Fas ligand expression and tumour immune evasion; the role of prostaglandin E2 and the EP1 receptor

Background and Aim: During carcinogenesis, tumours develop multiple mechanisms to evade the immune system and suppress the anti-tumour immune response. Upregulation of Fas Ligand (FasL/CD95L) expression may represent one such mechanism. FasL is a member of the tumour necrosis factor superfamily that triggers apoptotic cell death following ligation to its receptor Fas. Numerous studies have demonstrated upregulated FasL expression in tumor cells, with FasL expression associated with numerous pro-tumorigenic effects. However, little is known about the mechanisms that regulate FasL expression in tumours. The cyclooxgenase (COX) signalling pathway may play an important role in colon carcinogenesis, via the production of prostaglandins, in particular PGE2. PGE2 signals through four different receptor subtypes, EP1 – EP4. Thus, the aim of this study was to investigate the effect of targeting the PGE2-FasL signaling pathway. Results: (i) PGE2 induces FasL expression via the EP1 receptor in colon cancer cells. (ii) Suppression of FasL expression in colon tumour cells in vivo significantly delays and reduces tumour growth. (iii) Blocking EP1 receptor signaling, or suppression of the EP1 receptor in colon tumour cells, reduces tumour growth in vivo. Suppression of tumour growth correlates in part with suppression of FasL expression. (iv) The reduction in tumour growth is associated with an improved anti-tumour immune response. Tumour infiltration by Treg cells and macrophages was reduced, and the cytotoxic activity of CTL generated from splenocytes isolated from these mice increased. Conclusion: 1) Targeting FasL expression by blocking PGE2-EP1 receptor signalling reduces tumour development in vivo. 2) The mechanism is indirect but is associated with an increased anti-tumour immune response. Thus, unraveling the mechanisms regulating FasL expression and the pro-tumorigenic effects of the EP1 receptor may aid in the search for new therapeutic targets against colon cancer.

Epstein-Barr Virus Large Tegument Protein BPLF1 Contributes to Innate Immune Evasion through Interference with Toll-Like Receptor Signaling

Viral infection triggers an early host response through activation of pattern recognition receptors, including Toll-like receptors (TLR). TLR signaling cascades induce production of type I interferons and proinflammatory cytokines involved in establishing an anti-viral state as well as in orchestrating ensuing adaptive immunity. To allow infection, replication, and persistence, (herpes)viruses employ ingenious strategies to evade host immunity. The human gamma-herpesvirus Epstein-Barr virus (EBV) is a large, enveloped DNA virus persistently carried by more than 90% of adults worldwide. It is the causative agent of infectious mononucleosis and is associated with several malignant tumors. EBV activates TLRs, including TLR2, TLR3, and TLR9. Interestingly, both the expression of and signaling by TLRs is attenuated during productive EBV infection. Ubiquitination plays an important role in regulating TLR signaling and is controlled by ubiquitin ligases and deubiquitinases (DUBs). The EBV genome encodes three proteins reported to exert in vitro deubiquitinase activity. Using active site-directed probes, we show that one of these putative DUBs, the conserved herpesvirus large tegument protein BPLF1, acts as a functional DUB in EBV-producing B cells. The BPLF1 enzyme is expressed during the late phase of lytic EBV infection and is incorporated into viral particles. The N-terminal part of the large BPLF1 protein contains the catalytic site for DUB activity and suppresses TLR-mediated activation of NF-κB at, or downstream of, the TRAF6 signaling intermediate. A catalytically inactive mutant of this EBV protein did not reduce NF-κB activation, indicating that DUB activity is essential for attenuating TLR signal transduction. Our combined results show that EBV employs deubiquitination of signaling intermediates in the TLR cascade as a mechanism to counteract innate anti-viral immunity of infected hosts.

Chp8, a diguanylate cyclase from Pseudomonas syringae pv tomato DC3000 suppresses the PAMP flagellin, increases EPS and promotes plant immune evasion.

The bacterial plant pathogen Pseudomonas syringae causes disease in a wide range of plants. The associated decrease in crop yields results in economic losses and threatens global food security. Competition exists between the plant immune system and the pathogen, the basic principles of which can be applied to animal infection pathways. P. syringae uses a type III secretion system (T3SS) to deliver virulence factors into the plant that promote survival of the bacterium. The P. syringae T3SS is a product of the hypersensitive response and pathogenicity (hrp) and hypersensitive response and conserved (hrc) gene cluster, which is strictly controlled by the codependent enhancer-binding proteins HrpR and HrpS. Through a combination of bacterial gene regulation and phenotypic studies, plant infection assays, and plant hormone quantifications, we now report that Chp8 (i) is embedded in the Hrp regulon and expressed in response to plant signals and HrpRS, (ii) is a functional diguanylate cyclase, (iii) decreases the expression of the major pathogen-associated molecular pattern (PAMP) flagellin and increases extracellular polysaccharides (EPS), and (iv) impacts the salicylic acid/jasmonic acid hormonal immune response and disease progression. We propose that Chp8 expression dampens PAMP-triggered immunity during early plant infection.

Immune Evasion of Leptospira Species by Acquisition of Human Complement Regulator C4BP

Leptospirosis is a spirochetal zoonotic disease of global distribution with a high incidence in tropical regions. In the last 15 years it has been recognized as an important emerging infectious disease due to the occurrence of large outbreaks in warm-climate countries and, occasionally, in temperate regions. Pathogenic leptospires efficiently colonize target organs after penetrating the host. Their invasiveness is attributed to the ability to multiply in blood, adhere to host cells, and penetrate into tissues. Therefore, they must be able to evade the innate host defense. The main purpose of the present study was to evaluate how several Leptospira strains evade the protective function of the complement system. The serum resistance of six Leptospira strains was analyzed. We demonstrate that the pathogenic strain isolated from infected hamsters avoids serum bactericidal activity more efficiently than the culture-attenuated or the nonpathogenic Leptospira strains. Moreover, both the alternative and the classical pathways of complement seem to be responsible for the killing of leptospires. Serum-resistant and serum-intermediate strains are able to bind C4BP, whereas the serum-sensitive strain Patoc I is not. Surface-bound C4BP promotes factor I-mediated cleavage of C4b. Accordingly, we found that pathogenic strains displayed reduced deposition of the late complement components C5 to C9 upon exposure to serum. We conclude that binding of C4BP contributes to leptospiral serum resistance against host complement.

Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 1 mimics Epstein-Barr virus EBNA1 immune evasion through central repeat domain effects on protein processing

Kaposi's sarcoma-associated herpesvirus (KSHV/human herpesvirus 8 [HHV8]) and Epstein-Barr virus (EBV/HHV4) are distantly related gammaherpesviruses causing tumors in humans. KSHV latency-associated nuclear antigen 1 (LANA1) is functionally similar to the EBV nuclear antigen-1 (EBNA1) protein expressed during viral latency, although they have no amino acid similarities. EBNA1 escapes cytotoxic lymphocyte (CTL) antigen processing by inhibiting its own proteosomal degradation and retarding its own synthesis to reduce defective ribosomal product processing. We show here that the LANA1 QED-rich central repeat (CR) region, particularly the CR2CR3 subdomain, also retards LANA1 synthesis and markedly enhances LANA1 stability in vitro and in vivo. LANA1 isoforms have half-lives greater than 24 h, and fusion of the LANA1 CR2CR3 domain to a destabilized heterologous protein markedly decreases protein turnover. Unlike EBNA1, the LANA1 CR2CR3 subdomain retards translation regardless of whether it is fused to the 5′ or 3′ end of a heterologous gene construct. Manipulation of sequence order, orientation, and composition of the CR2 and CR3 subdomains suggests that specific peptide sequences rather than RNA structures are responsible for synthesis retardation. Although mechanistic differences exist between LANA1 and EBNA1, the primary structures of both proteins have evolved to minimize provoking CTL immune responses. Simple strategies to eliminate these viral inhibitory regions may markedly improve vaccine effectiveness by maximizing CTL responses. Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Exploring host-pathogen interactions through protein microarray. Large-scale protein microarray analysis revealed novel human receptors for the staphylococcal immune evasion protein FLIPr and for the neisserial adhesin NadA

Adhesion, immune evasion and invasion are key determinants during bacterial pathogenesis. Pathogenic bacteria possess a wide variety of surface exposed and secreted proteins which allow them to adhere to tissues, escape the immune system and spread throughout the human body. Therefore, extensive contacts between the human and the bacterial extracellular proteomes take place at the host-pathogen interface at the protein level. Recent researches emphasized the importance of a global and deeper understanding of the molecular mechanisms which underlie bacterial immune evasion and pathogenesis. Through the use of a large-scale, unbiased, protein microarray-based approach and of wide libraries of human and bacterial purified proteins, novel host-pathogen interactions were identified. This approach was first applied to Staphylococcus aureus, cause of a wide variety of diseases ranging from skin infections to endocarditis and sepsis. The screening led to the identification of several novel interactions between the human and the S. aureus extracellular proteomes. The interaction between the S. aureus immune evasion protein FLIPr (formyl-peptide receptor like-1 inhibitory protein) and the human complement component C1q, key players of the offense-defense fighting, was characterized using label-free techniques and functional assays. The same approach was also applied to Neisseria meningitidis, major cause of bacterial meningitis and fulminant sepsis worldwide. The screening led to the identification of several potential human receptors for the neisserial adhesin A (NadA), an important adhesion protein and key determinant of meningococcal interactions with the human host at various stages. The interaction between NadA and human LOX-1 (low-density oxidized lipoprotein receptor) was confirmed using label-free technologies and cell binding experiments in vitro. Taken together, these two examples provided concrete insights into S. aureus and N. meningitidis pathogenesis, and identified protein microarray coupled with appropriate validation methodologies as a powerful large scale tool for host-pathogen interactions studies.

Chlamydiae host cell interaction and immune evasion strategies

Chlamydiae are obligate intracellular bacteria with a strong global prevalence. They cause infections of the eye, lung and the genital tract and can either replicate in inclusion compartments or persist inside their host cell. In this thesis we focused on two aspects of chlamydiae infection. We hypothesize that transcription factor AP-1 is crucial for a replicative chlamydiae infection in epithelial cells. In addition we suggest that chlamydiae hide inside apoptotic blebs for a silent uptake by macrophages as immune evasion strategy.rnFocusing on AP-1, we could demonstrate that during Chlamydia pneumoniae infection, protein expression and phosphorylation of the AP-1 family member c-Jun significantly increased in a time and dose dependent manner. A siRNA knockdown of c-Jun in HEp-2 cells reduced chlamydial load, resulting in smaller inclusions and a significant lower chlamydial recovery. Furthermore, inhibition of the c-Jun containing AP-1 complexes, using Tanshinone IIA, changed the replicative infection into a persistent phenotype, characterized by (i) smaller, aberrant inclusions, (ii) a strong decrease in chlamydial load, as well as by (iii) its reversibility after removal of Tanshinone IIA. As chlamydiae are energy parasites, we investigated whether Tanshinone IIA interferes with energy/metabolism related processes. rnA role for autophagy or gene expression of glut-1 and c-jun in persistence could not be determined. However we could demonstrate Tanshinone IIA treatment to be accompanied by a significant decrease of ATP levels, probably causing a chlamydiae persistent phenotype.rnRegarding the chlamydial interaction with human primary cells we characterized infection of different chlamydiae species in either pro-inflammatory (type I) or anti-inflammatory (type II) human monocyte derived macrophages (hMDM). We found both phenotypes to be susceptible to chlamydiae infection. Furthermore, we observed that upon Chlamydia trachomatis and GFP-expressing Chlamydia trachomatis infection more hMDM type II were infected. However the chlamydial load was higher in hMDM type I and correspondingly, more replicative-like inclusions were found in this phenotype. Next, we focused on the chlamydial transfer using a combination of high speed live cell imaging and GFP-expressing Chlamydia trachomatis for optimal visualization. Thereby, we could successfully visualize the formation of apoptotic, chlamydiae-containing blebs and the interaction of hMDM with these blebs. Moreover, we observed the development of a replicative infection in hMDM. rnIn conclusion, we demonstrated a crucial role of AP-1 for C. pneumoniae development and preliminary time lapse data suggest that chlamydiae can be transferred to hMDMs via apoptotic blebs. In all, these data may contribute to a better understanding of chlamydial infection processes in humans.rn

Physiologic cold shock of Moraxella catarrhalis affects the expression of genes involved in the iron acquisition, serum resistance and immune evasion

Background Moraxella catarrhalis, a major nasopharyngeal pathogen of the human respiratory tract, is exposed to rapid downshifts of environmental temperature when humans breathe cold air. It was previously shown that the prevalence of pharyngeal colonization and respiratory tract infections caused by M. catarrhalis are greatest in winter. The aim of this study was to investigate how M. catarrhalis uses the physiologic exposure to cold air to upregulate pivotal survival systems in the pharynx that may contribute to M. catarrhalis virulence. Results A 26°C cold shock induces the expression of genes involved in transferrin and lactoferrin acquisition, and enhances binding of these proteins on the surface of M. catarrhalis. Exposure of M. catarrhalis to 26°C upregulates the expression of UspA2, a major outer membrane protein involved in serum resistance, leading to improved binding of vitronectin which neutralizes the lethal effect of human complement. In contrast, cold shock decreases the expression of Hemagglutinin, a major adhesin, which mediates B cell response, and reduces immunoglobulin D-binding on the surface of M. catarrhalis. Conclusion Cold shock of M. catarrhalis induces the expression of genes involved in iron acquisition, serum resistance and immune evasion. Thus, cold shock at a physiologically relevant temperature of 26°C induces in M. catarrhalis a complex of adaptive mechanisms that enables the bacterium to target their host cellular receptors or soluble effectors and may contribute to enhanced growth, colonization and virulence.

Impaired immune evasion in HIV through intracellular delays and multiple infection of cells

With its high mutation rate, HIV is capable of escape from recognition, suppression and/or killing by CD8(+) cytotoxic T lymphocytes (CTLs). The rate at which escape variants replace each other can give insights into the selective pressure imposed by single CTL clones. We investigate the effects of specific characteristics of the HIV life cycle on the dynamics of immune escape. First, it has been found that cells in HIV-infected patients can carry multiple copies of proviruses. To investigate how this process affects the emergence of immune escape, we develop a mathematical model of HIV dynamics with multiple infections of cells. Increasing the frequency of multiple-infected cells delays the appearance of immune escape variants, slows down the rate at which they replace the wild-type variant and can even prevent escape variants from taking over the quasi-species. Second, we study the effect of the intracellular eclipse phase on the rate of escape and show that escape rates are expected to be slower than previously anticipated. In summary, slow escape rates do not necessarily imply inefficient CTL-mediated killing of HIV-infected cells, but are at least partly a result of the specific characteristics of the viral life cycle.

To sense or not to sense viral RNA--essentials of coronavirus innate immune evasion.

An essential function of innate immunity is to distinguish self from non-self and receptors have evolved to specifically recognize viral components and initiate the expression of antiviral proteins to restrict viral replication. Coronaviruses are RNA viruses that replicate in the host cytoplasm and evade innate immune sensing in most cell types, either passively by hiding their viral signatures and limiting exposure to sensors or actively, by encoding viral antagonists to counteract the effects of interferons. Since many cytoplasmic viruses exploit similar mechanisms of innate immune evasion, mechanistic insight into the direct interplay between viral RNA, viral RNA-processing enzymes, cellular sensors and antiviral proteins will be highly relevant to develop novel antiviral targets and to restrict important animal and human infections.

Peroxisomes are platforms for cytomegalovirus’ evasion from the cellular immune response

The human cytomegalovirus developed distinct evasion mechanisms from the cellular antiviral response involving vMIA, a virally-encoded protein that is not only able to prevent cellular apoptosis but also to inhibit signalling downstream from mitochondrial MAVS. vMIA has been shown to localize at mitochondria and to trigger their fragmentation, a phenomenon proven to be essential for the signalling inhibition. Here, we demonstrate that vMIA is also localized at peroxisomes, induces their fragmentation and inhibits the peroxisomal-dependent antiviral signalling pathway. Importantly, we demonstrate that peroxisomal fragmentation is not essential for vMIA to specifically inhibit signalling downstream the peroxisomal MAVS. We also show that vMIA interacts with the cytoplasmic chaperone Pex19, suggesting that the virus has developed a strategy to highjack the peroxisomal membrane proteins' transport machinery. Furthermore, we show that vMIA is able to specifically interact with the peroxisomal MAVS. Our results demonstrate that peroxisomes constitute a platform for evasion of the cellular antiviral response and that the human cytomegalovirus has developed a mechanism by which it is able to specifically evade the peroxisomal MAVS-dependent antiviral signalling.

Hyperlipidemia impaired innate immune response to periodontal pathogen Porphyromonas gingivalis in apolipoprotein E knockout mice

A finely-tuned innate immune response plays a pivotal role in protecting host against bacterial invasion during periodontal disease progression. Hyperlipidemia has been suggested to exacerbate periodontal health condition. However, the underlying mechanism has not been addressed. In the present study, we investigated the effect of hyperlipidemia on innate immune responses to periodontal pathogen Porphyromonas gingivalis infection. Apolipoprotein E-deficient and wild-type mice at the age of 20 weeks were used for the study. Peritoneal macrophages were isolated and subsequently used for the study of viable P. gingivalis infection. ApoE−/− mice demonstrated inhibited iNOS production and impaired clearance of P. gingivalis in vitro and in vivo; furthermore, ApoE−/− mice displayed disrupted cytokine production pattern in response to P. gingivalis, with a decreased production of tumor necrosis factor-α, interleukin-6 (IL-6), IL-1β and monocyte chemotactic protein-1. Microarray data demonstrated that Toll-like receptor (TLR) and NOD-like receptor (NLR) pathway were altered in ApoE−/− mice macrophages; further analysis of pattern recognition receptors (PRRs) demonstrated that expression of triggering receptors on myeloid cells-1 (TREM-1), an amplifier of the TLR and NLR pathway, was decreased in ApoE−/− mice macrophages, leading to decreased recruitment of NF-κB onto the promoters of the TNF-α and IL-6. Our data suggest that in ApoE−/− mice hyperlipidemia disrupts the expression of PRRs, and cripples the host’s capability to generate sufficient innate immune response to P. gingivalis, which may facilitate immune evasion, subgingival colonization and establishment of P. gingivalis in the periodontal niche.

Src Homology 3-interacting Domain of Rv1917c of Mycobacterium tuberculosis Induces Selective Maturation of Human Dendritic Cells by Regulating PI3K-MAPK-NF-kappa B Signaling and Drives Th2 Immune Responses

Mycobacterium tuberculosis, an etiological agent of pulmonary tuberculosis, causes significant morbidity and mortality worldwide. Pathogenic mycobacteria survive in the host by subverting host innate immunity. Dendritic cells (DCs) are professional antigen-presenting cells that are vital for eliciting immune responses to infectious agents, including pathogenic mycobacteria. DCs orchestrate distinct Th responses based on the signals they receive. In this perspective, deciphering the interactions of the proline-glutamic acid/proline-proline-glutamic acid (PE/PPE) family of proteins of M. tuberculosis with DCs assumes significant pathophysiological attributes. In this study, we demonstrate that Rv1917c (PPE34), a representative member of the proline-proline-glutamic-major polymorphic tandem repeat family, interacts with TLR2 and triggers functional maturation of human DCs. Signaling perturbations implicated a critical role for integrated cross-talk among PI3K-MAPK and NF-kappa B signaling cascades in Rv1917c-induced maturation of DCs. However, this maturation of DCs was associated with a secretion of high amounts of anti-inflammatory cytokine IL-10, whereas Th1-polarizing cytokine IL-12 was not induced. Consistent with these results, Rv1917c-matured DCs favored secretion of IL-4, IL-5, and IL-10 from CD4(+) T cells and contributed to Th2-skewed cytokine balance ex vivo in healthy individuals and in patients with pulmonary tuberculosis. Interestingly, the Rv1917c-skewed Th2 immune response involved induced expression of cyclooxygenase-2 (COX-2) in DCs. Taken together, these results indicate that Rv1917c facilitates a shift in the ensuing immunity toward the Th2 phenotype and could aid in immune evasion by mycobacteria.

Transmission of single HIV-1 genomes and dynamics of early immune escape revealed by ultra-deep sequencing.

We used ultra-deep sequencing to obtain tens of thousands of HIV-1 sequences from regions targeted by CD8+ T lymphocytes from longitudinal samples from three acutely infected subjects, and modeled viral evolution during the critical first weeks of infection. Previous studies suggested that a single virus established productive infection, but these conclusions were tempered because of limited sampling; now, we have greatly increased our confidence in this observation through modeling the observed earliest sample diversity based on vastly more extensive sampling. Conventional sequencing of HIV-1 from acute/early infection has shown different patterns of escape at different epitopes; we investigated the earliest escapes in exquisite detail. Over 3-6 weeks, ultradeep sequencing revealed that the virus explored an extraordinary array of potential escape routes in the process of evading the earliest CD8 T-lymphocyte responses--using 454 sequencing, we identified over 50 variant forms of each targeted epitope during early immune escape, while only 2-7 variants were detected in the same samples via conventional sequencing. In contrast to the diversity seen within epitopes, non-epitope regions, including the Envelope V3 region, which was sequenced as a control in each subject, displayed very low levels of variation. In early infection, in the regions sequenced, the consensus forms did not have a fitness advantage large enough to trigger reversion to consensus amino acids in the absence of immune pressure. In one subject, a genetic bottleneck was observed, with extensive diversity at the second time point narrowing to two dominant escape forms by the third time point, all within two months of infection. Traces of immune escape were observed in the earliest samples, suggesting that immune pressure is present and effective earlier than previously reported; quantifying the loss rate of the founder virus suggests a direct role for CD8 T-lymphocyte responses in viral containment after peak viremia. Dramatic shifts in the frequencies of epitope variants during the first weeks of infection revealed a complex interplay between viral fitness and immune escape.

The impact of host immune status on the within-host and population dynamics of antigenic immune escape.

Antigenically evolving pathogens such as influenza viruses are difficult to control owing to their ability to evade host immunity by producing immune escape variants. Experimental studies have repeatedly demonstrated that viral immune escape variants emerge more often from immunized hosts than from naive hosts. This empirical relationship between host immune status and within-host immune escape is not fully understood theoretically, nor has its impact on antigenic evolution at the population level been evaluated. Here, we show that this relationship can be understood as a trade-off between the probability that a new antigenic variant is produced and the level of viraemia it reaches within a host. Scaling up this intra-host level trade-off to a simple population level model, we obtain a distribution for variant persistence times that is consistent with influenza A/H3N2 antigenic variant data. At the within-host level, our results show that target cell limitation, or a functional equivalent, provides a parsimonious explanation for how host immune status drives the generation of immune escape mutants. At the population level, our analysis also offers an alternative explanation for the observed tempo of antigenic evolution, namely that the production rate of immune escape variants is driven by the accumulation of herd immunity. Overall, our results suggest that disease control strategies should be further assessed by considering the impact that increased immunity--through vaccination--has on the production of new antigenic variants.