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Results for “Immunity, Innate”

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PubMed2002

Triggering of innate immune responses by schistosome egg glycolipids and their carbohydrate epitope GalNAc beta 1-4(Fuc alpha 1-2Fuc alpha 1-3)GlcNAc.

To investigate the interactions of glycoconjugates with the innate immune system, peripheral blood mononuclear cells were stimulated with glycolipids derived from Schistosoma mansoni eggs and worms and with biochemically synthesized neoglycoconjugates. Egg glycolipids stimulated the production of interleukin (IL)--10, IL-6, and tumor necrosis factor--alpha in monocytes, whereas worm glycolipids failed to do so. When monoclonal antibodies that specifically recognize defined carbohydrate epitopes were used, the binding of a GalNAc beta 1-4(Fuc alpha 1-2Fuc alpha 1-3)GlcNAc (LDN-DF) reactive antibody was pronounced on egg glycolipids but was absent on worm glycolipids. The binding of antibodies that recognize Gal beta 1-4(Fuc alpha 1-3)GlcNAc (LewisX), GalNAc beta 1-4GlcNAc (LDN), and GalNAc beta 1-4(Fuc alpha 1-3)GlcNAc (LDN-F) was comparable for both preparations. Cytokine production in response to neoglycoconjugates containing enzymatically synthesized glycans also was measured. The LDN-DF neoglycoconjugate was the most potent cytokine inducer, which indicates that this difucosylated glycan can act at the host-parasite interface and can trigger innate immune responses.

Animals↗

PubMed2003

Lipopolysaccharide binding protein is an essential component of the innate immune response to Escherichia coli peritonitis in mice.

Lipopolysaccharide (LPS) binding protein (LBP) is an acute-phase protein that enhances the responsiveness of immune cells to LPS by virtue of its capacity to transfer LPS to CD14. To determine the role of LBP in the innate immune response to peritonitis, LBP gene-deficient (LBP(-/-)) and normal wild-type mice were intraperitoneally infected with Escherichia coli, the most common causative pathogen of this disease. LBP was detected at low concentrations in peritoneal fluid of healthy wild-type mice, and the local LBP levels increased rapidly upon induction of peritonitis. LBP(-/-) mice were highly susceptible to E. coli peritonitis, as indicated by accelerated mortality, earlier bacterial dissemination to the blood, impaired bacterial clearance in the peritoneal cavity, and more severe remote organ damage. LBP(-/-) mice displayed diminished early tumor necrosis factor alpha, interleukin-6, cytokine-induced neutrophil chemoattractant, and macrophage inflammatory protein 2 production and attenuated recruitment of polymorphonuclear leukocytes to the site of infection, indicating that acute inflammation was promoted by LBP. Locally produced LBP is an essential component of an effective innate immune response to E. coli peritonitis.

Acute-Phase Proteins↗

PubMed2004

Innate immunity genes influence the severity of acute appendicitis.

OBJECTIVE: Using acute appendicitis as a model, we tested the hypothesis that polymorphisms in genes involved in host defense can be associated with the severity of local infection-inflammation in humans. SUMMARY BACKGROUND DATA: Innate immunity is the body's front-line system for antimicrobial host defense. Local inflammation is a major innate immune mechanism for containing and destroying microbes, but it may also contribute to tissue injury. METHODS: We studied 134 patients with acute appendicitis treated at an urban hospital. We looked for associations between the severity of appendicitis (uncomplicated vs. perforated or gangrenous), plasma and peritoneal cytokine concentrations, and single nucleotide polymorphisms in genes involved in recognizing bacterial molecules [CD14 (-159 C-->T); TLR4 (896 A-->G)] and in mounting an inflammatory response [IL-6 (-174 G-->C), TNF-alpha (-308 G-->A), IL-1beta (-31 C -->T)]. RESULTS: Ninety-one patients (68%) had uncomplicated appendicitis and 43 (32%) had complicated disease. The SNPs in the CD14, TLR4, IL-1beta, and TNF-alpha genes were not associated with the severity of appendicitis. A strong association was found between C-allele carriage at -174 in the IL-6 gene and decreased risk of complicated disease (adjusted odds ratio = 0.24, 95% CI = 0.07-0.76). Lower plasma and peritoneal fluid IL-6 concentrations in the IL-6 -174 C-carriers than in the GG homozygotes suggest that this polymorphism contributes to decreased IL-6 production in vivo. CONCLUSIONS: Polymorphism in the IL-6 gene was associated with the severity of appendicitis, even after adjustment for duration of symptoms. The risk for developing appendiceal perforation or gangrene may be determined, in part, by variation in the IL-6 gene.

Acute Disease↗

PubMedDate not supplied

Identification and quantification of innate immune system mediators in human breast milk.

Breast-feeding decreases the risk of breast cancer in mothers and infection, allergy, and autoimmunity in infants. The presence of mediators of the innate immune system in human milk, including soluble defensins, cathelicidins, and toll-like receptors (TLRs), has not been researched thoroughly. The whey fractions of colostrum and transitional and mature milk (n = 40) from normal mothers (n = 18) and from mothers with autoimmune or allergic diseases (n = 22) were analyzed for defensins by competitive enzyme-linked immunosorbent assay, and expression of messenger RNA (mRNA) for defensins, TLRs, and cathelicidin-derived antimicrobial peptide (LL-37) by cells in breast milk was determined by semiquantitative reverse-transcription-polymerase chain reaction. In whey, human neutrophil-derived a-defensin 1 (HNP-1) and human beta-defensin 2 (HBD-2) were present in the highest concentrations (median, 33.0 and 31.3 microg/mL, respectively), human alpha-defensin 6 (HD-6) was present in moderate amounts (3.1 microg/mL), and HD-5 and HBD-1 were present in the lowest concentrations (2.4 and 1.7 microg/mL, respectively). There was great variability of defensin levels between subjects, but there was no relation to autoimmune or allergic diagnosis. HNP-1, HD-5, and HD-6 were present in significantly higher levels in colostrum than in mature milk. Regarding defensin mRNA expression in the breast milk cells, 95% of the samples (n = 41) were positive for HBD-1, 68% were positive for HD-5, 22% were positive for HBD-3, 15% were positive for HBD-2, 5% were positive for HBD-4, and 2% were positive for HD-6; 88% (14/16) were positive for HNP-1. Breast milk cells also expressed mRNA for TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR9, CD14, and LL-37. Human breast milk contains high concentrations of multiple defensin proteins and cells in breast milk express mRNA for these defensins, multiple TLRs, and LL-37. The innate immune system in breast milk is complex and likely provides protection for maternal breast tissue and the developing digestive tract of newborns.

Adult↗

PubMed2006

Toll-like receptors TLR2 and TLR4 initiate the innate immune response of the renal tubular epithelium to bacterial products.

Renal tubular epithelial cells (TECs) respond diffusely to local infection, with the release of multiple cytokines, chemokines and other factors that are thought to orchestrate the cellular constituents of the innate immune response. We have investigated whether the Toll-like receptors TLR4 and TLR2, which are present on tubular epithelium and potentially detect a range of bacterial components, co-ordinate this inflammatory response acting through nuclear factor-kappa B (NF-kappaB). Primary cultures of TECs were grown from C57BL/6, C3H/HeN, C3H/HeJ, TLR2 and TLR4 knock-out mice. Cell monolayers were stimulated with lipopolysaccharide (LPS) and synthetic TLR2 and 4 agonists. The innate immune response was quantified by measurement of the cytokines tumour necrosis factor (TNF)-alpha and KC (IL-8 homologue) in cell supernatants by enzyme-linked immunosorbent assay. Cultured TECs grown from healthy mice produced the cytokines TNF-alpha and KC in response to stimulation by LPS and synthetic TLR2 and TLR4 agonists. Cells lacking the respective TLRs had a reduced response to stimulation. The TLR2- and TLR4-mediated response to stimulation was dependent on NF-kappaB signalling, as shown by curcumin pretreatment of TECs. Finally, apical stimulation of these TLRs elicited basal surface secretion of TNF-alpha and KC (as well as the reverse), consistent with the biological response in vivo. Our data highlight the potential importance of TLR-dependent mechanisms co-ordinating the innate immune response to upper urinary tract infection.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Gilthead seabream (Sparus aurata L.) innate immune response after dietary administration of heat-inactivated potential probiotics.

The effects of the dietary administration of two heat-inactivated whole bacteria from the Vibrionaceae family, singly or combined, on innate immune response of the seabream were studied. The two bacteria (Pdp11 and 51M6), which were obtained from the skin of gilthead seabream, showed in vitro characteristics that suggested they could be considered as potential fish probiotics. The fish were fed four different diets: control (non-supplemented), or diets supplemented with heat-inactivated bacteria at 10(8) cfu g(-1) Pdp11, 10(8) cfu g(-1) 51M6 or with 0.5 x 10(8) cfu g(-1) Pdp11 plus 0.5 x 10(8) cfu g(-1) 51M6 for 4 weeks. Six fish were sampled at weeks 1, 2, 3 and 4, when the main humoral (natural haemolytic complement activity and serum peroxidase content) and cellular innate immune responses (leucocyte peroxidase content, phagocytosis, respiratory burst and cytotoxicity) were evaluated. The serum peroxidase content and the natural haemolytic complement activity increased with time, reaching the highest values in the third and fourth weeks of feeding, respectively. The phagocytic ability of specimens fed the mixture of the two inactivated bacteria was significantly higher than in the controls after 2 and 3 weeks of treatment. The same activity increased significantly in seabream fed the Pdp11 diet for 2 weeks or the 51M6 diet for 3 weeks. Respiratory burst activity was unaffected by all the experimental diets at all times assayed. Cytotoxic activity had significantly increased after 3 weeks in fish fed the 51M6 diet. These results are discussed in terms of the usefulness of incorporating inactivated probiotic bacteria into fish diets.

Analysis of Variance↗

PubMed2005

Helicobacter pylori and the innate immune system.

Since its discovery, Helicobacter pylori surprises us by its ability for life-long chronic persistence, proliferation, and probably active adaptation in the unfavourable niche of the human stomach, without being eliminated by the defence systems of the human body. This minireview highlights recent developments about the interaction of H. pylori with the innate immune system, and makes a case that evasion and possibly suppression of innate immune responses play an important role for the active survival in its local mucosal environment.

Defensins↗

PubMed2004

Intact gram-negative Helicobacter pylori, Helicobacter felis, and Helicobacter hepaticus bacteria activate innate immunity via toll-like receptor 2 but not toll-like receptor 4.

Molecular and genetic studies have demonstrated that members of the Toll-like receptor (TLR) family are critical innate immune receptors. TLRs are recognition receptors for a diverse group of microbial ligands including bacteria, fungi, and viruses. This study demonstrates that distinct TLRs are responsible for the recognition of Helicobacter lipopolysaccharide (LPS) versus intact Helicobacter bacteria. We show that the cytokine-inducing activity of Helicobacter LPS was mediated by TLR4; i.e., TLR4-deficient macrophages were unresponsive to Helicobacter pylori LPS. Surprisingly, the cytokine response to whole Helicobacter bacteria (H. pylori, H. hepaticus, and H. felis) was mediated not by TLR4 but rather by TLR2. Studies of HEK293 transfectants revealed that expression of human TLR2 was sufficient to confer responsiveness to intact Helicobacter bacteria, but TLR4 transfection was not sufficient. Our studies further suggest that cag pathogenicity island genes may modulate the TLR2 agonist activity of H. pylori as cagA+ bacteria were more active on a per-cell basis compared to cagA mutant bacteria for interleukin-8 (IL-8) cytokine secretion. Consistent with the transfection studies, analysis of knockout mice demonstrated that TLR2 was required for the cytokine response to intact Helicobacter bacteria. Macrophages from both wild-type and TLR4-deficient mice produced a robust cytokine secretion response (IL-6 and MCP-1) when stimulated with intact Helicobacter bacteria. In contrast, macrophages from TLR2-deficient mice were profoundly unresponsive to intact Helicobacter stimulation, failing to secrete cytokines even at high (100:1) bacterium-to-macrophage ratios. Our studies suggest that TLR2 may be the dominant innate immune receptor for recognition of gastrointestinal Helicobacter species.

Animals↗

PubMed2006

Innate immunity of the bovine mammary gland.

Understanding the immune defenses of the mammary gland is instrumental in devising and developing measures to control mastitis, the major illness of dairy ruminants. Innate immunity is an extremely broad field for investigation, and despite decades of research, our present knowledge of the innate defenses of the udder is incomplete. Yet, information is being gained on the recognition of pathogens by the mammary gland, and on several locally inducible defenses. The contribution of mammary epithelial cells to local defenses and to the mobilization of leucocytes is under growing scrutiny. Interactions of mastitis-causing bacteria such as Escherichia coli or Staphylococcus aureus and the mammary gland represents a suitable model for studies on innate immunity at an epithelium frontier. Powerful new research tools are radically modifying the prospects for the understanding of the interplay between the mammary gland innate defenses and mastitis-causing bacteria: genetic dissection of the immune response, microarray gene technology, transcriptomic methodologies and gene silencing by RNA interference will make possible the discovery of several of the key defense mechanisms which govern the susceptibility/resistance to mastitis at the molecular and genetic levels. It should then be possible to enhance the resistance of dairy ruminants to mastitis through immunomodulation and genetic improvement.

Animals↗

PubMed2005

Yersinia outer protein P suppresses TGF-beta-activated kinase-1 activity to impair innate immune signaling in Yersinia enterocolitica-infected cells.

Pathogenic Yersinia spp. use a panel of virulence proteins that antagonize signal transduction processes in infected cells to undermine host defense mechanisms. One of these proteins, Yersinia enterocolitica outer protein P (YopP), down-regulates the NF-kappaB and MAPK signaling pathways, which suppresses the proinflammatory host immune response. In this study, we explored the mechanism by which YopP succeeds to simultaneously disrupt several of these key signaling pathways of innate immunity. Our data show that YopP operates upstream of its characterized eukaryotic binding partner IkappaB kinase-beta to shut down the NF-kappaB signaling cascade. Accordingly, YopP efficiently impaired the activities of TGF-beta-activated kinase-1 (TAK1) in infected cells. TAK1 is an important activator of the IkappaB kinase complex in the TLR signaling cascade. The repression of TAK1 activities correlated with reduced activation of NF-kappaB- as well as AP-1-dependent reporter gene expression in Yersinia-infected murine macrophages. This suggests that the impairment of the TAK1 enzymatic activities by Yersinia critically contributes to down-regulate activation of NF-kappaB and of MAPK members in infected host cells. The inhibition of TAK1 potentially results from the blockade of signaling events that control TAK1 induction. This process could involve the attenuation of ubiquitination of the upstream signal transmitter TNFR-associated factor-6. Together, these results indicate that, by silencing the TAK1 signaling complex, Yersinia counteracts the induction of several conserved signaling pathways of innate immunity, which aids the bacterium in subverting the host immune response.

Animals↗

PubMed2006

The innate immune modulators staphylococcal complement inhibitor and chemotaxis inhibitory protein of Staphylococcus aureus are located on beta-hemolysin-converting bacteriophages.

Two newly discovered immune modulators, chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) and staphylococcal complement inhibitor (SCIN), cluster on the conserved 3' end of beta-hemolysin (hlb)-converting bacteriophages (betaC-phis). Since these betaC-phis also carry the genes for the immune evasion molecules staphylokinase (sak) and enterotoxin A (sea), this 8-kb region at the 3' end of betaC-phi represents an innate immune evasion cluster (IEC). By PCR and Southern analyses of 85 clinical Staphylococcus aureus strains and 5 classical laboratory strains, we show that 90% of S. aureus strains carry a betaC-phi with an IEC. Seven IEC variants were discovered, carrying different combinations of chp, sak, or sea (or sep), always in the same 5'-to-3' orientation and on the 3' end of a betaC-phi. From most IEC variants we could isolate active bacteriophages by mitomycin C treatment, of which lysogens were generated in S. aureus R5 (broad phage host). All IEC-carrying bacteriophages integrated into hlb, as was measured by Southern blotting of R5 lysogens. Large quantities of the different bacteriophages were obtained by mitomycin C treatment of the lysogens, and bacteriophages were collected and used to reinfect all lysogenic R5 strains. In total, five lytic families were found. Furthermore, phage DNA was isolated and digested with EcoR1, revealing that one IEC variant can be found on different betaI-phis. In conclusion, the four human-specific innate immune modulators SCIN, CHIPS, SAK, and SEA form an IEC that is easily transferred among S. aureus strains by a diverse group of beta-hemolysin-converting bacteriophages.

3' Flanking Region↗

PubMed2006

Neutrophil elastase, an innate immunity effector molecule, represses flagellin transcription in Pseudomonas aeruginosa.

Recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors triggers an innate immune response to colonizing or invading bacteria. Conversely, many bacteria have evolved mechanisms to dampen this response by downregulating the synthesis of such PAMPs. We have previously demonstrated that Pseudomonas aeruginosa growing in mucopurulent human respiratory mucus from cystic fibrosis patients represses the expression of its flagellin, a potent stimulant of the innate immune response. Here we demonstrate that this phenomenon occurs in response to the presence of neutrophil elastase in such mucus. Nonpurulent mucus from animals had no such repressive effect. Furthermore, lysed neutrophils from human blood reproduced the flagellin-repressive effect ex mucus and, significantly, had no effect on the viability of this organism. Neutrophil elastase, a component of the innate host defense system, has been described to be bactericidal for gram-negative bacteria and to degrade bacterial virulence factors. Thus, the resistance of P. aeruginosa to the bactericidal effect of neutrophil elastase, as well as this organism's ability to sense this enzyme's presence and downregulate the synthesis of a PAMP, may be the key factors in allowing P. aeruginosa to colonize the lungs. These findings demonstrate the dynamic nature of this bacterium's response to host defenses that ensures its success as a colonizer and also highlights the dual nature of defense molecules that confer advantages and disadvantages to both hosts and pathogens.

Cell Extracts↗

PubMed2006

GM-CSF and the impaired pulmonary innate immune response following hyperoxic stress.

We have previously demonstrated that mice exposed to sublethal hyperoxia (an atmosphere of >95% oxygen for 4 days, followed by return to room air) have significantly impaired pulmonary innate immune response. Alveolar macrophages (AM) from hyperoxia-exposed mice exhibit significantly diminished antimicrobial activity and markedly reduced production of inflammatory cytokines in response to stimulation with LPS compared with AM from control mice in normoxia. As a consequence of these defects, mice exposed to sublethal hyperoxia are more susceptible to lethal pneumonia with Klebsiella pneumoniae than control mice. Granulocyte/macrophage colony-stimulating factor (GM-CSF) is a growth factor produced by normal pulmonary alveolar epithelial cells that is critically involved in maintenance of normal AM function. We now report that sublethal hyperoxia in vivo leads to greatly reduced alveolar epithelial cell GM-CSF expression. Systemic treatment of mice with recombinant murine GM-CSF during hyperoxia exposure preserved AM function, as indicated by cell surface Toll-like receptor 4 expression and by inflammatory cytokine secretion following stimulation with LPS ex vivo. Treatment of hyperoxic mice with GM-CSF significantly reduced lung bacterial burden following intratracheal inoculation with K. pneumoniae, returning lung bacterial colony-forming units to the level of normoxic controls. These data point to a critical role for continuous GM-CSF activity in the lung in maintenance of normal AM function and demonstrate that lung injury due to hyperoxic stress results in significant impairment in pulmonary innate immunity through suppression of alveolar epithelial cell GM-CSF expression.

Animals↗

PubMed2001

Role of antigen-presenting cells in innate immune system.

Activation of antigen-presenting cells (APC) and natural killer (NK) cells initiates the production of various proinflammatory cytokines including interleukin 12 (IL-12), interferon gamma (IFN-gamma) and nitric oxide (NO), which are important in the innate immune response for controlling infection by intracellular pathogens. In this review, we focus on these cytokines produced by APC and summarize the current understanding of how APC functions are regulated by cytokines in innate immunity.

Animals↗

PubMed2003

Intestinal myofibroblasts in innate immune responses of the intestine.

BACKGROUND & AIMS: Intestinal myofibroblasts are known to respond to inflammatory signals and may play a role in Crohn's disease-associated fibrosis. However, putative involvement by myofibroblasts in innate immune responses as part of intestinal host defense has not been characterized. We therefore analyzed expression and regulation of toll-like receptors (TLRs) in colonic human myofibroblasts (CCD-18) and primary human colonic myofibroblasts in comparison with human lung myofibroblasts (CCD-37). METHODS: Expression of TLRs (1-10) and NOD 1 and 2 was assessed before and after stimulation with either lipopolysaccharide (LPS) or lipoteichoic acid (LTA) by using a custom microarray, reverse-transcription polymerase chain reaction, Northern blot and Western blot analysis, and immunohistochemistry. Activation of signaling pathways, translocation of p65, and secretion of interleukin (IL)-8 were determined. RESULTS: Messenger RNAs encoding for TLR1-9, as well as NOD1 and NOD2, were amplified from cultured and primary human intestinal myofibroblasts. After stimulation with LPS or LTA, a 1.5-4.2-fold up-regulation of TLRs (2, 3, 4, 6, 7) and elements of the signaling cascade (MyD88, TIR domain-containing adapter protein [TIRAP]) was observed. CCD-18 and CCD-37 cells expressed TLR 2 and 4 protein, which were located primarily on the cell membrane. Stimulation with LTA or LPS resulted in activation of the mitogen-activated protein kinases pathway, nuclear translocation of p65, and significantly increased IL-8 secretion. CONCLUSIONS: Bacterial components directly activate intestinal myofibroblasts expressing TLRs. These cells may therefore participate in innate immune responses by sensing and responding to bacterial products that have penetrated into the subepithelial compartment.

Cells, Cultured↗

PubMed2000

A protective role for innate immunity in autoimmune disease.

Systemic lupus erythematosus is an incurable autoimmune disease characterized by autoantibodies directed against highly conserved nuclear proteins and DNA. While the cause of lupus is not known, B-lymphocytes are essential for disease. Most striking is that deficiency in components of the innate immune system such as complement is a major risk factor in disease. One explanation for the involvement of complement and innate immunity is that they are important in the induction and maintenance of B cell tolerance to lupus antigens.

Animals↗

PubMed2024

TMPRSS2-mediated SARS-CoV-2 uptake boosts innate immune activation, enhances cytopathology, and drives convergent virus evolution.

The accessory protease transmembrane protease serine 2 (TMPRSS2) enhances severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uptake into ACE2-expressing cells, although how increased entry impacts downstream viral and host processes remains unclear. To investigate this in more detail, we performed infection assays in engineered cells promoting ACE2-mediated entry with and without TMPRSS2 coexpression. Electron microscopy and inhibitor experiments indicated TMPRSS2-mediated cell entry was associated with increased virion internalization into endosomes, and partially dependent upon clathrin-mediated endocytosis. TMPRSS2 increased panvariant uptake efficiency and enhanced early rates of virus replication, transcription, and secretion, with variant-specific profiles observed. On the host side, transcriptional profiling confirmed the magnitude of infection-induced antiviral and proinflammatory responses were linked to uptake efficiency, with TMPRSS2-assisted entry boosting early antiviral responses. In addition, TMPRSS2-enhanced infections increased rates of cytopathology, apoptosis, and necrosis and modulated virus secretion kinetics in a variant-specific manner. On the virus side, convergent signatures of cell-uptake-dependent innate immune induction were recorded in viral genomes, manifesting as switches in dominant coupled Nsp3 residues whose frequencies were correlated to the magnitude of the cellular response to infection. Experimentally, we demonstrated that selected Nsp3 mutations conferred enhanced interferon antagonism. More broadly, we show that TMPRSS2 orthologues from evolutionarily diverse mammals facilitate panvariant enhancement of cell uptake. In summary, our study uncovers previously unreported associations, linking cell entry efficiency to innate immune activation kinetics, cell death rates, virus secretion dynamics, and convergent selection of viral mutations. These data expand our understanding of TMPRSS2's role in the SARS-CoV-2 life cycle and confirm its broader significance in zoonotic reservoirs and animal models.

SARS-CoV-2↗

PubMed2004

Dynamic changes in the localization of MAPK cascade components controlling pathogenesis-related (PR) gene expression during innate immunity in parsley.

The activation of mitogen-activated protein kinase (MAPK) cascades is an important mechanism for stress adaptation through the control of gene expression in mammals, yeast, and plants. MAPK activation has emerged as a common mechanism by which plants trigger pathogen defense responses following innate immune recognition of potential microbial pathogens. We are studying the non-host plant defense response of parsley to attempted infection by Phytophthora species using an experimental system of cultured parsley cells and the Phytophthora-derived Pep-13 peptide elicitor. Following receptor-mediated recognition of this peptide, parsley cells trigger a multifaceted innate immune response, involving the activation of three MAPKs that have been shown to function in the oxidative burst-independent activation of defense gene expression. Using this same experimental model we now report the identification of a MAPK kinase (MAPKK) that functions upstream in this pathway. This kinase, referred to as PcMKK5 based on sequence similarity to Arabidopsis thaliana AtMKK5, is activated in parsley cells following Pep-13 treatment and functions as an in vivo activator of all three MAPKs previously shown to be involved in this response. Gain- and loss-of-function mutant versions of PcMKK5, when used in protoplast co-transfection assays, demonstrated that kinase activity of PcMKK5 is required for PR gene promoter activation following Pep-13 treatment. Furthermore, using specific antibodies and immunofluorescent labeling, we demonstrate that activation of MAPKs in parsley cells correlates with an increase in their nuclear localization, which is not detectable for activated PcMKK5. These results suggest that activation of gene expression through MAPK cascades during innate immune responses in plants involves dynamic changes in the localization of the proteins involved, which may reflect the distribution of key protein substrates for the activated MAPKs.

Arabidopsis↗