals. This bacterium is highly adaptable to its environments, which frequently select for traits that promote bacterial persistence. A clinically significant temporal adaptation is the formation of surface- or cell-adhered bacterial biofilms that are associated with increased resistance to immune and antibiotic clearance. Extensive research has shown that bacterial flagellar motility promotes formation of such biofilms, whereupon the bacteria subsequently become nonmotile. However, recent evidence shows that antibiotic-tolerant nonattached bacterial aggregates, distinct from surface-adhered biofilms, can form, and these have been reported in the context of lung infections, otitis media, nonhealing wounds, and soft tissue fillers. It is unclear whether the same bacterial traits are required for aggregate formation as for biofilm formation. In this report, using isogenic mutants, we demonstrate that P. aeruginosa aggregates in liquid cultures are spontaneously formed independent of bacterial flagellar motility and independent of an exogenous scaffold. This contrasts with the role of the flagellum to initiate surface-adhered biofilms. Similarly to surface-attached biofilms, these aggregates exhibit increased antibiotic tolerance compared to planktonic cultures. These findings provide key insights into the requirements for aggregate formation that contrast with those for biofilm formation and that may have relevance for the persistence and dissemination of nonmotile bacteria found within chronic clinical infections. IMPORTANCE In this work, we have investigated the role of bacterial motility with regard to antibiotic-tolerant bacterial aggregate formation. Previous work has convincingly demonstrated that P. aeruginosa flagellar motility promotes the formation of surface-adhered biofilms in many systems. In contrast, aggregate formation by P. aeruginosa was observed for nonmotile but not for motile cells in the presence of an exogenous scaffold. Here, we demonstrate that both wild-type P. aeruginosa and mutants that genetically lack motility spontaneously form antibiotic-tolerant aggregates in the absence of an exogenously added scaffold. Additionally, we also demonstrate that wild-type (WT) and nonmotile P. aeruginosa bacteria can coaggregate, shedding light on potential physiological interactions and heterogeneity of aggregates.

Pseudomonas aeruginosa is a Gram-negative bacterium that, as an opportunistic pathogen, substantially contributes to high morbidity and mortality rates in susceptible individuals such as those with cystic fibrosis or neutropenia. Previous studies identified that the downregulation or loss of bacterial flagellar motility, typically observed within chronic infections, enables bacteria to evade interactions with phagocytic cells that would result in phagocytic uptake. Our recent work demonstrated that exogenous addition of a negatively charged lipid, PIP3, induces binding and phagocytosis of non-motile strains of P. aeruginosa. Based on this work, we hypothesized that the engagement of P. aeruginosa by host innate cells, and subsequent phagocytosis, is mediated by motility-dependent interactions with cell-surface polyanions. We now report that endogenous polyanionic N-linked glycans and heparan sulfate mediate bacterial binding of P. aeruginosa by human monocytic cells. These specific cell-surface interactions result in P. aeruginosa phagocytosis, bacterial type 3 secretion system (T3SS)-mediated cellular intoxication and the IL-1β inflammatory response of the host innate immune cells. Concomitantly, inhibition of host cell N-glycan synthesis reduces T3SS-mediated cytotoxicity and the IL-1β response induced by the bacteria. Importantly, the bacterial interactions with the glycans were motility-dependent and could be recapitulated with purified, immobilized glycans. Therefore, this work describes novel interactions of P. aeruginosa with specific phagocyte cell-surface glycans that modulate relevant host innate immune responses to the bacteria, including phagocytosis, inflammation and cytotoxicity.

Epithelial ovarian cancer (EOC) is a deadly gynecologic malignancy, but animal models for the study of EOC pathophysiology and drug efficacy are limited. Based on the finding that women with EOC are at risk for metastasis at a trocar site after laparoscopy, we developed a syngeneic murine model of port-site metastasis of EOC. We leveraged the ID8 murine EOC cell line to induce intra-peritoneal tumors in mice. Once durable intraperitoneal tumor was confirmed with bioluminescence imaging, intra-abdominal wall tumors were induced by abdominal wall puncture with a hollow bore needle. This resulted in a robust system in which C57BL/6 mice developed metastatic deposits at a rate of 66.7% ± 10.77; no intra-abdominal wall metastases were seen in control samples (P = .0003, CI 41.16–90.84). Immunodeficient NOD SCID gamma mice developed puncture site metastases in 70% ± 10.0 of mice and also had no metastases documented in control sites (P = .002, CI 42.24–97.76). In addition we were able to demonstrate the presence of immune infiltrates within the metastatic deposits of C57BL/6 mice via IHC. Therefore, in this study we demonstrate the predictable development of invasive abdominal wall metastases in a syngeneic mouse model of EOC. This model enables studies of the metastatic process and provides a novel system in which to test the effect of therapies on a clinically-relevant model in an immune competent mouse.

Pathogenic bacteria that establish chronic infections in immunocompromised patients frequently undergo adaptation or selection for traits that are advantageous for their growth and survival. Clinical isolates of Pseudomonas aeruginosa, a Gram-negative, opportunistic bacterial pathogen, exhibit a temporal transition from a motile to a nonmotile phenotype through loss of flagellar motility during the course of chronic infection. This progressive loss of motility is associated with increased resistance to both antibiotic and immune clearance. We have previously shown that loss of bacterial motility enables P. aeruginosa to evade phagocytic clearance both in vitro and in vivo and fails to activate the phosphatidylinositol 3-kinase (PI3K)/Akt-dependent phagocytic pathway. Therefore, we tested the hypothesis that clearance of phagocytosis-resistant bacteria could be induced by exogenously pretreating innate immune cells with the Akt-activating molecule phosphatidylinositol-(3,4,5)-trisphosphate (PIP3). Here, we demonstrate that PIP3 induces the uptake of nonmotile P. aeruginosa by primary human neutrophils >25-fold, and this effect is phenocopied with the use of murine phagocytes. However, surprisingly, mechanistic studies revealed that the induction of phagocytosis by PIP3 occurs because polyphosphoinositides promote bacterial binding by the phagocytes rather than bypassing the requirement for PI3K. Moreover, this induction was selective since the uptake of other nonmotile Gram-negative, but not Gram-positive, bacteria can also be induced by PIP3 Since there is currently no treatment that effectively eradicates chronic P. aeruginosa infections, these findings provide novel insights into a potential methodology by which to induce clearance of nonmotile pathogenic bacteria and into the endogenous determinants of phagocytic recognition of P. aeruginosa.

Detection of bacterial ligands is a pre-requisite for inflammasome activation. During Pseudomonas aeruginosa infection, flagellin which is secreted through the T3SS is detected by the NLRC4 inflammasome. Activation of the NLRC4 inflammasome is believed to contribute to high IL-1β production and pathogenicity in cystic fibrosis patients with chronic P. aeruginosa infection. Interestingly, the majority of P. aeruginosa isolated from cystic fibrosis patients with chronic airway infection are non-motile and T3SS-negative, suggesting that yet un-characterized inflammasome pathways regulate IL-1β production in cystic fibrosis patients. Here we demonstrate the role of guanylate-binding proteins (GBPs) in regulating bacterial proliferation and inflammasome activation in response to T3SS-negative P. aeruginosa. Bacterial ligands liberated by the action of GBP2 and IRGB10 activate caspase-11 and regulate non-canonical NLRP3 inflammasome activation and IL-1β release. Overall, our results reveal the role of caspase-11 in inhibiting bacterial proliferation and promoting IL-1β secretion during T3SS-negative P. aeruginosa infection. This study suggests that non canonical inflammasomes might have co-evolved to detect Gram-negative bacterial pathogens that have evolved to bypass detection by canonical NLRs.

Infection by Pseudomonas aeruginosa , and bacteria in general, frequently promotes acidification of the local microenvironment, and this is reinforced by pulmonary exertion and exacerbation. However, the consequence of an acidic environment on the host inflammatory response to P. aeruginosa infection is poorly understood. Here we report that the pivotal cellular and host proinflammatory interleukin-1β (IL-1β) response, which enables host clearance of the infection but can produce collateral inflammatory damage, is increased in response to P. aeruginosa infection within an acidic environment. Synergistic mechanisms that promote increased IL-1β release in response to P. aeruginosa infection in an acidic environment are increased pro-IL-1β induction and increased caspase-1 activity, the latter being dependent upon a functional type III secretion system of the bacteria and the NLRC4 inflammasome of the host. Using an in vivo peritonitis model, we have validated that the IL-1β inflammatory response is increased in mice in response to P. aeruginosa infection within an acidic microenvironment. These data reveal novel insights into the regulation and exacerbation of inflammatory responses to P. aeruginosa .

Purpose To evaluate the thrombus maturity noninvasively providing the promise of much earlier and more accurate diagnosis of diseases ranging from stroke to myocardial infarction to deep vein thrombosis. Methods Magnetic spectroscopy of nanoparticle Brownian rotation (MSB), a form of magnetic particle spectroscopy sensitive to Brownian rotation of magnetic nanoparticles, was used for the detection and characterization of blood clots. The nanoparticles' relaxation time was quantified by scaling the MSB spectra in frequency to match the spectra from nanoparticles in a reference state. The nanoparticles' relaxation time, in the bound state, was used to characterize the nanoparticle binding to thrombin on the blood clot. The number of nanoparticles bound to the clot was also estimated. Both the relaxation time and the weight of bound nanoparticles were obtained for clots of several ages, reflecting different stages of development and organization. The impact of clot development was explored using functionalized nanoparticles present during clot formation. Results The relaxation time of the bound nanoparticles decreases for more mature, organized clots. The number of nanoparticles able to bind the clot diminishes quantitatively with clot age. On mature clots, the nanoparticles bind the thrombin on the surface while for developing clots the nanoparticles bind several thrombin molecules or become trapped in the clot matrix during formation. Conclusions By estimating the magnetic nanoparticles' relaxation time the clot age and organization can be predicted. The purposed methods are quick and minimally invasive for in vivo applications.

Bacterial infection can lead to acidosis of the local microenvironment, which is believed to exacerbate disease pathogenesis; however, the mechanisms by which changes in pH alter disease progression are poorly understood. We test the hypothesis that acidosis enhances respiratory epithelial cell death in response to infection with Pseudomonas aeruginosa. Our findings support the idea that acidosis in the context of P. aeruginosa infection results in increased epithelial cell cytotoxicity due to ExoU intoxication. Importantly, enforced maintenance of neutral pH during P. aeruginosa infection demonstrates that cytotoxicity is dependent on the acidosis. Investigation of the underlying mechanisms revealed that host cell cytotoxicity correlated with increased bacterial survival during an acidic infection that was due to reduced bactericidal activity of host-derived antimicrobial peptides. These findings extend previous reports that the activities of antimicrobial peptides are pH-dependent and provide novel insights into the consequences of acidosis on infection-derived pathology. Therefore, this report provides the first evidence that physiological levels of acidosis increase the susceptibility of epithelial cells to acute Pseudomonas infection and demonstrates the benefit of maintaining pH homeostasis during a bacterial infection.

Arsenic is the number one contaminant of concern with regard to human health according to the World Health Organization. Epidemiological studies on Asian and South American populations have linked arsenic exposure with an increased incidence of lung disease, including pneumonia, and chronic obstructive pulmonary disease, both of which are associated with bacterial infection. However, little is known about the effects of low dose arsenic exposure, or the contributions of organic arsenic to the innate immune response to bacterial infection. This study examined the effects on Pseudomonas aeruginosa (P. aeruginosa) induced cytokine secretion by human bronchial epithelial cells (HBEC) by inorganic sodium arsenite (iAsIII) and two major metabolites, monomethylarsonous acid (MMAIII) and dimethylarsenic acid (DMAV), at concentrations relevant to the U.S. population. Neither iAsIII nor DMAV altered P. aeruginosa induced cytokine secretion. By contrast, MMAIII increased P. aeruginosa induced secretion of IL-8, IL-6 and CXCL2. A combination of iAsIII, MMAIII and DMAV (10 pbb total) reduced IL-8 and CXCL1 secretion. These data demonstrate for the first time that exposure to MMAIII alone, and a combination of iAsIII, MMAIII and DMAV at levels relevant to the U.S. may have negative effects on the innate immune response of human bronchial epithelial cells to P. aeruginosa.

Pseudomonas aeruginosa is a pathogenic Gram-negative bacterium that causes severe opportunistic infections in immunocompromised individuals; in particular, severity of infection with P. aeruginosa positively correlates with poor prognosis in cystic fibrosis (CF) patients. Establishment of chronic infection by this pathogen is associated with downregulation of flagellar expression and of other genes that regulate P. aeruginosa motility. The current paradigm is that loss of flagellar expression enables immune evasion by the bacteria due to loss of engagement by phagocytic receptors that recognize flagellar components and loss of immune activation through flagellin-mediated Toll-like receptor (TLR) signaling. In this work, we employ bacterial and mammalian genetic approaches to demonstrate that loss of motility, not the loss of the flagellum per se , is the critical factor in the development of resistance to phagocytosis by P. aeruginosa . We demonstrate that isogenic P. aeruginosa mutants deficient in flagellar function, but retaining an intact flagellum, are highly resistant to phagocytosis by both murine and human phagocytic cells at levels comparable to those of flagellum-deficient mutants. Furthermore, we show that loss of MyD88 signaling in murine phagocytes does not recapitulate the phagocytic deficit observed for either flagellum-deficient or motility-deficient P. aeruginosa mutants. Our data demonstrate that loss of bacterial motility confers a dramatic resistance to phagocytosis that is independent of both flagellar expression and TLR signaling. These findings provide an explanation for the well-documented observation of nonmotility in clinical P. aeruginosa isolates and for how this phenotype confers upon the bacteria an advantage in the context of immune evasion.

The ovarian cancer microenvironment recruits an array of immune cells to the site of tumor growth. Within the peritoneal ascites of both humans and mice, the predominant population of tumor-infiltrating leukocytes is a CD11c+CD11b+ population variably referred to as vascular leukocytes (VLCs), tumor-associated macrophages, and immature dendritic cells. We have previously shown that these cells are critical for tumor growth because their selective elimination from the peritoneal tumor microenvironment inhibited tumor progression. However, the underlying mechanism by which this therapy was efficacious is poorly understood. Here, we use the murine ID8 ovarian tumor model to demonstrate that the tumor microenvironment induces in vivo immunosuppression of T cells and that the SR-A+ VLCs mediate this suppression. Importantly, the elimination of SR-A+ VLCs from the peritoneum of tumor-bearing mice relieves the T cell suppression. Moreover, the profound changes that VLC elimination has on the immune system are T cell-dependent because the protective antitumor effect of VLC elimination does not occur when CD8 T cells are concomitantly depleted. These results were confirmed and extended with the use of a genetic model for VLC depletion, which demonstrated that short-term therapeutic depletion of VLCs alleviates immunosuppression and allows for efficacious vaccination against model tumor antigens in tumor-bearing mice. These studies provide a mechanistic explanation for how leukocytes contribute to ovarian tumor progression and, correspondingly, how leukocyte depletion inhibits tumor growth.

During infection, signals from the periphery are known to reach draining lymph nodes (DLNs), but how these molecules, such as inflammatory cytokines, traverse the significant distances involved without dilution or degradation remains unclear. We show that peripheral mast cells, upon activation, release stable submicrometer heparin-based particles containing tumor necrosis factor and other proteins. These complexes enter lymphatic vessels and rapidly traffic to the DLNs. This physiological drug delivery system facilitates communication between peripheral sites of inflammation and remote secondary lymphoid tissues.

Unresolved infection by gram-negative bacteria can result in the potentially lethal condition known as endotoxic shock, whereby uncontrolled inflammation can lead to multiple organ failure and death of the infected host. Previous results have demonstrated that animals deficient in class A scavenger receptor (SRA), a trafficking receptor for bacteria and bacterium-derived molecules, are more susceptible to endotoxic shock. This has been proposed to be a result of impaired SRA-dependent phagocytic clearance of bacteria resulting in stronger proinflammatory stimuli. In this report, we test the hypothesis that there is an obligate reciprocal relationship between SRA-mediated phagocytosis of bacteria and susceptibility to endotoxic shock. Here, we demonstrate that both SRA-dependent and -independent gram-negative bacterial strains elicit SRA-dependent increased cytokine production in vitro and in vivo and increased susceptibility to endotoxic shock in SRA-deficient mice. This is the first evidence showing that SRA-mediated clearance of LPS is functionally distinct from the role of SRA in bacterial phagocytosis and is a formal demonstration that the SRA-dependent cytokine responses and the resultant endotoxic shock are not coupled to SRA-mediated clearance of bacteria.

Class-A scavenger receptors (SR-A) and TLR mediate early immune responses against pathogenic bacteria. SR-A and TLR molecules are expressed on phagocytes and interact with common ligands from Gram-negative and Gram-positive bacteria; however, the contribution of TLR activity to SR-A-mediated phagocytosis has not been assessed directly. Herein, we provide genetic and functional evidence that ligand- and TLR-specific stimuli synergize with SR-A to mediate bacterial phagocytosis. Although complete loss of SR-A (SR-A−/−) is known to impair bacterial clearance, here we identify the first deficiency attributable to SR-A heterozygosity: SR-A+/−TLR4+/− cells and mice are impaired significantly in the clearance of Gram-negative Escherichia coli. This phenotype is specific to the TLR signaling event, as SR-A+/−TLR4+/− cells are not deficient for the clearance of Gram-positive Staphylococcus aureus bacteria, which contain cell-surface TLR2 ligands but lack TLR4 ligands. We demonstrate that this is a global, phagocytic mechanism, regulated independently by multiple TLRs, as analogous to the SR-A+/−TLR4+/− deficit, SR-A+/−TLR2+/− cells are impaired for S. aureus uptake. In support of this, we show that SR-A+/−MyD88+/− cells recapitulate the phagocytosis defect observed in SR-A+/−TLR4+/− cells. These data identify for the first time that TLR-driven innate immune responses, via a MyD88 signaling mechanism, regulate SR-A-dependent phagocytosis of bacteria. These findings provide novel insights into how innate immune cells control SR-A-mediated trafficking and are the first demonstration that subtle changes in the expression of SR-A and TLRs can substantially affect host bacterial clearance.

CD8(+) T cells (T(CD8+)) differentiate into effector cells following recognition of specific peptide-major histocompatibility complex (MHC) class I complexes (pMHC-I) on the surface of professional APCs (pAPCs), such as dendritic cells. Antigenic pMHC-I can be generated from two spatially distinct sources. The direct presentation pathway involves generation of peptide from protein substrate synthesized within the cell that is presenting the pMHC-I. Alternatively, the cross presentation pathway involves presentation of antigen that is not synthesized within the presenting cell, but is derived from exogenous proteins synthesized within other donor cells. The mechanisms by which cross presentation of exogenous antigens occur in vivo remain controversial. The C-type lectin scavenger receptor A (SR-A) has been implicated in a number of potential cross presentation pathways, including the presentation of peptide bound to heat shock proteins, such as glycoprotein 96 (gp96), and the transfer of pMHC-I from a donor cell to the pAPC. We demonstrate here that initiation of T(CD8+) responses is normal in mice lacking SR-A, and that the redundancy of ligand binding exhibited by the SR family is likely to be an important mechanism that ensures cross presentation in vivo. These observations emphasize the requirement to target multiple receptors and antigen-processing pathways during the rational design of vaccines aimed at eliciting protective T(CD8+).

The predominant leukocyte population present in both human and murine peritoneal ovarian tumors is the Vascular Leukocyte (VLC). VLCs are recruited en masse to the ovarian tumor microenvironment whereupon they promote tumor progression. Importantly, the presence of VLCs is requisite for peritoneal ovarian cancer progression: selective elimination of VLCs inhibits tumor burden and ascites accumulation. Despite the critical importance of VLCs to ovarian tumors, their derivation and the mechanisms by which they facilitate tumor progression are not well understood. Here we demonstrate in vivo that the murine ID8 ovarian tumor model can usurp the host peritoneal macrophage pathway to elicit and recruit VLCs. Moreover, we demonstrate that VLCs express CD11b and Gr-1, a characteristic phenotype shared amongst heterogeneous populations of leukocytes referred to as myeloid-derived suppressor cells (MDSCs). In accord with their MDSC phenotype, both murine and human VLCs express arginase-1 (ARG1). Importantly, we demonstrate that the VLCs suppress both CD8(+) and CD4(+) T cells responses and that this immunosuppression is ARG1-dependent, since blockade of VLC ARG1 activity with nor-NOHA reversed the immunosuppression. These data further characterize the tumor-associated leukocytes in ovarian cancer and provide insights into the mechanisms by which they promote tumor growth.

Molecular chaperones stimulate the immune system to induce both protective immune responses and therapeutic tumor rejection. However, the underlying basis for this immunogenic activity is not well understood. A variety of chaperones, including calreticulin, hsp70 and grp94, function as vehicles to efficiently traffic associated peptides into professional antigen presenting cells. Importantly, these chaperones have also been proposed to function as adjuvants by stimulating the dendritic cell activation and co-stimulatory responses required to elicit peptide-specific CD8(+) T cell cytolytic activity. The efficacy of chaperone-mediated tumor rejection has been attributed to the ability of chaperones to function in both of these capacities. However, purified calreticulin has not previously been assessed for its ability to elicit DC maturation and, moreover, recent data indicates that it is not efficient at inducing Nf-kappaB activity which often accompanies or stimulates DC maturation. Here we use two complementary methods to produce endotoxin-free calreticulin and demonstrate that it does not measurably mature or activate dendritic cells both in vitro and in vivo. Additionally, a calreticulin/peptide complex required the addition of an exogenous adjuvant to elicit in vivo cytotoxic CD8(+) T cell responses. These data are discussed with respect to current models for chaperone-derived immune responses and in regard to rational vaccine design.

The NLRC4 inflammasome is responsible for IL-1β processing by macrophages in response to Pseudomonas aeruginosa infection. We therefore hypothesized that mice that lack ASC, an NLRC4 inflammasome adaptor protein necessary for in vitro IL-1β production by macrophages, would be preferentially protected from a hyperinflammatory lethal challenge that is dependent on bacterial type three secretion system (T3SS) activity. We report herein that lack of ASC does not confer preferential protection in response to P. aeruginosa acute infection and that ASC−/−mice are capable of producing robust amounts of IL-1β comparable with C57BL/6 mice. We now identify that neutrophils represent the ASC-independent source of IL-1β production during the acute phases of infection both in models of acute pneumonia and peritonitis. Consequently, depletion of neutrophils in ASC−/−mice leads to a marked deficit in IL-1β production in vivo. The pulmonary neutrophil IL-1β response is predominantly dependent on caspase-1, which contrasts with data derived from ocular infection. These studies therefore identify a noncanonical mechanism of IL-1β production by neutrophils independent of ASC and demonstrate the first physiological contribution of neutrophils as an important source of IL-1β in response to acute P. aeruginosa infection during acute pneumonia and peritonitis.

Calreticulin (CRT) interaction with cell-surface receptors is integral to its function in escorting associated peptides into the antigen-presenting cell (APC) antigen presentation pathway. Additionally, extracellular CRT is proposed to be required for lung APC interaction with collectins. In both cases, CD91 has been proposed to act as the APC cell-surface receptor requisite for mediating these processes. However, the evidence for a CRT interaction with CD91 is indirect, predicated on partial competition of cellular binding by gp96, of which CD91 has been proposed as the unique endocytic receptor, and by the CD91 ligand α2-macroglobulin. Here, we directly investigate the function of CD91 in binding and trafficking CRT. We find that the ability of CRT to interact with APC does not correlate with cellular CD91 expression or function. Additionally, in the first genetic test of CD91 function regarding CRT, CD91 expression neither conferred CRT association nor did CD91-deficient (CD91–/–) and CD91-expressing cells differ in their ability to traffic CRT. Finally, cellular CRT trafficking did not parallel that of Pseudomonas exotoxin-A, an obligate CD91 ligand, by the criteria of CD91 dependence, cell-type specificity and endocytic itinerary. These data identify that CRT trafficking is not, as previously hypothesized, CD91 dependent and indicate usage of alternative cellular trafficking pathways.

The molecular chaperone heat shock protein 90 (Hsp90) serves essential roles in the regulation of signaling protein function, trafficking, and turnover. Hsp90 function is intimately linked to intrinsic ATP binding and hydrolysis activities, the latter of which is under the regulatory control of accessory factors. Glucose-regulated protein of 94 kDa (GRP94), the endoplasmic reticulum Hsp90, is highly homologous to cytosolic Hsp90. However, neither accessory factors nor adenosine nucleotides have been clearly implicated in the regulation of GRP94-client protein interactions. In the current study, the structural and regulatory consequences of adenosine nucleotide binding to GRP94 were investigated. We report that apo-GRP94 undergoes a time- and temperature-dependent tertiary conformational change that exposes a site(s) of protein-protein interaction; ATP, ADP, and radicicol markedly suppress this conformational change. In concert with these findings, ATP and ADP act identically to suppress GRP94 homooligomerization, as well as both local and global conformational activity. To identify a role(s) for ATP or ADP in the regulation of GRP94-client protein interactions, immunoglobulin (Ig) heavy chain folding intermediates containing bound GRP94 and immunoglobulin binding protein (BiP) were isolated from myeloma cells, and the effects of adenosine nucleotides on chaperone-Ig heavy chain interactions were examined. Whereas ATP elicited efficient release of BiP from both wild-type and mutant Ig heavy chain intermediates, GRP94 remained in stable association with Ig heavy chains in the presence of ATP or ADP. On the basis of these data, we propose that structural maturation of the client protein substrate, rather than ATP binding or hydrolysis, serves as the primary signal for dissociation of GRP94-client protein complexes.

Vaccination of mice with GRP94/gp96, the endoplasmic reticulum Hsp90, elicits a variety of immune responses sufficient for tumor rejection and the suppression of metastatic tumor progression. Macrophages are a prominent GRP94/gp96 target, with GRP94/gp96 reported to activate macrophage NF-kappa B signaling and nitric oxide production, as well as the MAP kinase p38, JNK, and ERK signaling cascades. However, recent studies report that heat shock protein elicited macrophage activation is due, in large part, to contaminating endotoxin. To examine the generality of this finding, we have investigated the role of endotoxin in GRP94/gp96-elicited macrophage activation. We report that GRP94/gp96 binds endotoxin in a high-affinity, saturable, and specific manner. Low endotoxin calreticulin and GRP94/gp96 were purified, the latter using a novel method of depyrogenation; this resulted in GRP94/gp96 and calreticulin preparations with endotoxin levels substantially lower than those of previously reported preparations. Low endotoxin GRP94/gp96 retained its native conformation, ligand binding activity, and in vitro chaperone function, yet did not activate macrophage NF-kappa B signaling, nitric oxide production or inducible nitric-oxide synthase production. Low endotoxin GRP94/gp96 and calreticulin did, however, elicit a marked increase in ERK phosphorylation at protein concentrations as low as 2 microg/ml. These results are discussed with respect to current understanding of the contributions of endotoxin and heat shock/chaperone proteins to the stimulation of innate immune responses.

GRP94 (gp96)-associated peptides can elicit cellular immune responses, an activity thought to reflect the presence of a cell surface receptor (CD91) on antigen-presenting cells that mediates GRP94 internalization and trafficking to an amenable site for peptide transfer to major histocompatibility complex class I molecules. We report that GRP94 internalized by receptor-mediated endocytosis is trafficked to a Rab5a, CD1 and transferrin-negative, Fc receptor and major histocompatibility complex class I-positive endocytic compartment. Receptor-internalized GRP94 did not access the endoplasmic reticulum of antigen-presenting cells. To identify the site of re-presentation of GRP94-associated peptides, kinetic analyses were performed utilizing GRP94-OVA (SIINFEKL) peptide complexes, with peptide re-presentation assayed with the Kb-SIINFEKL-specific MAb, 25-D1.16. Analyses of the kinetics of re-presentation of GRP94-associated peptides, under conditions in which de novo synthesis of major histocompatibility complex class I molecules was inhibited, identified a post-endoplasmic reticulum compartment, accessed by mature major histocompatibility complex class I, as the predominant site of GRP94-associated peptide exchange onto major histocompatibility complex class I.

Deep vein thrombosis, the development of blood clots in the peripheral veins, is a very serious, life threatening condition that is prevalent in the elderly. To deliver proper treatment that enhances the survival rate, it is very important to detect thrombi early and at the point of care. We explored the ability of magnetic particle spectroscopy (MSB) to detect thrombus via specific binding of aptamer functionalized magnetic nanoparticles with the blood clot. MSB uses the harmonics produced by nanoparticles in an alternating magnetic field to measure the rotational freedom and, therefore, the bound state of the nanoparticles. The nanoparticles' relaxation time for Brownian rotation increases when bound [A.M. Rauwerdink and J. B. Weaver, Appl. Phys. Lett. 96, 1 (2010)]. The relaxation time can therefore be used to characterize the nanoparticle binding to thrombin in the blood clot. For longer relaxation times, the approach to saturation is more gradual reducing the higher harmonics and the harmonic ratio. The harmonic ratios of nanoparticles conjugated with anti-thrombin aptamers (ATP) decrease significantly over time with blood clot present in the sample medium, compared with nanoparticles without ATP. Moreover, the blood clot removed from the sample medium produced a significant MSB signal, indicating the nanoparticles are immobilized on the clot. Our results show that MSB could be a very useful non-invasive, quick tool to detect blood clots at the point of care so proper treatment can be used to reduce the risks inherent in deep vein thrombosis.

Molecular chaperones, both endoplasmic reticulum and cytosol derived, have been identified as tumor rejection antigens; in animal models, they can elicit prophylactic and therapeutic immune responses against their tumor of origin. Chaperone immunogenic activity derives from three principal characteristics: they bind an array of immunogenic (poly)peptides, they can be efficiently internalized by professional antigen-presenting cells, and once internalized, they traffic to a subcellular compartment(s) where peptide release can occur. Within the antigen-presenting cell, chaperone-derived peptides can be assembled onto major histocompatibility class I molecules for presentation at the antigen-presenting cell surface, thereby yielding the requisite and specific CD8+ T-cell responses that contribute to the process of tumor rejection. Though it is clear that chaperones, in particular GRP94 (gp96), calreticulin and Hsp70, can elicit cellular immune responses, the subcellular basis of chaperone processing by antigen-presenting cells remains mysterious. In this review, we discuss recent reports describing the identification of a chaperone internalization receptor and the physiological release of chaperones from necrotic cells, and we present views on the trafficking pathways within antigen-presenting cells that may function to deliver the chaperone-associated peptides to subcellular organelles for their subsequent exchange onto major histocompatibility complex molecules.

Calcium-activated protein for secretion (CAPS) is proposed to play an essential role in Ca2+-regulated dense-core vesicle exocytosis in vertebrate neuroendocrine cells. Here we report the cloning, mutation, and characterization of the Drosophila ortholog (dCAPS). Null dCAPS mutants display locomotory deficits and complete embryonic lethality. The mutant NMJ reveals a 50% loss in evoked glutamatergic transmission, and an accumulation of synaptic vesicles at active zones. Importantly, dCAPS mutants display a highly specific 3-fold accumulation of dense-core vesicles in synaptic terminals, which was not observed in mutants that completely arrest synaptic vesicle exocytosis. Targeted transgenic CAPS expression in identified motoneurons fails to rescue dCAPS neurotransmission defects, demonstrating a cell nonautonomous role in synaptic vesicle fusion. We conclude that dCAPS is required for dense-core vesicle release and that a dCAPS-dependent mechanism modulates synaptic vesicle release at glutamatergic synapses.

Phagocytosis of Borrelia burgdorferi, the causative agent of Lyme disease, is mediated partly by the interaction of the spirochete with Complement Receptor (CR) 3. CR3 requires the GPI-anchored protein, CD14, in order to efficiently internalize CR3-B. burgdorferi complexes. GPI-anchored proteins reside in cholesterol-rich membrane microdomains, and through its interaction with partner proteins, help initiate signaling cascades. Here, we investigated the role of CD14 on the internalization of B. burgdorferi mediated by CR3. We show that CR3 partly colocalizes with CD14 in lipid rafts. The use of the cholesterol-sequestering compound methyl-β-cyclodextran completely prevents the internalization of the spirochete in CHO cells that co-express CD14 and CR3, while no effect was observed in CD11b-deficient macrophages. These results show that lipid rafts are required for CR3-dependent, but not independent, phagocytosis of B. burgdorferi. Our results also suggest that CD14 interacts with the C-lectin domain of CR3, favoring the formation of multi-complexes that allow their internalization, and the use of β-glucan, a known ligand for the C-lectin domain of CR3, can compensate for the lack of CD14 in CHO cells that express CR3. These results provide evidence to understand the mechanisms that govern the interaction between CR3 and CD14 during the phagocytosis of B. burgdorferi.

Pivotal components of the IFN response to virus infection include the IFN receptors (IFNR), and the downstream factor signal transducer and activator of transcription 1 (Stat1). Mice deficient for Stat1 and IFNR (Stat1(-/-) and IFNαßγR(-/-) mice) lack responsiveness to IFN and exhibit high sensitivity to various pathogens. Here we examined herpes simplex virus type 1 (HSV-1) pathogenesis in Stat1(-/-) mice and in IFNαßγR(-/-) mice following corneal infection and bioluminescent imaging. Two divergent and paradoxical patterns of infection were observed. Mice with an N-terminal deletion in Stat1 (129Stat1(-/-) (N-term)) had transient infection of the liver and spleen, but succumbed to encephalitis by day 10 post-infection. In stark contrast, infection of IFNαßγR(-/-) mice was rapidly fatal, with associated viremia and fulminant infection of the liver and spleen, with infected infiltrating cells being primarily of the monocyte/macrophage lineage. To resolve the surprising difference between Stat1(-/-) and IFNαßγR(-/-) mice, we infected an additional Stat1(-/-) strain deleted in the DNA-binding domain (129Stat1(-/-) (DBD)). These 129Stat1(-/-) (DBD) mice recapitulated the lethal pattern of liver and spleen infection seen following infection of IFNαßγR(-/-) mice. This lethal pattern was also observed when 129Stat1(-/-) (N-term) mice were infected and treated with a Type I IFN-blocking antibody, and immune cells derived from 129Stat1(-/-) (N-term) mice were shown to be responsive to Type I IFN. These data therefore show significant differences in viral pathogenesis between two commonly-used Stat1(-/-) mouse strains. The data are consistent with the hypothesis that Stat1(-/-) (N-term) mice have residual Type I IFN receptor-dependent IFN responses. Complete loss of IFN signaling pathways allows viremia and rapid viral spread with a fatal infection of the liver. This study underscores the importance of careful comparisons between knockout mouse strains in viral pathogenesis, and may also be relevant to the causation of HSV hepatitis in humans, a rare but frequently fatal infection.

The Moloney murine leukemia virus (M-MuLV) repressor binding site (RBS) mediates cell-type-specific repression in embryonal carcinoma (EC) cells of expression from several different promoters, including the M-MuLV long terminal repeat promoter. Silencing has been shown to depend on an element normally located in the proviral 5' noncoding region and occurs at the DNA level in the absence of retroviral proteins. Using fragments of the RBS region, we now show that the minimal size of the silencer corresponds to M-MuLV nt 147-163 and overlaps with the retroviral primer binding site region by 17 of its 18 bp. A panel of point mutations within the RBS has been examined to yield a consensus RBS sequence which is consistent with the notion that a previously identified nuclear factor (binding factor A) mediates RBS repression. Viral vectors using neomycin, beta-galactosidase, and luciferase reporters have been employed to show that RBS-mediated repression occurs in EC and embryonal stem, but not in other tested cell types. Repression was observed to occur within 48 hr of infection, prior to when global methylation of proviruses has been reported to occur. Repression also occurred after azacytidine treatment of EC cells, supporting the notion that the RBS functions independently of provirus methylation. However, levels of provirus methylation in selected cells were increased in the presence of a wild-type RBS, and methylation correlated with a secondary stage of virus repression. Thus, the M-MuLV RBS acts directly to control expression in EC cells and also appears to trigger a secondary level of repression which is coincident with provirus methylation.

Immunosuppressive leukocytes are emerging as a critical factor in facilitating tumor progression. These leukocytes are converted by the tumor microenvironment to become tolerogenic, facilitate metastasis, and to aid in neovascularization. The predominant variety of suppressive leukocytes found in human and murine ovarian cancer are called vascular leukocytes (VLC), due to sharing functions and cell surface markers of both dendritic cells and endothelial cells. Using the ID8 murine model of ovarian cancer, the aim of this study was to test the efficacy of VLC elimination as an ovarian tumor therapy. We show that carrageenan-mediated depletion of peritoneal tumor-associated leukocytes inhibits ovarian tumor progression. We then identified scavenger receptor-A (SR-A) as a cell surface receptor that is robustly and specifically expressed within human and murine ovarian tumor ascites upon VLCs. Administration of anti–SR-A immunotoxin to mice challenged with peritoneal ID8 tumors eliminated tumor-associated VLCs and, importantly, substantially inhibited peritoneal tumor burden and ascites accumulation. Moreover, the toxin required targeting to SR-A because mice that received untargeted toxin did not exhibit inhibition of tumor progression. We conclude that SR-A constitutes a novel and specific target for efficacious immunotherapeutic treatment of peritoneal ovarian cancer. [Cancer Res 2007;67(10):4783–9]

Class-A scavenger receptors (SR-A) are cellular pattern recognition receptors that bind and traffic a variety of endogenous and microbial ligands. However, despite an emerging role for SR-A as a contributor to the innate immune system, little is known of the regulation or function of SR-A on dendritic cells (DCs). Here we show that SR-A expression is upregulated during murine DC differentiation and that SR-A expression levels correlate with the expression of the murine DC marker CD11c. Using bone marrow-derived DCs (BMDCs) from SR-A knockout (SR-A(-/-)) mice, we investigated the contribution of SR-A to BMDC particulate phagocytosis. Functional analyses demonstrated that SR-A is a critical phagocytic receptor for BMDC internalization of the gram-negative bacteria E. coli. SR-A(-/-) BMDCs were impaired in their ability to phagocytose bacteria, and this deficit varied with the bacteria:BMDC cell ratio. Microscopic and biochemical analyses revealed that SR-A is broadly distributed on the surface of BMDCs and is not physically associated with lipid rafts. However, cholesterol depletion demonstrated dependence of SR-A-mediated phagocytosis upon lipid rafts. These data demonstrate a functional contribution for SR-A in the BMDC phagocytic pathway.

Calreticulin and gp96 (GRP94) traffic associated peptides into the major histocompatibility complex class-I cross-presentation pathway of antigen-presenting cells (APCs). Efficient accession of the cross-presentation pathway requires APC receptor-mediated endocytosis of the chaperone/peptide complexes. Previously, scavenger receptor class-A (SRA) was shown to play a substantial role in trafficking gp96 and calreticulin into macrophages, accounting for half of total receptor-mediated uptake. However, the scavenger receptor ligand fucoidin competed the chaperone uptake beyond that accounted for by SRA, indicating that another scavenger receptor(s) may also contribute. Consistent with this hypothesis, we showed that the residual calreticulin uptake into SRA(-/-) macrophages is competed by the scavenger receptor ligand acetylated low density lipoprotein (LDL). We now report that an additional scavenger receptor, SREC-I (scavenger receptor expressed by endothelial cell-I), mediates the endocytosis of calreticulin and gp96. Ectopic expression of SREC-I in Chinese hamster ovary cells yielded chaperone recognition and uptake, and these processes were competed by the inhibitory ligands fucoidin and acetylated (Ac)LDL. Although AcLDL competes for the chaperone interactions with SRA and SREC, we showed that not all of the scavenger receptors, which bind AcLDL, bind calreticulin or gp96. The overexpression of SREC-I in macrophages increased chaperone endocytosis, indicating that SREC-I functions in APCs and that the cytosolic components necessary for the endocytosis of SREC-I and its cargo are present and not limiting in APCs. These data identify a novel class of ligands for SREC-I and provide insight into the mechanisms by which APCs and potentially endothelial cells traffic chaperone/antigen complexes.

Vaccination of mice with GRP94/gp96, the endoplasmic reticulum Hsp90, elicits a variety of immune responses sufficient for tumor rejection and the suppression of metastatic tumor progression. Macrophages are a prominent GRP94/gp96 target, with GRP94/gp96 reported to activate macrophage NF-kappa B signaling and nitric oxide production, as well as the MAP kinase p38, JNK, and ERK signaling cascades. However, recent studies report that heat shock protein elicited macrophage activation is due, in large part, to contaminating endotoxin. To examine the generality of this finding, we have investigated the role of endotoxin in GRP94/gp96-elicited macrophage activation. We report that GRP94/gp96 binds endotoxin in a high-affinity, saturable, and specific manner. Low endotoxin calreticulin and GRP94/gp96 were purified, the latter using a novel method of depyrogenation; this resulted in GRP94/gp96 and calreticulin preparations with endotoxin levels substantially lower than those of previously reported preparations. Low endotoxin GRP94/gp96 retained its native conformation, ligand binding activity, and in vitro chaperone function, yet did not activate macrophage NF-kappa B signaling, nitric oxide production or inducible nitric-oxide synthase production. Low endotoxin GRP94/gp96 and calreticulin did, however, elicit a marked increase in ERK phosphorylation at protein concentrations as low as 2 microg/ml. These results are discussed with respect to current understanding of the contributions of endotoxin and heat shock/chaperone proteins to the stimulation of innate immune responses.

GRP94(gp96) elicits CD8+ T cell responses against its bound peptides, a process requiring access of its associated peptides into the MHC class I cross-presentation pathway of APCs. Entry into this pathway requires receptor-mediated endocytosis, and CD91 (low-density lipoprotein receptor-related protein) has been reported to be the receptor mediating GRP94 uptake into APC. However, a direct role for CD91 in chaperone-based peptide Ag re-presentation has not been demonstrated. We investigated the contribution of CD91 to GRP94 cell surface binding, internalization, and trafficking in APCs. Whereas a clear role for CD91 in α2-macroglobulin binding and uptake was readily obtained, the addition of excess CD91 ligand, activated α2-macroglobulin, or receptor-associated protein, an antagonist of all known CD91 ligands, did not affect GRP94 cell surface binding, receptor-mediated endocytosis, or peptide re-presentation. These data identify a CD91-independent, GRP94 internalization pathway that functions in peptide Ag re-presentation.

Necrotic cell death yields the release of cellular components that can function in the initiation of cellular immune responses. Given the established capacity of the endoplasmic reticulum chaperone GRP94 (gp96) to elicit CD8(+) T cell activation, we have investigated the cellular fate and antigenicity of GRP94 in differing scenarios of cell death. Virally induced cell death or mechanical cell death, elicited by freeze/thaw treatment of cell suspensions, yielded GRP94 release into the extracellular space; apoptotic cell death occurring in response to serum deprivation did not elicit GRP94 release. To assess the antigenicity of GRP94 released following virally induced cell death (lethal infection of cells with rVV ES-OVA(Met258-265), a recombinant, ovalbumin epitope-expressing vaccinia virus) or mechanical cell death (freeze/thaw of ovalbumin-expressing cells), tissue culture supernatant fractions were pulsed onto antigen-presenting cells, and antigen re-presentation was assayed as activation of an ovalbumin-specific T cell hybridoma. For both cell death scenarios, released GRP94 elicited a dose-dependent, ovalbumin-specific, hybridoma activation. In contrast, calreticulin derived from rVV ES-OVA(Met258-265)-infected cell extracts did not stimulate B3Z activity. These data identify GRP94 as an antigenic component released upon pathological, but not apoptotic, cell death and provide an assay system for the identification of cellular components of related activity.

Using modified nuclear lysis and binding conditions, we have examined the binding of an embryonal carcinoma (EC) cell factor, binding factor A, to a stem cell-specific silencer which acts at the DNA level and overlaps the Moloney murine leukemia virus (M-MuLV) proline primer binding site (PBS). Following our protocol, we found that in vitro binding of factor A correlated with the in vivo activity of the M-MuLV silencer. Factor A bound specifically to the wild-type silencer element at room temperature and 30 degrees C, but not at 4 degrees C, and bound 10-fold better to the full-length silencer than to a minimal silencer core element. The factor was enriched in nuclear compared with cytosolic extracts and in undifferentiated EC cells compared with differentiated cells in which the silencer is nonfunctional. Salt and ion requirements for factor A binding were investigated, and partial purification steps indicated the factor to be a heparin-Sepharose-binding moiety of greater than 100 kDa. To examine possible relationships between silencer and PBS activities, sequences representing phenylalanine, isoleucine, lysine-1,2, lysine-3, methionine, and tryptophan PBS DNA fragments were tested in vivo for stem cell-specific repression of M-MuLV expression and in vitro in DNA binding assays. Of these PBS elements, only the lysine-1,2 PBS DNA fragment showed consistently high levels of repression. Interestingly, the lysine-1,2 PBS DNA fragment also formed a complex with an EC cell factor with characteristics similar to those of factor A. However, the two factors did not cross-compete in binding studies, suggesting that they may be different but related factors. Our results suggest that expression of Mason-Pfizer monkey virus, visna virus, and spumavirus, which use the lysine-1,2 PBS, may be inhibited in undifferentiated stem cells.