Incoming Pathogens Research Articles

Cold activation of a plasma membrane-tethered NAC transcription factor induces a pathogen resistance response in Arabidopsis

Cold signals interact with other environmental cues to modulate plant developmental processes. Recent studies have shown that many Pathogenesis-Related (PR) genes are induced and disease resistance is enhanced after exposure to low temperatures, linking cold signals with pathogenesis in plants. However, the underlying molecular mechanisms and signaling schemes are largely unknown. Here, we demonstrate that cold stimulates proteolytic activation of a plasma membrane-tethered NAC (NAM/ATAF1/2/CUC2) transcription factor NTL6. The transcriptionally active NTL6 protein enters the nucleus, where it induces a subset of PR genes by directly binding to a conserved sequence in the promoters of cold-responsive PR genes, such as PR1, PR2, and PR5. While transgenic plants overexpressing an active NTL6 form exhibited enhanced disease resistance, RNAi plants with reduced NTL6 activity were more susceptible to pathogen infection at low temperatures. Accordingly, cold induction of PR1 disappeared in the RNAi plants. Consistent with the close relationship between cold and pathogenesis, cold-acclimated plants showed enhanced resistance to pathogen infection. In this signaling cascade, controlled activation of the membrane-tethered, dormant NTL6 transcription factor serves as a molecular link that incorporates cold signals into pathogen resistance responses. However, the NTL6-mediated cold induction of the PR genes is independent of salicylic acid (SA). The PR genes were still induced by SA in the NTL6 RNAi plants. Cold regulation of the PR genes through the membrane-mediated transcriptional control is thought to be an adaptive process that ensures quick plant responses to incoming pathogens that frequently occur during cold seasons.

HSV-1 upregulates the ARE-binding protein tristetraprolin in a STAT1- and p38-dependent manner in mature dendritic cells

Dendritic cells are the sentinels of the immune system and as such represent the first-line of defense against incoming pathogens. Upon encounter with harmful antigens, these antigen-presenting cells start to mature and migrate towards the draining lymph nodes to display the antigen to T-lymphocytes, thereby eliciting the immune response of the host. Viruses, including human herpesvirus type I (HSV-1), seek to avoid such immune reactions. Therefore, they developed an arsenal of immune evasion strategies, some of which have been described earlier by our group and others. The secretion of tumor necrosis factor (TNF) represents a typical defense line of the host and it has been shown that this cytokine contributes to the inhibition of viral replication and augments the proliferation of cytotoxic T-lymphocytes. Here we report, that upon infection of mature dendritic cells, HSV-1 very strongly induces the expression of the AU-rich elements (ARE)-binding protein tristetraprolin (TTP), an mRNA-destabilizing protein. One of the best described targets of TTP is the TNF mRNA. This induction is dependent on the phosphorylation of both signal transducer and activator of transcription (STAT1) and p38 in a collaborative manner. By repressing this phosphorylation with specific inhibitors, we were able to reduce TTP mRNA levels. At the same time TNF mRNA levels were increased, suggesting that TNF mRNA is indeed a target of TTP in this setting. In summary, these data underline that HSV-1 induces TTP transcription in order to reduce TNF levels generated by infected mature dendritic cell.

Salmonella and Escherichia coli O157:H7 Contamination on Hides and Carcasses of Cull Cattle Presented for Slaughter in the United States: an Evaluation of Prevalence and Bacterial Loads by Immunomagnetic Separation and Direct Plating Methods

The hide and carcass hygiene of cull cattle at slaughter in four geographically distant regions of the United States was examined from July 2005 to April 2006 by measuring the aerobic plate counts (APC) and the prevalences and loads of Salmonella and Escherichia coli O157:H7. The geometric mean log(10) APC CFU/100 cm(2) levels on hides and preevisceration and postintervention carcasses ranged from 6.17 to 8.19, 4.24 to 6.47, and 1.46 to 1.96, respectively, and were highest in the summer (P < 0.0001). The average prevalences of Salmonella on hides and preevisceration and postintervention carcasses were 89.6% (95% confidence interval [CI], 85.1 to 94.0), 50.2% (95% CI, 40.9 to 59.5), and 0.8% (95% CI, 0.18 to 1.42), respectively. The prevalences of E. coli O157:H7 were 46.9% (95% CI, 37.3 to 56.6) and 16.7% (95% CI, 9.8 to 23.6) on hides and preevisceration carcasses, respectively. Examination of the concomitant incidence of Salmonella and E. coli O157:H7 showed that, on average, 33.3% (95% CI, 15.9 to 69.8) of cattle hide and 4.1% (95% CI, 0.98 to 17.3) of preevisceration carcass samples were contaminated with both pathogens. The pathogen prevalence on hides and carcasses was not significantly affected by the season; however, significant differences were observed between plants with respect to the incoming pathogen load and the ability to mitigate hide-to-carcass transfer. In spite of these differences, postintervention carcass contamination was significantly reduced (P < 0.001), likely as a result of the use of one or more of the processing interventions employed at each of the four processing plants examined.

The C-Type Lectin Macrophage Galactose-Type Lectin Impedes Migration of Immature APCs

Dendritic cells (DCs) are the most potent APCs of the immune system that seed the peripheral tissues and lymphoid organs. In an immature state, DCs sample their surroundings for incoming pathogens. Upon Ag encounter, DCs mature and migrate to the lymph node to induce adaptive immune responses. The C-type macrophage galactose-type lectin (MGL), expressed in immature DCs, mediates binding to glycoproteins carrying GalNAc moieties. In the present study, we demonstrate that MGL ligands are present on the sinusoidal and lymphatic endothelium of lymph node and thymus, respectively. MGL binding strongly correlated with the expression of the preferred MGL ligand, alpha-GalNAc-containing glycan structures, as visualized by staining with the alpha-GalNAc-specific snail lectin Helix pomatia agglutinin. MGL(+) cells were localized in close proximity of the endothelial structures that express the MGL ligand. Strikingly, instead of inducing migration, MGL mediated retention of human immature DCs, as blockade of MGL interactions enhanced DC trafficking and migration. Thus, MGL(+) DCs are hampered in their migratory responses and only upon maturation, when MGL expression is abolished; these DCs will be released from their MGL-mediated restraints.

APOBEC3 proteins and reverse transcription

The ability of members of the APOBEC3 (A3) family of proteins to confer intrinsic immunity to retroviral infection was recognized in several studies. More specifically, A3 proteins are cytidine deaminases (CDAs) that cause hypermutations of nascent retroviral genomes by deamination of cytidine residues. Although A3 proteins can restrict the replication of HIV, this inhibition is overcome by the viral infectivity factor (Vif). Inhibitory effects of APOBEC proteins are not limited to HIV but extend to other viruses and endogenous mobile genetic elements that share a reverse transcription process analogous to that of exogenous retroviruses. In sharp contrast, another conundrum of A3 proteins is that they inhibit viral replication even in the absence of CDA activity and recent advances have defined the inhibition of reverse transcriptase (RT) catalyzed DNA elongation reactions by A3 proteins. Together, these proteins provide strong and immediate intracellular immunity against incoming pathogens and restrict the movement of mobile genetic elements protecting the genome.

Cholera toxin, E. coli heat-labile toxin, and non-toxic derivatives induce dendritic cell migration into the follicle-associated epithelium of Peyer's patches

The follicle-associated epithelium (FAE) of Peyer's patches (PPs) transports antigens and microorganisms into mucosal lymphoid tissues where they are captured by subepithelial dendritic cells (DCs). Feeding of cholera toxin (CT) induced migration of subepithelial DCs to interfollicular T-cell areas within 24 h. This study investigated short-term effects of CT, Escherichia coli heat-labile toxin, and non-toxic derivatives on DC migration. CT or CTB injected into ligated intestinal loops induced significant increase in CD11c+ DCs within the FAE within 90 min. In mice fed CT intragastrically, DC numbers in the FAE increased by 1 h, were maximal by 2 h, declined between 8 and 12 h, and were reversed by 24 h. Feeding of native LT, recombinant CTB, dibutyryl cyclic AMP, and to a lesser extent mutated CT(E29H) or mutated LT(R192G) had the same effect. Thus, both A and B subunits of enterotoxins, presumably acting through distinct signaling pathways, may promote capture of incoming antigens and pathogens by PP DCs.

Factor VIII-Specific Memory B-Cells in Patients with Hemophilia A.

The quick anamnestic antibody response seen after recurrent exposure to antigen involves memory B- cells that, helped by T-cells, undergo rigid proliferation and subsequent differentiation into antibody producing cells. The presence of a pool of memory B-cells allows for a rapid response to antigens which quickly eliminates incoming pathogens. In the context of immune responses to therapeutic agents such as blood coagulation factor VIII (FVIII), antigenic re-stimulation of specific memory B-cells is undesirable. In approximately 25% of hemophilia A patients replacement therapy is hampered by inhibitory antibodies that bind to FVIII. Currently, the FVIII-specific memory B-cell compartment in patients with hemophilia A has remained poorly characterized. We have developed a protocol that allows for identification and quantification of circulating memory B-cells in patients with hemophilia A. CD19+ B-cells were sorted on a layer of irradiated EL4B5 thymoma cells expressing CD40L in the presence of the supernatant of mitogen-stimulated T-cells. These experimental conditions, that mimic the interaction of B-cells with an activating helper CD4+ T-cell, induce proliferation of memory B-cells and allow them to differentiate into antibody secreting cells (ASC) in an antigen-independent manner. After 9–10 days of culture, total IgG and FVIII-specific IgG was determined by ELISA and number of ASC was determined by ELISpot. We analyzed blood samples of five multi-transfused patients (>50 FVIII administrations) who never experienced any inhibitor episode, five patients who experienced inhibitory antibodies in the past but were successfully treated with immune tolerance induction and 6 patients with an inhibitor at the time of blood sampling. The ELISA set-up appeared to be more sensitive than ELISpot showing ASC producing anti-FVIII antibodies varying from 0.2–50 ng/ml. In contrast, ELISpot analysis only allowed for detection of B-cell clones producing over ~4 ng/ml of FVIII-specific IgG. Frequencies of FVIII-specific memory B-cells varied from 0–0.027% of total number of circulating peripheral B-cells. The relative amount of circulating memory B-cells did not correspond to inhibitor titers as measured in a Bethesda assay. The highest frequencies were observed in patients suffering from anamnestic response to FVIII suggesting the importance of antigenic stimulation for maintenance of memory B-cell levels. This is further supported by the low frequency that was observed in a high-titer inhibitor patient who had not been treated with FVIII for several months prior to blood sampling. Surprisingly, we detected FVIII-specific memory B-cells in two multi-transfused patients who did not experience any inhibitor episode in the past. These B-cells were present in a low frequency however and developed into ASC producing only limited amounts of anti-FVIII antibodies. These observations suggest that peripheral blood memory B-cells can develop in the absence of clinically relevant inhibitors.

Dendritic Cells and HIV Infection: Activating Dendritic Cells to Boost Immunity

Dendritic cells (DCs) are white blood cells that coordinate innate and adaptive immunity. They are distributed within epithelia and mucosal-associated lymphoid tissues, positioned to entrap incoming pathogens or vaccines. Human immunodeficiency virus (HIV) and the non-human primate equivalent (SIV) exploit DCs to amplify infection, underscoring the need to harness strategies that promote presentation of virus by DCs to stimulate potent anti-viral immunity instead of virus transmission. Two main subsets of DCs need to be considered: myeloid (MDC) and plasmacytoid (PDC) subsets. Using the SIV-macaque system to advance oral vaccine research, we examined macaque PDC and MDC biology, identifying ways to activate DCs and boost antiviral immunity. Immunostimulatory oligodeoxyribonucleotides (ISS-ODNs) stimulated PDC/MDC mixtures to up-regulate co-stimulatory molecule expression and to secrete both IFN-alpha and IL-12. Additionally, ISS-ODNs augmented SIV-specific IFN-gamma responses induced by virus-bearing DCs. ISS-ODN-driven DC activation is being pursued to improve oral/nasopharyngeal mucosal vaccines and therapies against HIV.

High Expression of Antioxidant Proteins in Dendritic Cells

Dendritic cells (DCs) display the unique ability to activate naive T cells and to initiate primary T cell responses revealed in DC-T cell alloreactions. DCs frequently operate under stress conditions. Oxidative stress enhances the production of inflammatory cytokines by DCs. We performed a proteomic analysis to see which major changes occur, at the protein expression level, during DC differentiation and maturation. Comparative two-dimensional gel analysis of the monocyte, immature DC, and mature DC stages was performed. Manganese superoxide dismutase (Mn-SOD) reached 0.7% of the gel-displayed proteins at the mature DC stage. This important amount of Mn-SOD is a primary antioxidant defense system against superoxide radicals, but its product, H(2)O(2), is also deleterious for cells. Peroxiredoxin (Prx) enzymes play an important role in eliminating such peroxide. Prx1 expression level continuously increased during DC differentiation and maturation, whereas Prx6 continuously decreased, and Prx2 peaked at the immature DC stage. As a consequence, DCs were more resistant than monocytes to apoptosis induced by high amounts of oxidized low density lipoproteins containing toxic organic peroxides and hydrogen peroxide. Furthermore DC-stimulated T cells produced high levels of receptor activator of nuclear factor kappaB ligand, a chemotactic and survival factor for monocytes and DCs. This study provides insights into the original ability of DCs to express very high levels of antioxidant enzymes such as Mn-SOD and Prx1, to detoxify oxidized low density lipoproteins, and to induce high levels of receptor activator of nuclear factor kappaB ligand by the T cells they activate and further emphasizes the role that DCs might play in atherosclerosis, a pathology recognized as a chronic inflammatory disorder.

Intracellular immunity to HIV-1: newly defined retroviral battles inside infected cells

Studies of the human immunodeficiency virus type 1 (HIV-1) continue to enrich eukaryotic biology and immunology. Recent advances have defined factors that function after viral entry and prevent the replication of proviruses in the infected cell. Some of these attack directly viral structures whereas others edit viral genetic material during reverse transcription. Together, they provide strong and immediate intracellular immunity against incoming pathogens. These processes also offer a tantalizing glimpse at basic cellular mechanisms that might restrict the movement of mobile genetic elements and protect the genome.

Monocyte-Derived Dendritic Cells: A Promising Armament for Immunotherapy in Human Malignancies

Monocyte-Derived Dendritic Cells: A Promising Armament for Immunotherapy in Human Malignancies

Phagocyte sabotage: disruption of macrophage signalling by bacterial pathogens.

Macrophages function at the front line of immune defences against incoming pathogens. But the ability of macrophages to internalize bacteria, migrate, recruit other immune cells to the site of infection and influence the nature of the immune response can provide unintended benefits for bacterial pathogens that are able to subvert or co-opt these normally effective defences. This review highlights recent advances in our understanding of the many interference strategies that are used by bacterial pathogens to undermine macrophage signalling.

The enigma of dendritic cell-immunodeficiency virus interplay.

A dendritic cell (DC) encountering an immunodeficiency virus should pose a threat to the virus, by efficiently processing and presenting viral antigenic determinants to activate specific anti-viral T and B cell immunity. While this may occur in vivo, it is apparent that DC-entrapped viruses can freely spread between cells, move to distal tissues, and proliferate rapidly particularly upon meeting CD4+ T cells. In fact, the latter is further augmented when the T cells are activated. Thus, it seems that immunodeficiency viruses exploit the unique ability of DCs to survey the periphery and capture incoming pathogens, traffic around the body often targeting the lymphoid tissues, and efficiently communicate with naïve and memory T cells. Combined with the fact that DCs are likely the first leukocytes interacting with virions crossing the mucosae, these features provide the basis on which the virus maximizes its chance to establish infection even in the face of immune activation. How this is actually achieved by the virus is still an enigma. Herein, we intend to summarize what is known about how distinct DC subsets and immunodeficiency viruses interact, what cellular and viral factors influence these events, and how this drives virus replication versus stimulation of protective immunity. Clarifying these issues is necessary to define the exact role of DCs in the transmission and dissemination of HIV infection, to facilitate the development of methods to improve the immune-activating capacity of DCs as well as the design of strategies to prevent DC-driven infection.

Human immature dendritic cells efficiently bind and take up secretory IgA without the induction of maturation.

Immature dendritic cells (DC) reside in peripheral tissues, where they pick up and process incoming pathogens via scavenger receptors or FcR such as FcgammaR and FcepsilonR. At mucosal surfaces, IgA is the main Ig to protect the body from incoming pathogens. In addition, DC are present in high numbers at these sites. We detected expression of FcalphaR (CD89) on the CD14+ population of CD34+ progenitor-derived DC and on monocyte-derived DC (MoDC). However, CD89 expression was strongly decreased upon differentiation from monocyte to DC. We found only minimal binding of serum IgA to MoDC but strong binding of secretory IgA (SIgA). The SIgA binding to MoDC could not be blocked by anti-CD89 blocking Abs. DC efficiently internalized SIgA, but not serum IgA, and uptake of SIgA could be blocked by specific sugars or partially by Ab reactive with mannose receptor. Importantly, binding and uptake of SIgA was not accompanied by signs of DC maturation, such as increased expression of CD86 and CD83 or induction of cytokine secretion. These data indicate that SIgA can interact with DC not via CD89, but via carbohydrate-recognizing receptors like mannose receptor and suggest that uptake of SIgA-containing immune complexes by immature DC may be a mechanism to modulate mucosal immune responses.

Immunohistological study of infiltrated cells and cytokines in murine herpetic keratitis.

To identify localization and kinetics of infiltrated cells and cytokines in murine herpetic keratitis. HSV-1 was inoculated onto the scarified BALB/c corneas. At given times post infection (PI), eyes were removed and studied immunohistochemically using monoclonal antibodies against several infiltrated cells and cytokines. Neutrophils and NK cells infiltrated as early as 1 day PI reaching a maximum number at 2 day PI in initial stage. gamma delta TCR positive cells were observed in the corneal stroma from 1 day PI to 8 day PI. IL-2 and IFN-gamma were positive in the cell-infiltrated areas of the epithelial and stromal lesions, whereas IL-4 was negative throughout the experiment. Our results indicated that cytokine profile upon herpes infection on the cornea is Th1 dominant. Together with neutrophils in the early phase of infection, gamma delta positive T cells may play an additional role in protecting the cornea against incoming pathogens.

Precursors to neonatal lymph nodes: LT beta+CD45+CD4+CD3- cells are found in fetal liver.

Lymph nodes are strategically positioned throughout the body. All lymph, draining from tissues and organs, passes through lymph nodes before entering the bloodstream, which allows for efficient screening of incoming pathogens or tissue aberrations. It is now well established that the highly organized structure of lymph nodes in various compartments is essential for an optimal interaction of lymphocytes and antigen presenting cells to induce immune responses.

Antigen-presenting cells in nasal allergy.

Antigen-presenting cells in nasal allergy.

Dendritic cells at the end of the millennium.

We have recently proposed a dual role for dendritic cells (DC) in the amplification of innate immune responses and in the activation of adaptive immune responses. The DC are localized along the major routes of entry of micro-organisms, where they perform a sentinel function for incoming pathogens. Soon after interaction with appropriate stimuli, DC undergo a coordinated process of maturation and respond to danger signals by re- programming their functions. The DC first regulate leucocyte recruitment at the site of inflammation, through the production of chemokines, inflammatory cytokines and interferons, and then they acquire migratory properties and undergo a rapid switch in chemokine receptor expression. This allows them to leave the inflamed tissue and to reach the lymph node T cell area. During this migration, DC complete their maturation process and acquire the ability to prime T cell responses. Thus, DC bridge innate and adaptive immunity.

Functional studies on dendritic cells in the respiratory tract and related mucosal tissues.

Recent reports indicate that mucosal tissues contain dendritic cell (DC) populations that exhibit phenotypic features distinct from those at more studied sites such as skin. In particular, mucosal DC populations display very rapid baseline turnover rates, which increase in response to local inflammatory stimuli. This property is likely to be central to the role of mucosal DC in surveillance of these front-line tissues for incoming microbial pathogens. As we discuss, it may also indirectly account for the "Th2 default," which is the recognized hallmark of mucosal immune system in the steady state.

Dendritic cells are recruited into the airway epithelium during the inflammatory response to a broad spectrum of stimuli.

A key rate-limiting step in the adaptive immune response at peripheral challenge sites is the transmission of antigen signals to T cells in regional lymph nodes. Recent evidence suggests that specialized dendritic cells (DC) fulfill this surveillance function in the resting state, but their relatively slow turnover in most peripheral tissues brings into question their effectiveness in signaling the arrival of highly pathogenic sources of antigen which require immediate mobilization of the full range of host defenses for maintenance of homeostasis. However, the present report demonstrates that recruitment of a wave of DC into the respiratory tract mucosa is a universal feature of the acute cellular response to local challenge with bacterial, viral, and soluble protein antigens. Consistent with this finding, we also demonstrate that freshly isolated respiratory mucosal DC respond in vitro to a variety of CC chemokines as well as complementary cleavage products and N-formyl-methionyl-leucine-phenylalanine. This suggests that rapid amplification of specific antigen surveillance at peripheral challenge sites is an integral feature of the innate immune response at mucosal surfaces, and serves as an "early warning system" to alert the adaptive immune system to incoming pathogens.