Checkpoint inhibition through small molecule-induced internalization of programmed death-ligand 1.

Programmed death-ligand 1 is a glycoprotein expressed on antigen presenting cells, hepatocytes, and tumors which upon interaction with programmed death-1, results in inhibition of antigen-specific T cell responses. Here, we report a mechanism of inhibiting programmed death-ligand 1 through small molecule-induced dimerization and internalization. This represents a mechanism of checkpoint inhibition, which differentiates from anti-programmed death-ligand 1 antibodies which function through molecular disruption of the programmed death 1 interaction. Testing of programmed death ligand 1 small molecule inhibition in a humanized mouse model of colorectal cancer results in a significant reduction in tumor size and promotes T cell proliferation. In addition, antigen-specific T and B cell responses from patients with chronic hepatitis B infection are significantly elevated upon programmed death ligand 1 small molecule inhibitor treatment. Taken together, these data identify a mechanism of small molecule-induced programmed death ligand 1 internalization with potential therapeutic implications in oncology and chronic viral infections.

Publisher Correction: Circulating serum HBsAg level is a biomarker for HBV-specific T and B cell responses in chronic hepatitis B patients.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Circulating serum HBsAg level is a biomarker for HBV-specific T and B cell responses in chronic hepatitis B patients.

Chronic hepatitis B (CHB) infection functional cure is defined as sustained loss of HBsAg and several therapeutic strategies are in clinical development designed to pharmacologically reduce serum HBsAg, break immune tolerance, and increase functional cure rates. However, little is known about pre-treatment HBsAg levels as an indicator of HBV immune potential. Here, we compared the phenotypes and HBV-specific response of lymphocytes in CHB patients stratified by serum HBsAg levels <500 (HBslo) or >50,000 IU/ml (HBshi) using immunological assays (flow cytometry, ICS, ELISPOT). HBshi patients had significantly higher expression of inhibitory PD-1 on CD4+ T cells, particularly among TEMRA subset, and higher FcRL5 expression on B cells. Upon HBcAg(core) or HBsAg(env)-stimulation, 85% and 60% of HBslo patients had IFNγ+TNFα+ and IFNγ+ IL2+ CD4+ T cell responses respectively, in comparison to 33% and 13% of HBshi patients. Checkpoint blockade with αPD-1 improved HBV-specific CD4+ T cell function only in HBslo patients. HBsAg-specific antibody-secreting cells (ASCs) response was not different between these groups, yet αPD-1 treatment resulted in significantly higher fold change in ASCs among patients with HBsAg <100 IU/ml compared to patients with HBsAg >5,000 IU/ml. Thus, serum HBsAg correlates with inhibitory receptor expression, HBV-specific CD4+ T cell responses, and augmentation by checkpoint blockade.

Discovery of selective small molecule type III phosphatidylinositol 4-kinase alpha (PI4KIIIα) inhibitors as anti hepatitis C (HCV) agents.

Hepatitis C virus (HCV) assembles many host cellular proteins into unique membranous replication structures as a prerequisite for viral replication, and PI4KIIIα is an essential component of these replication organelles. RNA interference of PI4KIIIα results in a breakdown of this replication complex and cessation of HCV replication in Huh-7 cells. PI4KIIIα is a lipid kinase that interacts with the HCV nonstructural 5A protein (NS5A) and enriches the HCV replication complex with its product, phosphoinositol 4-phosphate (PI4P). Elevated levels of PI4P at the endoplasmic reticulum have been linked to HCV infection in the liver of HCV infected patients. We investigated if small molecule inhibitors of PI4KIIIα could inhibit HCV replication in vitro. The synthesis and structure-activity relationships associated with the biological inhibition of PI4KIIIα and HCV replication are described. These efforts led directly to identification of quinazolinone 28 that displays high selectivity for PI4KIIIα and potently inhibits HCV replication in vitro.

Primary ear fibroblast derivation from mice.

Mouse embryonic fibroblasts (MEFs) are commonly utilized as a primary cell culture model and have several advantages over other types of ex vivo-derived cells. However, the successful generation of MEFs is time consuming and requires a certain level of mouse expertise to successfully complete. Thus, primary ear-derived fibroblasts offer an acceptable alternative to MEFs. Fibroblasts derived from the pinna of adult mice are easily attainable with minimal skill, proliferate rapidly, and are easy to manipulate. Likewise, because they are derived from adult mice, other organs can be concurrently harvested for the isolation of additional types of primary cells. Similar to MEFs, ear fibroblasts are an excellent ex vivo model system to study mechanisms associated with virus infection and produce a diverse array of inflammatory mediators, such as cytokines and interferon. Here, we describe a highly versatile and simple method for the derivation, maintenance, and viral challenge of primary ear-derived fibroblasts from mice.

The mitochondrial proteins NLRX1 and TUFM form a complex that regulates type I interferon and autophagy.

The mitochondrial protein MAVS (also known as IPS-1, VISA, and CARDIF) interacts with RIG-I-like receptors (RLRs) to induce type I interferon (IFN-I). NLRX1 is a mitochondrial nucleotide-binding, leucine-rich repeats (NLR)-containing protein that attenuates MAVS-RLR signaling. Using Nlrx1(-/-) cells, we confirmed that NLRX1 attenuated IFN-I production, but additionally promoted autophagy during viral infection. This dual function of NLRX1 paralleled the previously described functions of the autophagy-related proteins Atg5-Atg12, but NLRX1 did not associate with Atg5-Atg12. High-throughput quantitative mass spectrometry and endogenous protein-protein interaction revealed an NLRX1-interacting partner, mitochondrial Tu translation elongation factor (TUFM). TUFM interacted with Atg5-Atg12 and Atg16L1 and has similar functions as NLRX1 by inhibiting RLR-induced IFN-I but promoting autophagy. In the absence of NLRX1, increased IFN-I and decreased autophagy provide an advantage for host defense against vesicular stomatitis virus. This study establishes a link between an NLR protein and the viral-induced autophagic machinery via an intermediary partner, TUFM.

NLRX1 protein attenuates inflammatory responses to infection by interfering with the RIG-I-MAVS and TRAF6-NF-κB signaling pathways.

The nucleotide-binding domain and leucine-rich-repeat-containing (NLR) proteins regulate innate immunity. Although the positive regulatory impact of NLRs is clear, their inhibitory roles are not well defined. We showed that Nlrx1(-/-) mice exhibited increased expression of antiviral signaling molecules IFN-ß, STAT2, OAS1, and IL-6 after influenza virus infection. Consistent with increased inflammation, Nlrx1(-/-) mice exhibited marked morbidity and histopathology. Infection of these mice with an influenza strain that carries a mutated NS-1 protein, which normally prevents IFN induction by interaction with RNA and the intracellular RNA sensor RIG-I, further exacerbated IL-6 and type I IFN signaling. NLRX1 also weakened cytokine responses to the 2009 H1N1 pandemic influenza virus in human cells. Mechanistically, Nlrx1 deletion led to constitutive interaction of MAVS and RIG-I. Additionally, an inhibitory function is identified for NLRX1 during LPS activation of macrophages where the MAVS-RIG-I pathway was not involved. NLRX1 interacts with TRAF6 and inhibits NF-κB activation. Thus, NLRX1 functions as a checkpoint of overzealous inflammation.

The NLR adaptor ASC/PYCARD regulates DUSP10, mitogen-activated protein kinase (MAPK), and chemokine induction independent of the inflammasome.

ASC/PYCARD is a common adaptor for a diverse set of inflammasomes that activate caspase-1, most prominently the NLR-based inflammasome. Mounting evidence indicates that ASC and these NLRs also elicit non-overlapping functions, but the molecular basis for this difference is unclear. To address this, we performed microarray and network analysis of ASC shRNA knockdown cells. In pathogen-infected cells, an ASC-dependent interactome is centered on the mitogen-activated protein kinase (MAPK) ERK and on multiple chemokines. ASC did not affect the expression of MAPK but affected its phosphorylation by pathogens and Toll-like receptor agonists via suppression of the dual-specificity phosphatase, DUSP10/MKP5. Chemokine induction, DUSP function, and MAPK phosphorylation were independent of caspase-1 and IL-1ß. MAPK activation by pathogen was abrogated in Asc(-/-) but not Nlrp3(-/-), Nlrc4(-/-), or Casp1(-/-) macrophages. These results demonstrate a function for ASC that is distinct from the inflammasome in modulating MAPK activity and chemokine expression and further identify DUSP10 as a novel ASC target.

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.

Short hairpin RNA (shRNA): design, delivery, and assessment of gene knockdown.

Shortly after the cellular mechanism of RNA interference (RNAi) was first described, scientists began using this powerful technique to study gene function. This included designing better methods for the successful delivery of small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) into mammalian cells. While the simplest method for RNAi is the cytosolic delivery of siRNA oligonucleotides, this technique is limited to cells capable of transfection and is primarily utilized during transient in vitro studies. The introduction of shRNA into mammalian cells through infection with viral vectors allows for stable integration of shRNA and long-term knockdown of the targeted gene; however, several challenges exist with the implementation of this technology. Here we describe some well-tested protocols which should increase the chances of successful design, delivery, and assessment of gene knockdown by shRNA. We provide suggestions for designing shRNA targets and controls, a protocol for sequencing through the secondary structure of the shRNA hairpin structure, and protocols for packaging and delivery of shRNA lentiviral particles. Using real-time PCR and functional assays we demonstrate the successful knockdown of ASC, an inflammatory adaptor molecule. These studies demonstrate the practicality of including two shRNAs with different efficacies of knockdown to provide an additional level of control and to verify dose dependency of functional effects. Along with the methods described here, as new techniques and algorithms are designed in the future, shRNA is likely to include further promising application and continue to be a critical component of gene discovery.

Drosophila MUS312 and the vertebrate ortholog BTBD12 interact with DNA structure-specific endonucleases in DNA repair and recombination.

DNA recombination and repair pathways require structure-specific endonucleases to process DNA structures that include forks, flaps, and Holliday junctions. Previously, we determined that the Drosophila MEI-9-ERCC1 endonuclease interacts with the MUS312 protein to produce meiotic crossovers, and that MUS312 has a MEI-9-independent role in interstrand crosslink (ICL) repair. The importance of MUS312 to pathways crucial for maintaining genomic stability in Drosophila prompted us to search for orthologs in other organisms. Based on sequence, expression pattern, conserved protein-protein interactions, and ICL repair function, we determined that the mammalian ortholog of MUS312 is BTBD12. Orthology between these proteins and S. cerevisiae Slx4 helped identify a conserved interaction with a second structure-specific endonuclease, SLX1. Genetic and biochemical evidence described here and in related papers suggest that MUS312 and BTBD12 direct Holliday junction resolution by at least two distinct endonucleases in different recombination and repair contexts.

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.

The NLRP3 inflammasome mediates in vivo innate immunity to influenza A virus through recognition of viral RNA.

The nucleotide-binding domain and leucine-rich-repeat-containing (NLR) family of pattern-recognition molecules mediate host immunity to various pathogenic stimuli. However, in vivo evidence for the involvement of NLR proteins in viral sensing has not been widely investigated and remains controversial. As a test of the physiologic role of the NLR molecule NLRP3 during RNA viral infection, we explored the in vivo role of NLRP3 inflammasome components during influenza virus infection. Mice lacking Nlrp3, Pycard, or caspase-1, but not Nlrc4, exhibited dramatically increased mortality and a reduced immune response after exposure to the influenza virus. Utilizing analogs of dsRNA (poly(I:C)) and ssRNA (ssRNA40), we demonstrated that an NLRP3-mediated response could be activated by RNA species. Mechanistically, NLRP3 inflammasome activation by the influenza virus was dependent on lysosomal maturation and reactive oxygen species (ROS). Inhibition of ROS induction eliminated IL-1beta production in animals during influenza infection. Together, these data place the NLRP3 inflammasome as an essential component in host defense against influenza infection through the sensing of viral RNA.

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.

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.

Regulation of mitochondrial antiviral signaling pathways.

Mitochondrial antiviral immunity involves the detection of viral RNA by intracellular pattern-recognition receptors (PRRs) belonging to the RIG-I-like helicase family. The convergence of these and other signaling molecules to the outer mitochondrial membrane results in the rapid induction of antiviral cytokines including type-1 interferon. Here, we discuss recent studies describing new molecules implicated in the regulation of this antiviral response.

NLRX1 is a regulator of mitochondrial antiviral immunity.

The RIG-like helicase (RLH) family of intracellular receptors detect viral nucleic acid and signal through the mitochondrial antiviral signalling adaptor MAVS (also known as Cardif, VISA and IPS-1) during a viral infection. MAVS activation leads to the rapid production of antiviral cytokines, including type 1 interferons. Although MAVS is vital to antiviral immunity, its regulation from within the mitochondria remains unknown. Here we describe human NLRX1, a highly conserved nucleotide-binding domain (NBD)- and leucine-rich-repeat (LRR)-containing family member (known as NLR) that localizes to the mitochondrial outer membrane and interacts with MAVS. Expression of NLRX1 results in the potent inhibition of RLH- and MAVS-mediated interferon-beta promoter activity and in the disruption of virus-induced RLH-MAVS interactions. Depletion of NLRX1 with small interference RNA promotes virus-induced type I interferon production and decreases viral replication. This work identifies NLRX1 as a check against mitochondrial antiviral responses and represents an intersection of three ancient cellular processes: NLR signalling, intracellular virus detection and the use of mitochondria as a platform for anti-pathogen signalling. This represents a conceptual advance, in that NLRX1 is a modulator of pathogen-associated molecular pattern receptors rather than a receptor, and identifies a key therapeutic target for enhancing antiviral responses.

ATP binding by monarch-1/NLRP12 is critical for its inhibitory function.

The recently discovered nucleotide binding domain-leucine rich repeat (NLR) gene family is conserved from plants to mammals, and several members are associated with human autoinflammatory or immunodeficiency disorders. This family is defined by a central nucleotide binding domain that contains the highly conserved Walker A and Walker B motifs. Although the nucleotide binding domain is a defining feature of this family, it has not been extensively studied in its purified form. In this report, we show that purified Monarch-1/NLRP12, an NLR protein that negatively regulates NF-kappaB signaling, specifically binds ATP and exhibits ATP hydrolysis activity. Intact Walker A/B motifs are required for this activity. These motifs are also required for Monarch-1 to undergo self-oligomerization, Toll-like receptor- or CD40L-activated association with NF-kappaB-inducing kinase (NIK) and interleukin-1 receptor-associated kinase 1 (IRAK-1), degradation of NIK, and inhibition of IRAK-1 phosphorylation. The stable expression of a Walker A/B mutant in THP-1 monocytes results in increased production of proinflammatory cytokines and chemokines to an extent comparable to that in cells in which Monarch-1 is silenced via short hairpin RNA. The results of this study are consistent with a model wherein ATP binding regulates the anti-inflammatory activity of Monarch-1.

Downregulation of immune signaling genes in patients with large surface burn injury.

We sought to evaluate the temporal pattern of expression of important immune signaling genes in patients with varied TBSA burn injury during the first week after burn. Peripheral blood mononuclear cell fractions were collected from each patient (N = 10) at two time points, one immediately following burn injury, and the other 1 week later. The change in gene expression was correlated with all clinical data including burn size. It was found that gene expression was indirectly proportional with burn size. Smaller burns (<30%) TBSA resulted in an up-regulation of several genes measured, while larger burns (>30%) TBSA resulted in significant down-regulation. In conclusion, a larger burn establishes conditions for severe immunosuppression by down-regulating key immune signaling genes and could be one explanation for the increased susceptibility of major burns to infection. These data shed light on the etiology of burn-induced immune dysfunction in humans and support a more comprehensive genomic profile study in burn patients.

Monarch-1 suppresses non-canonical NF-kappaB activation and p52-dependent chemokine expression in monocytes.

CATERPILLER (NOD, NBD-LRR) proteins are rapidly emerging as important mediators of innate and adaptive immunity. Among these, Monarch-1 operates as a novel attenuating factor of inflammation by suppressing inflammatory responses in activated monocytes. However, the molecular mechanisms by which Monarch-1 performs this important function are not well understood. In this report, we show that Monarch-1 inhibits CD40-mediated activation of NF-kappaB via the non-canonical pathway in human monocytes. This inhibition stems from the ability of Monarch-1 to associate with and induce proteasome-mediated degradation of NF-kappaB inducing kinase. Congruently, silencing Monarch-1 with shRNA enhances the expression of p52-dependent chemokines.