Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.     

Phenoloxidase Activity Acts as a Mosquito Innate Immune Response against Infection with Semliki Forest Virus

Several components of the mosquito immune system including the RNA interference (RNAi), JAK/STAT, Toll and IMD pathways have previously been implicated in controlling arbovirus infections. In contrast, the role of the phenoloxidase (PO) cascade in mosquito antiviral immunity is unknown. Here we show that conditioned medium from the Aedes albopictus-derived U4.4 cell line contains a functional PO cascade, which is activated by the bacterium Escherichia coli and the arbovirus Semliki Forest virus (SFV) (Togaviridae; Alphavirus). Production of recombinant SFV expressing the PO cascade inhibitor Egf1.0 blocked PO activity in U4.4 cell- conditioned medium, which resulted in enhanced spread of SFV. Infection of adult female Aedes aegypti by feeding mosquitoes a bloodmeal containing Egf1.0-expressing SFV increased virus replication and mosquito mortality. Collectively, these results suggest the PO cascade of mosquitoes plays an important role in immune defence against arboviruses.     

Innate Immune Response to Arenaviral Infection: A Focus on the Highly Pathogenic New World Hemorrhagic Arenaviruses

Arenaviruses are enveloped, negative-stranded RNA viruses that belong to the family Arenaviridae. This diverse family can be further classified into OW (Old World) and NW (New World) arenaviruses based on their antigenicity, phylogeny, and geographical distribution. Many of the NW arenaviruses are highly pathogenic viruses that cause systemic human infections characterized by hemorrhagic fever and/or neurological manifestations, constituting public health problems in their endemic regions. NW arenavirus infection induces a variety of host innate immune responses, which could contribute to the viral pathogenesis and/or influence the final outcome of virus infection in vitro and in vivo. On the other hand, NW arenaviruses have also developed several strategies to counteract the host innate immune response. We will review current knowledge regarding the interplay between the host innate immune response and NW arenavirus infection in vitro and in vivo, with emphasis on viral-encoded proteins and their effect on the type I interferon response.     

The Innate Immune Response Elicited by Group A Streptococcus Is Highly Variable among Clinical Isolates and Correlates with the emm Type

Group A Streptococcus (GAS) infections remain a significant health care problem due to high morbidity and mortality associated with GAS diseases, along with their increasing worldwide prevalence. Macrophages play a key role in the control and clearance of GAS infections. Moreover, pro-inflammatory cytokines production and GAS persistence and invasion are related. In this study we investigated the correlation between the GAS clinical isolates genotypes, their known clinical history, and their ability to modulate innate immune response. We constituted a collection of 40 independent GAS isolates representative of the emm types currently prevalent in France and responsible for invasive (57.5%) and non-invasive (42.5%) clinical manifestations. We tested phagocytosis and survival in mouse bone marrow-derived macrophages and quantified the pro-inflammatory mediators (IL-6, TNF-α) and type I interferon (INF-β) production. Invasive emm89 isolates were more phagocytosed than their non-invasive counterparts, and emm89 isolates more than the other isolates. Regarding the survival, differences were observed depending on the isolate emm type, but not between invasive and non-invasive isolates within the same emm type. The level of inflammatory mediators produced was also emm type-dependent and mostly invasiveness status independent. Isolates of the emm1 type were able to induce the highest levels of both pro-inflammatory cytokines, whereas emm89 isolates induced the earliest production of IFN-β. Finally, even within emm types, there was a variability of the innate immune responses induced, but survival and inflammatory mediator production were not linked.     

Role of the Innate Immune Response and Tumor Immunity Associated with Simian Virus 40 Large Tumor Antigen

We examined properties of the innate immune response against the tumor-specific antigen simian virus 40 (SV40) large tumor antigen (Tag) following experimental pulmonary metastasis in naive mice. Approximately 14 days after mKSA tumor cell challenge, expression of inflammatory mediators such as tumor necrosis factor alpha (TNF-α), interleukin-2 (IL-2), and RANTES was upregulated in splenocytes harvested from mice, as assessed by flow cytometry and antibody array assays. This response was hypothesized to activate and induce tumor-directed NK cell lysis since IL-2-stimulated NK cells mediated tumor cell destruction in vitro. The necessary function of NK cells was further validated in vivo through selected antibody depletion of NK cells, which resulted in an overwhelming lung tumor burden relative to that in animals receiving a control rabbit IgG depletion regimen. Interestingly, mice achieved increased protection from experimental pulmonary metastasis when NK cells were further activated indirectly through in vivo administration of poly(I:C), a Toll-like receptor 3 (TLR3) agonist. In a separate study, mice receiving treatments of poly(I:C) and recombinant SV40 Tag protein immunization mounted effective tumor immunity in an established experimental pulmonary metastasis setting. Initiating broad-based immunity with poly(I:C) was observed to induce a Th1 bias in the SV40 Tag antibody response that led to successful antitumor responses not observed in animals treated only with poly(I:C) or SV40 Tag. These data have direct implications for immunotherapeutic strategies incorporating methods to elicit inflammatory reactions, particularly NK cell-driven lysis, against malignant cell types that express a tumor-specific antigen such as SV40 Tag.Considerable interest has been directed toward the role innate immunity plays in reducing malignant growth and progression. Although the innate system by broad definition is not endowed with the antigen specificity and memory recall of adaptive immunity, natural killer (NK) cells are an innate effector population that shares most properties with the adaptive arm of the immune system, excluding receptor rearrangement (28). Interestingly, NK cells can be employed to directly target and destroy malignant cell types through diverse pathways that include tumor major histocompatibility complex class I (MHC-I) loss and upregulation of stress-inducible protein ligands for the NK cell activating receptor NKG2D (24, 29). Much effort is under way in human clinical trials to manipulate NK cell properties for directed therapies against cancer (13, 29).One strategy in eliciting innate immunity in general involves activating the Toll-like receptor (TLR) family, which are preferentially expressed by innate effectors such as NK cells, macrophages, and dendritic cells (DCs) (26). TLR ligands include a variety of pathogen-associated molecular patterns with differing downstream responses based on the cell type involved and specific TLR activated. In TLR-expressing cells, signal transduction pathways follow a MyD88-independent course to produce type I interferons (IFNs) (e.g., TLR3) or a MyD88-dependent pathway that results in the production of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), and IL-6 and expression of costimulatory molecules such as CD40, CD80, and CD86 (e.g., TLR4 and TLR9) (2, 12, 23, 26). In the case of TLR3, activation by poly(I:C) causes DCs and additional accessory cells to secrete type I interferons and IL-12, activating NK cells and prompting NK cell secretion of IFN-γ among other effects (14, 20). Ultimately, modulation of TLR activation results in the generation of a range of cytokines that promote inflammation, Th1 bias, and NK cell-directed killing that can be utilized in a beneficial manner for tumor treatment strategies.TLR agonist incorporation alongside vaccine strategies has resulted in promising results in mouse models of cancer (12). Indeed, the TLR7 agonist imiquimod is an effective FDA-approved topical compound used to treat superficial basal-cell carcinoma and external genital warts (9). However, to our knowledge, modulating TLR activity while also incorporating recombinant simian virus 40 (SV40) large tumor antigen (Tag) protein immunizations in a therapeutic tumor setting has not been previously reported. SV40 Tag is a clinically relevant tumor-specific antigen that has been shown to be expressed by a number of human malignancies, including malignant pleural mesothelioma (MPM), and represents a potential target for immunotherapeutic strategies.Our laboratory has previously defined a unique role for antibody-dependent cell-mediated cytotoxicity (ADCC) reactions—specific against SV40 Tag—promoting cytotoxic T-lymphocyte (CTL) activity in response to neoantigens through cross-presentation of tumor cell debris in a model of experimental pulmonary metastasis (16, 17). In this report, we analyze the role of innate immunity in mediating tumor cell lysis during the early course of tumorigenesis in the absence of vaccination. Overall, we find that activated NK cells are necessary effector cells in achieving antitumor reactions and providing partial tumor immunity during the onset of tumorigenesis and that these functioning NK cells are likely activated in vivo due to inflammation as a result of tumor growth and progression. The burden of tumor challenge could be further reduced in naive animals with the indirect activation of NK cells using poly(I:C) as a TLR3 agonist prior to and during malignant dissemination. Interestingly, in an established pulmonary tumor setting, therapeutic treatment of mice with poly(I:C) and recombinant SV40 Tag resulted in enhanced protection that was not observed using poly(I:C) or SV40 Tag alone. One effect of instituting poly(I:C) treatment alongside SV40 Tag immunizations was a Th1 skewing of the SV40 Tag IgG antibody response that correlated with therapeutic tumor protection.Our results have direct implications for the prevention and treatment of malignancies, such as MPM, that express the SV40 Tag oncoprotein. Combining specific aspects of innate and adaptive immunity by targeting both NK cells and humoral activity against SV40 Tag, respectively, represents a novel and clinically significant immunotherapeutic strategy for potential use in patients.     

Human Coronavirus HKU1 Infection of Primary Human Type II Alveolar Epithelial Cells: Cytopathic Effects and Innate Immune Response

Because they are the natural target for respiratory pathogens, primary human respiratory epithelial cells provide the ideal in vitro system for isolation and study of human respiratory viruses, which display a high degree of cell, tissue, and host specificity. Human coronavirus HKU1, first discovered in 2005, has a worldwide prevalence and is associated with both upper and lower respiratory tract disease in both children and adults. Research on HCoV-HKU1 has been difficult because of its inability to be cultured on continuous cell lines and only recently it was isolated from clinical specimens using primary human, ciliated airway epithelial cells. Here we demonstrate that HCoV-HKU1 can infect and be serially propagated in primary human alveolar type II cells at the air-liquid interface. We were not able to infect alveolar type I-like cells or alveolar macrophages. Type II alveolar cells infected with HCoV-HKU1 demonstrated formation of large syncytium. At 72 hours post inoculation, HCoV-HKU1 infection of type II cells induced increased levels of mRNAs encoding IL29,CXCL10, CCL5, and IL-6 with no significant increases in the levels of IFNβ. These studies demonstrate that type II cells are a target cell for HCoV-HKU1 infection in the lower respiratory tract, that type II alveolar cells are immune-competent in response to infection exhibiting a type III interferon and proinflammatory chemokine response, and that cell to cell spread may be a major factor for spread of infection. Furthermore, these studies demonstrate that human alveolar cells can be used to isolate and study novel human respiratory viruses that cause lower respiratory tract disease.     

NADH Oxidase Functions as an Adhesin in Streptococcus pneumoniae and Elicits a Protective Immune Response in Mice

The initial event in disease caused by S. pneumoniae is adhesion of the bacterium to respiratory epithelial cells, mediated by surface expressed molecules including cell-wall proteins. NADH oxidase (NOX), which reduces free oxygen to water in the cytoplasm, was identified in a non-lectin enriched pneumococcal cell-wall fraction. Recombinant NOX (rNOX) was screened with sera obtained longitudinally from children and demonstrated age-dependent immunogenicity. NOX ablation in S. pneumoniae significantly reduced bacterial adhesion to A549 epithelial cells in vitro and their virulence in the intranasal or intraperitoneal challenge models in mice, compared to the parental strain. Supplementation of Δnox WU2 with the nox gene restored its virulence. Saturation of A549 target cells with rNOX or neutralization of cell-wall residing NOX using anti-rNOX antiserum decreased adhesion to A549 cells. rNOX-binding phages inhibited bacterial adhesion. Moreover, peptides derived from the human proteins contactin 4, chondroitin 4 sulfotraferase and laminin5, homologous to the insert peptides in the neutralizing phages, inhibited bacterial adhesion to the A549 cells. Furthermore, rNOX immunization of mice elicited a protective immune response to intranasal or intraperitoneal S. pneumoniae challenge, whereas pneumococcal virulence was neutralized by anti-rNOX antiserum prior to intraperitoneal challenge. Our results suggest that in addition to its enzymatic activity, NOX contributes to S. pneumoniae virulence as a putative adhesin and thus peptides derived from its target molecules may be considered for the treatment of pneumococcal infections. Finally, rNOX elicited a protective immune response in both aerobic and anaerobic environments, which renders NOX a candidate for future pneumococcal vaccine.     

A Teleost Bactericidal Permeability-Increasing Protein Kills Gram-Negative Bacteria,Modulates Innate Immune Response,and Enhances Resistance against Bacterial and Viral Infection

Bactericidal/permeability-increasing protein (BPI) is an important factor of innate immunity that in mammals is known to take part in the clearance of invading Gram-negative bacteria. In teleost, the function of BPI is unknown. In the present work, we studied the function of tongue sole (Cynoglossus semilaevis) BPI, CsBPI. We found that CsBPI was produced extracellularly by peripheral blood leukocytes (PBL). Recombinant CsBPI (rCsBPI) was able to bind to a number of Gram-negative bacteria but not Gram-positive bacteria. Binding to bacteria led to bacterial death through membrane permeabilization and structural destruction, and the bound bacteria were more readily taken up by PBL. In vivo, rCsBPI augmented the expression of a wide arrange of genes involved in antibacterial and antiviral immunity. Furthermore, rCsBPI enhanced the resistance of tongue sole against bacterial as well as viral infection. These results indicate for the first time that a teleost BPI possesses immunoregulatory effect and plays a significant role in antibacterial and antiviral defense.     

NKLP27: A Teleost NK-Lysin Peptide that Modulates Immune Response,Induces Degradation of Bacterial DNA,and Inhibits Bacterial and Viral Infection

NK-lysin is an antimicrobial protein produced by cytotoxic T lymphocytes and natural killer cells. In this study, we examined the biological property of a peptide, NKLP27, derived from tongue sole (Cynoglossus semilaevis) NK-lysin. NKLP27 is composed of 27 amino acids and shares little sequence identity with known NK-lysin peptides. NKLP27 possesses bactericidal activity against both Gram-negative and Gram-positive bacteria including common aquaculture pathogens. The bactericidal activity of NKLP27 was dependent on the C-terminal five residues, deletion of which dramatically reduced the activity of NKLP27. During its interaction with the target bacterial cells, NKLP27 destroyed cell membrane integrity, penetrated into the cytoplasm, and induced degradation of genomic DNA. In vivo study showed that administration of tongue sole with NKLP27 before bacterial and viral infection significantly reduced pathogen dissemination and replication in tissues. Further study revealed that fish administered with NKLP27 exhibited significantly upregulated expression of the immune genes including those that are known to be involved in antibacterial and antiviral defense. These results indicate that NKLP27 is a novel antimicrobial against bacterial and viral pathogens, and that the observed effect of NKLP27 on bacterial DNA and host gene expression adds new insights to the action mechanism of fish antimicrobial peptides.     

Toll-like Receptor Signaling Activation by Entamoeba histolytica Induces Beta Defensin 2 in Human Colonic Epithelial Cells: Its Possible Role as an Element of the Innate Immune Response

Background

Entamoeba histolytica, a protozoan parasite of humans, produces dysenteric diarrhea, intestinal mucosa damage and extraintestinal infection. It has been proposed that the intestinal microbiota composition could be an important regulatory factor of amebic virulence and tissue invasion, particularly if pathogenic bacteria are present. Recent in vitro studies have shown that Entamoeba histolytica trophozoites induced human colonic CaCo2 cells to synthesize TLR-2 and TLR-4 and proinflammatory cytokines after binding to the amebic Gal/GalNac lectin carbohydrate recognition domain. The magnitude of the inflammatory response induced by trophozoites and the subsequent cell damage were synergized when cells had previously been exposed to pathogenic bacteria.

Methodology/Principal Findings

We show here that E. histolytica activation of the classic TLR pathway in CaCo2 cells is required to induce β defensin-2 (HBD2) mRNA expression and production of a 5-kDa cationic peptide with similar properties to the antimicrobial HBD2 expressed by CaCo2 cells exposed to enterotoxigenic Escherichia coli. The induced peptide showed capacity to permeabilize membranes of bacteria and live trophozoites. This activity was abrogated by inhibition of TLR2/4-NFκB pathway or by neutralization with an anti-HBD2 antibody.

Conclusions/Significance

Entamoeba histolytica trophozoites bind to human intestinal cells and induce expression of HBD2; an antimicrobial molecule with capacity to destroy pathogenic bacteria and trophozoites. HDB2''s possible role as a modulator of the course of intestinal infections, particularly in mixed ameba/bacteria infections, is discussed.     

An Immediate Innate Immune Response Occurred In the Early Stage of E.granulosus Eggs Infection in Sheep: Evidence from Microarray Analysis

Background

Cystic Echinococcosis(CE), caused by infection with the larval stage of the cestode Echinococcus granulosus (E. granulosus), is a chronic parasitic zoonosis, with highly susceptible infection in sheep. However, the comprehensive molecular mechanisms that underlie the process of E. granulosus infection in the early stage remain largely unknown. The objective of this present study was to gain a cluster of genes expression profiles in the intestine tissue of sheep infected with CE.

Methods

Nine healthy sheep were divided into infection group and healthy controls, with six infected perorally 5000 E. granulosus eggs suspended in 1000μl physiological saline and three controls perorally injected 1000μl physiological saline. All animals were sacrificed at 4 hours post-infection, respectively. The intestine tissue was removed and the RNA was extracted. In the infection group, the biology replicates were designed to make sure the accuracy of the data. The ovine microarrays were used to analyze changes of gene expression in the intestine tissue between CE infected sheep and healthy controls. Real-time PCR was used to assess reliability of the microarray data.

Results

By biology repeats, a total of 195 differentially expressed genes were identified between infected group and controls at 4 hours post-infection, with 105 genes related to immune responses, while 90 genes associated with functions including energy metabolism, fat soluble transport, etc. Among the 105 immunity genes, 72 genes showed up-regulated expression levels while 33 showed down-regulation levels. Function analysis showed that most of up-regulated genes were related to innate immune responses, such as mast cell, NK cell, cytokines, chemokines and complement. In addition, Real-time PCR analysis of a random selection of nine genes confirmed the reliability of the microarray data.

Conclusion

To our knowledge, this is the first report describing gene expression profiles in the intestine tissue of CE infection sheep. These results suggested that the innate immune system was activated to elicit immediate defense in the intestine tissue where E. granulosus invaded in at 4 hour-post infection. Furthermore, future interest will also focus on unraveling similar events, especially for the function of adaptive immunity, but at late stage infection.     

Vaginal Challenge with an SIV-Based Dual Reporter System Reveals That Infection Can Occur throughout the Upper and Lower Female Reproductive Tract

The majority of new HIV infections occur in women as a result of heterosexual intercourse, overcoming multiple innate barriers to infection within the mucosa. However, the avenues through which infection is established, and the nature of bottlenecks to transmission, have been the source of considerable investigation and contention. Using a high dose of a single round non-replicating SIV-based vector containing a novel dual reporter system, we determined the sites of infection by the inoculum using the rhesus macaque vaginal transmission model. Here we show that the entire female reproductive tract (FRT), including the vagina, ecto- and endocervix, along with ovaries and local draining lymph nodes can contain transduced cells only 48 hours after inoculation. The distribution of infection shows that virions quickly disseminate after exposure and can access target cells throughout the FRT, with an apparent preference for infection in squamous vaginal and ectocervical mucosa. JRFL enveloped virions infect diverse CD4 expressing cell types, with T cells resident throughout the FRT representing the primary target. These findings establish a new perspective that the entire FRT is susceptible and virus can reach as far as the ovary and local draining lymph nodes. Based on these findings, it is essential that protective mechanisms for prevention of HIV acquisition must be present at protective levels throughout the entire FRT to provide complete protection.     

Identification of Hemopexin as an Anti-Inflammatory Factor That Inhibits Synergy of Hemoglobin with HMGB1 in Sterile and Infectious Inflammation

Hemoglobin is released from lysed RBCs in numerous clinical settings. High mobility group box 1 (HMGB1) is a nuclear and cytosolic DNA-binding protein released from injured cells that has been shown to play an important role in inducing inflammation. Because both of these endogenous molecules are frequently present in sites of necrosis and inflammation, we studied their interaction on the activation of macrophages. We report in this article that hemoglobin and HMGB1 synergize to activate mouse macrophages to release significantly increased proinflammatory cytokines. Addition of microbial ligands that activate through TLR2 or TLR4 resulted in further significant increases, in a "three-way" synergy between endogenous and microbial ligands. The synergy was strongly suppressed by hemopexin (Hx), an endogenous heme-binding plasma protein. The findings suggest that hemoglobin may play an important role in sterile and infectious inflammation, and that endogenous Hx can modulate this response. Administration of Hx may be beneficial in clinical settings characterized by elevated extracellular hemoglobin and HMGB1.     

Cyclophilin A Associates with Enterovirus-71 Virus Capsid and Plays an Essential Role in Viral Infection as an Uncoating Regulator

Viruses utilize host factors for their efficient proliferation. By evaluating the inhibitory effects of compounds in our library, we identified inhibitors of cyclophilin A (CypA), a known immunosuppressor with peptidyl-prolyl cis-trans isomerase activity, can significantly attenuate EV71 proliferation. We demonstrated that CypA played an essential role in EV71 entry and that the RNA interference-mediated reduction of endogenous CypA expression led to decreased EV71 multiplication. We further revealed that CypA directly interacted with and modified the conformation of H-I loop of the VP1 protein in EV71 capsid, and thus regulated the uncoating process of EV71 entry step in a pH-dependent manner. Our results aid in the understanding of how host factors influence EV71 life cycle and provide new potential targets for developing antiviral agents against EV71 infection.