Brian P. O'Connor

Neisseria gonorrhoeae Activates the Proteinase Cathepsin B to Mediate the Signaling Activities of the NLRP3 and ASC-Containing Inflammasome

Neisseria gonorrhoeae is a common sexually transmitted pathogen that significantly impacts female fertility, neonatal health, and transmission of HIV worldwide. N. gonorrhoeae usually causes localized inflammation of the urethra and cervix by inducing production of IL-1β and other inflammatory cytokines. Several NLR (nucleotide-binding domain, leucine-rich repeat) proteins are implicated in the formation of pro-IL-1β-processing complexes called inflammasomes in response to pathogens. We demonstrate that NLRP3 (cryopyrin, NALP3) is the primary NLR required for IL-1β/IL-18 secretion in response to N. gonorrhoeae in monocytes. We also show that N. gonorrhoeae infection promotes NLRP3-dependent monocytic cell death via pyronecrosis, a recently described pathway with morphological features of necrosis, including release of the strong inflammatory mediator HMBG1. Additionally, N. gonorrhoeae activates the cysteine protease cathepsin B as measured by the breakdown of a cathepsin B substrate. Inhibition of cathepsin B shows that this protease is an apical controlling step in the downstream activities of NLRP3 including IL-1β production, pyronecrosis, and HMGB1 release. Nonpathogenic Neisseria strains (Neisseria cinerea and Neisseria flavescens) do not activate NLRP3 as robustly as N. gonorrhoeae. Conditioned medium from N. gonorrhoeae contains factors capable of initiating the NLRP3-mediated signaling events. Isolated N. gonorrhoeae lipooligosaccharide, a known virulence factor from this bacterium that is elaborated from the bacterium in the form of outer membrane blebs, activates both NLRP3-induced IL-1β secretion and pyronecrosis. Our findings indicate that activation of NLRP3-mediated inflammatory response pathways is an important venue associated with host response and pathogenesis of N. gonorrhoeae.

MAVS-mediated apoptosis and its inhibition by viral proteins.

Background Host responses to viral infection include both immune activation and programmed cell death. The mitochondrial antiviral signaling adaptor, MAVS (IPS-1, VISA or Cardif) is critical for host defenses to viral infection by inducing type-1 interferons (IFN-I), however its role in virus-induced apoptotic responses has not been elucidated. Principal Findings We show that MAVS causes apoptosis independent of its function in initiating IFN-I production. MAVS-induced cell death requires mitochondrial localization, is caspase dependent, and displays hallmarks of apoptosis. Furthermore, MAVS−/− fibroblasts are resistant to Sendai virus-induced apoptosis. A functional screen identifies the hepatitis C virus NS3/4A and the Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) nonstructural protein (NSP15) as inhibitors of MAVS-induced apoptosis, possibly as a method of immune evasion. Significance This study describes a novel role for MAVS in controlling viral infections through the induction of apoptosis, and identifies viral proteins which inhibit this host response.

Cutting Edge: NLRP12 Controls Dendritic and Myeloid Cell Migration To Affect Contact Hypersensitivity

Nucleotide-binding domain leucine-rich repeat (NLR) proteins are regulators of inflammation and immunity. Although first described 8 y ago, a physiologic role for NLRP12 has remained elusive until now. We find that murine Nlrp12, an NLR linked to atopic dermatitis and hereditary periodic fever in humans, is prominently expressed in dendritic cells (DCs) and neutrophils. Nlrp12-deficient mice exhibit attenuated inflammatory responses in two models of contact hypersensitivity that exhibit features of allergic dermatitis. This cannot be attributed to defective Ag processing/presentation, inflammasome activation, or measurable changes in other inflammatory cytokines. Rather, Nlrp12−/− DCs display a significantly reduced capacity to migrate to draining lymph nodes. Both DCs and neutrophils fail to respond to chemokines in vitro. These findings indicate that NLRP12 is important in maintaining neutrophils and peripheral DCs in a migration-competent state.

Critical Role of Apoptotic Speck Protein Containing a Caspase Recruitment Domain (ASC) and NLRP3 in Causing Necrosis and ASC Speck Formation Induced by Porphyromonas gingivalis in Human Cells

Periodontal disease is a chronic inflammatory disorder that leads to the destruction of tooth-supporting tissue and affects 10–20 million people in the U.S. alone. The oral pathogen Porphyromonas gingivalis causes inflammatory host response leading to periodontal and other secondary inflammatory diseases. To identify molecular components that control host response to P. gingivalis in humans, roles for the NLR (NBD-LRR) protein, NLRP3 (cryopyrin, NALP3), and its adaptor apoptotic speck protein containing a C-terminal caspase recruitment domain (ASC) were studied. P. gingivalis strain A7436 induces cell death in THP1 monocytic cells and in human primary peripheral blood macrophages. This process is ASC and NLRP3 dependent and can be replicated by P. gingivalis LPS and Escherichia coli. P. gingivalis-induced cell death is caspase and IL-1 independent and exhibits morphological features consistent with necrosis including loss of membrane integrity and release of cellular content. Intriguingly, P. gingivalis-induced cell death is accompanied by the formation of ASC aggregation specks, a process not previously described during microbial infection. ASC specks are observed in P. gingivalis-infected primary human mononuclear cells and are dependent on NLRP3. This work shows that P. gingivalis causes ASC- and NLRP3-dependent necrosis, accompanied by ASC speck formation.

C-C chemokine receptor 5 on pulmonary fibrocytes facilitates migration and promotes metastasis via matrix metalloproteinase 9.

Previously, our group has used a B16-F10 melanoma model to show that C-C chemokine receptor 5 (CCR5) knockout (CCR5(-/-)) mice form fewer pulmonary metastases than wild-type mice. This advantage can be eliminated by injecting CCR5(-/-) mice with wild-type pulmonary mesenchymal cells before tumor injection. In this article, we present the mechanisms underlying this finding. First, we demonstrate that wild-type mesenchymal cells migrate to CCL4 more efficiently in vitro than CCR5(-/-) cells. Wild-type mesenchymal cells were also 3.6 (1.85 to 5.85) times more efficient than CCR5(-/-) cells at migrating into the lung after intravenous injection (P < 0.01). The injection of wild-type but not CCR5(-/-) mesenchymal cells led to a 7.0 +/- 1.6 (P < 0.05)-fold induction of matrix metalloproteinase 9 (MMP9) in the host lung. Neither wild-type nor CCR5(-/-) cells caused significant increases in MMP2, MMP3, or MMP8. Inhibition of the gelatinase activity of MMP9 decreased the number of metastases and restored the advantage that CCR5(-/-) mice have over wild-type mice. Further analysis showed that the CCR5(+) mesenchymal cells expressed CD45(+) and CD13(+) but did not express alpha-smooth muscle actin. This phenotype is characteristic of a subset of mesenchymal cells called fibrocytes. Together, these data suggest a novel role for CCR5 in the migration of pulmonary fibrocytes and the promotion of metastasis.

Semaphorin 6D regulates the late phase of CD4+ T cell primary immune responses

The semaphorin and plexin family of ligand and receptor proteins provides important axon guidance cues required for development. Recent studies have expanded the role of semaphorins and plexins in the regulation of cardiac, circulatory and immune system function. Within the immune system, semaphorins and plexins regulate cell–cell interactions through a complex network of receptor and ligand pairs. Immune cells at different stages of development often express multiple semaphorins and plexins, leading to multivariate interactions, involving more than one ligand and receptor within each functional group. Because of this complexity, the significance of semaphorin and plexin regulation on individual immune cell types has yet to be fully appreciated. In this work, we examined the regulation of T cells by semaphorin 6D. Both in vitro and in vivo T cell stimulation enhanced semaphorin 6D expression. However, semaphorin 6D was only expressed by a majority of T cells during the late phases of activation. Consequently, the targeted disruption of semaphorin 6D receptor–ligand interactions inhibited T cell proliferation at late but not early phases of activation. This proliferation defect was associated with reduced linker of activated T cells protein phosphorylation, which may reflect semaphorin 6D regulation of c-Abl kinase activity. Semaphorin 6D disruption also inhibited expression of CD127, which is required during the multiphase antigen-presenting cell and T cell interactions leading to selection of long-lived lymphocytes. This work reveals a role for semaphorin 6D as a regulator of the late phase of primary immune responses.

Lymphotoxin β Receptor (LtβR): Dual Roles in Demyelination and Remyelination and Successful Therapeutic Intervention Using LtβR–Ig Protein

Inflammation mediated by macrophages is increasingly found to play a central role in diseases and disorders that affect a myriad of organs, prominent among these are diseases of the CNS. The neurotoxicant-induced, cuprizone model of demyelination is ideally suited for the analysis of inflammatory events. Demyelination on exposure to cuprizone is accompanied by predictable microglial activation and astrogliosis, and, after cuprizone withdrawal, this activation reproducibly diminishes during remyelination. This study demonstrates enhanced expression of lymphotoxin β receptor (LtβR) during the demyelination phase of this model, and LtβR is found in areas enriched with microglial and astroglial cells. Deletion of the LtβR gene (LtβR−/−) resulted in a significant delay in demyelination but also a slight delay in remyelination. Inhibition of LtβR signaling by an LtβR–Ig fusion decoy protein successfully delayed demyelination in wild-type mice. Unexpectedly, this LtβR–Ig decoy protein dramatically accelerated the rate of remyelination, even after the maximal pathological disease state had been reached. This strongly indicates the beneficial role of LtβR–Ig in the delay of demyelination and the acceleration of remyelination. The discrepancy between remyelination rates in these systems could be attributed to developmental abnormalities in the immune systems of LtβR−/− mice. These findings bode well for the use of an inhibitory LtβR–Ig as a candidate biological therapy in demyelinating disorders, because it is beneficial during both demyelination and remyelination.

Cutting Edge: Rho Activation and Actin Polarization Are Dependent on Plexin-A1 in Dendritic Cells

We recently identified expression of the semaphorin receptor, plexin-A1, in dendritic cells (DCs); however, its function in these cells remains to be elucidated. To investigate function and maximize physiological relevance, we devised a retroviral approach to ablate plexin-A1 gene expression using small hairpin RNA (shRNA) in primary bone marrow-derived DCs. We show that plexin-A1 localizes within the cytoplasm of immature DCs, becomes membrane-associated, and is enriched at the immune synapse in mature DCs. Reducing plexin-A1 expression with shRNA greatly reduced actin polarization as well as Rho activation without affecting Rac or Cdc42 activation. A Rho inhibitor, C3, also reduced actin polarization. These changes were accompanied by the near-ablation of T cell activation. We propose a mechanism of adaptive immune regulation in which plexin-A1 controls Rho activation and actin cytoskeletal rearrangements in DCs that is associated with enhanced DC-T cell interactions.

Plexin-B2 Negatively Regulates Macrophage Motility, Rac, and Cdc42 Activation

Plexins are cell surface receptors widely studied in the nervous system, where they mediate migration and morphogenesis though the Rho family of small GTPases. More recently, plexins have been implicated in immune processes including cell-cell interaction, immune activation, migration, and cytokine production. Plexin-B2 facilitates ligand induced cell guidance and migration in the nervous system, and induces cytoskeletal changes in overexpression assays through RhoGTPase. The function of Plexin-B2 in the immune system is unknown. This report shows that Plexin-B2 is highly expressed on cells of the innate immune system in the mouse, including macrophages, conventional dendritic cells, and plasmacytoid dendritic cells. However, Plexin-B2 does not appear to regulate the production of proinflammatory cytokines, phagocytosis of a variety of targets, or directional migration towards chemoattractants or extracellular matrix in mouse macrophages. Instead, Plxnb2−/− macrophages have greater cellular motility than wild type in the unstimulated state that is accompanied by more active, GTP-bound Rac and Cdc42. Additionally, Plxnb2−/− macrophages demonstrate faster in vitro wound closure activity. Studies have shown that a closely related family member, Plexin-B1, binds to active Rac and sequesters it from downstream signaling. The interaction of Plexin-B2 with Rac has only been previously confirmed in yeast and bacterial overexpression assays. The data presented here show that Plexin-B2 functions in mouse macrophages as a negative regulator of the GTPases Rac and Cdc42 and as a negative regulator of basal cell motility and wound healing.

TNF superfamily member TWEAK exacerbates inflammation and demyelination in the cuprizone-induced model.

Inflammatory cytokines have been implicated in the pathology of multiple neurologic diseases, including multiple sclerosis. We examined the role of the TNF family member TWEAK in neuroinflammation. Cuprizone-fed mice undergo neuroinflammation and demyelination in the brain, but upon removal of cuprizone from the diet, inflammation is resolved and remyelination occurs. Using this model, we demonstrate that mice lacking TWEAK exhibit a significant delay in demyelination and microglial infiltration. During remyelination, mice lacking the TWEAK gene demonstrate only a marginal delay in remyelination. Thus, this study identifies a primary role of TWEAK in promoting neuroinflammation and exacerbating demyelination during cuprizone-induced damage.