IL-10 Immunomodulation of Myeloid Cells Regulates a Murine Model of Ovarian Cancer

Elevated levels of IL-10 in the microenvironment of human ovarian cancer and murine models of ovarian cancer are well established and correlate with poor clinical prognosis. However, amongst a myriad of immunosuppressive factors, the actual contribution of IL-10 to the ovarian tumor microenvironment, the mechanisms by which it acts, and its possible functional redundancy are unknown. We previously demonstrated that elimination of the myeloid-derived suppressor cell (MDSC) compartment within the ovarian tumor ascites inhibited tumor progression and, intriguingly, significantly decreased local IL-10 levels. Here we identify a novel pathway in which the tumor-infiltrating MDSC are the predominant producers of IL-10 and, importantly, require it to develop their immunosuppressive function in vivo. Importantly, we demonstrate that the role of IL-10 is critical, and not redundant with other immunosuppressive molecules, to in vivo tumor progression: blockade of the IL-10 signaling network results in alleviation of MDSC-mediated immunosuppression, altered T cell phenotype and activity, and improved survival. These studies define IL-10 as a fundamental modulator of both MDSC and T cells within the ovarian tumor microenvironment. Importantly, IL-10 signaling is shown to be necessary to the development and maintenance of a permissive tumor microenvironment and represents a viable target for anti-tumor strategies

Flagellar Motility Is a Key Determinant of the Magnitude of the Inflammasome Response to Pseudomonas aeruginosa

We previously demonstrated that bacterial flagellar motility is a fundamental mechanism by which host phagocytes bind and ingest bacteria. Correspondingly, loss of bacterial motility, consistently observed in clinical isolates from chronic Pseudomonas aeruginosa infections, enables bacteria to evade association and ingestion of P. aeruginosa by phagocytes both in vitro and in vivo. Since bacterial interactions with the phagocyte cell surface are required for type three secretion system-dependent NLRC4 inflammasome activation by P. aeruginosa, we hypothesized that reduced bacterial association with phagocytes due to loss of bacterial motility, independent of flagellar expression, will lead to reduced inflammasome activation. Here we report that inflammasome activation is reduced in response to nonmotile P. aeruginosa. Nonmotile P. aeruginosa elicits reduced IL-1β production as well as caspase-1 activation by peritoneal macrophages and bone marrow-derived dendritic cells in vitro. Importantly, nonmotile P. aeruginosa also elicits reduced IL-1β levels in vivo in comparison to those elicited by wild-type P. aeruginosa. This is the first demonstration that loss of bacterial motility results in reduced inflammasome activation and antibacterial IL-1β host response. These results provide a critical insight into how the innate immune system responds to bacterial motility and, correspondingly, how pathogens have evolved mechanisms to evade the innate immune system

Mast Cell–Derived Particles Deliver Peripheral Signals to Remote Lymph Nodes

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

Acidosis Potentiates the Host Proinflammatory Interleukin-1β Response to Pseudomonas Aeruginosa Infection

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

Step-Wise Loss of Bacterial Flagellar Torsion Confers Progressive Phagocytic Evasion

Phagocytosis of bacteria by innate immune cells is a primary method of bacterial clearance during infection. However, the mechanisms by which the host cell recognizes bacteria and consequentially initiates phagocytosis are largely unclear. Previous studies of the bacterium Pseudomonas aeruginosa have indicated that bacterial flagella and flagellar motility play an important role in colonization of the host and, importantly, that loss of flagellar motility enables phagocytic evasion. Here we use molecular, cellular, and genetic methods to provide the first formal evidence that phagocytic cells recognize bacterial motility rather than flagella and initiate phagocytosis in response to this motility. We demonstrate that deletion of genes coding for the flagellar stator complex, which results in non-swimming bacteria that retain an initial flagellar structure, confers resistance to phagocytic binding and ingestion in several species of the gamma proteobacterial group of Gram-negative bacteria, indicative of a shared strategy for phagocytic evasion. Furthermore, we show for the first time that susceptibility to phagocytosis in swimming bacteria is proportional to mot gene function and, consequently, flagellar rotation since complementary genetically- and biochemically-modulated incremental decreases in flagellar motility result in corresponding and proportional phagocytic evasion. These findings identify that phagocytic cells respond to flagellar movement, which represents a novel mechanism for non-opsonized phagocytic recognition of pathogenic bacteria

Monomethylarsonous Acid (MMAIII) Has an Adverse Effect on the Innate Immune Response of Human Bronchial Epithelial Cells to Pseudomonas Aeruginosa

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

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

Assessment of the Glycan-Binding Profile of Pseudomonas aeruginosa PAO1

ABSTRACT Pseudomonas aeruginosa is an opportunistic pathogen that can establish acute and chronic infections in individuals who lack fully functional innate immunity. In particular, phagocytosis by neutrophils and macrophages is a key mechanism that modulates host control and clearance of P. aeruginosa. Individuals with neutropenia or cystic fibrosis are highly susceptible to P. aeruginosa infection, 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 the binding and subsequent phagocytosis of P. aeruginosa cells. However, the suite of glycans that P. aeruginosa cells bind to on host phagocytic cells remains poorly characterized. Here, we demonstrate, with the use of exogenous N-linked glycans and a glycan array, that P. aeruginosa PAO1 cells preferentially attach to a subset of glycans, including a bias toward 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 disaccharide glycans. We discuss our findings in the context of previous reports of P. aeruginosa glycan binding. IMPORTANCE P. aeruginosa cells bind to a variety of glycans as part of their interaction with host cells, and a number of P. 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 by P. aeruginosa PAO1 cells to bind to phagocytic cells and by using a glycan array to characterize the suite of such molecules that can facilitate host cell binding by this microbe. This study provides an increased understanding of the glycans bound by P. aeruginosa and furthermore provides a useful data set for future studies of P. aeruginosa-glycan interactions

Ovarian Tumor-Induced T Cell Suppression Is Alleviated by Vascular Leukocyte Depletion

AbstractThe 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

Cisplatin increases immune activity of monocytes and cytotoxic T-cells in a murine model of epithelial ovarian cancer.

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 CD11