Brent Berwin

Cutting Edge: CD91-Independent Cross-Presentation of GRP94(gp96)-Associated Peptides

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.

Pseudomonas aeruginosa Evasion of Phagocytosis Is Mediated by Loss of Swimming Motility and Is Independent of Flagellum Expression

ABSTRACT 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.

Pivotal Advance: Toll-like receptor regulation of scavenger receptor-A-mediated phagocytosis.

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.

Scavenger Receptor-A–Targeted Leukocyte Depletion Inhibits Peritoneal Ovarian Tumor Progression

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.

Murine ovarian cancer vascular leukocytes require arginase-1 activity for T cell suppression.

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.

Bioluminescent imaging reveals divergent viral pathogenesis in two strains of Stat1-deficient mice, and in αßγ interferon receptor-deficient mice.

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.

Differential CD91 dependence for calreticulin and Pseudomonas exotoxin-A endocytosis.

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.

Uncoupling Scavenger Receptor A-Mediated Phagocytosis of Bacteria from Endotoxic Shock Resistance

ABSTRACT 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.

Redundancy renders the glycoprotein 96 receptor scavenger receptor A dispensable for cross priming in vivo.

CD8+ T cells (TCD8+) 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 TCD8+ 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 TCD8+.

Differential ASC requirements reveal a key role for neutrophils and a noncanonical IL-1β response to Pseudomonas aeruginosa

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.