Dissection of transcriptional events in graft incompatible reactions of "Bearss" lemon (Citrus limon) and "Valencia" sweet orange (C. sinensis) on a novel citrandarin (C. reticulata × Poncirus trifoliata) rootstock.

Citrus is commercially propagated via grafting, which ensures trees have consistent fruit traits combined with favorable traits from the rootstock such as soil adaptability, vigor, and resistance to soil pathogens. Graft incompatibility can occur when the scion and rootstock are not able to form a permanent, healthy union. Understanding and preventing graft incompatibility is of great importance in the breeding of new fruit cultivars and in the choice of scion and rootstock by growers. The rootstock US-1283, a citrandarin generated from a cross of "Ninkat" mandarin (Citrus reticulata) and "Gotha Road" #6 trifoliate orange (Poncirus trifoliata), was released after years of field evaluation because of its superior productivity and good fruit quality on "Hamlin" sweet orange (C. sinensis) under Florida's growing conditions. Subsequently, it was observed that trees of "Bearss" lemon (C. limon) and "Valencia" sweet orange (C. sinensis) grafted onto US-1283 exhibited unhealthy growth near the graft union. The incompatibility manifested as stem grooving and necrosis underneath the bark on the rootstock side of the graft. Another citrandarin rootstock, US-812 (C. reticulata "Sunki" × P. trifoliata "Benecke"), is fully graft compatible with the same scions. Transcriptome analysis was performed on the vascular tissues above and below the graft union of US-812 and US-1283 graft combinations with "Bearss" and "Valencia" to identify expression networks associated with incompatibility and help understand the processes and potential causes of incompatibility. Transcriptional reprogramming was stronger in the incompatible rootstock than in the grafted scions. Differentially expressed genes (DEGs) in US-1283, but not the scions, were associated with oxidative stress and plant defense, among others, similar to a pathogen-induced immune response localized to the rootstock; however, no pathogen infection was detected. Therefore, it is hypothesized that this response could have been triggered by signaling miscommunications between rootstock and scion either through (1) unknown molecules from the scion that were perceived as danger signals by the rootstock, (2) missing signals from the scion or missing receptors in the rootstock necessary for the formation of a healthy graft union, (3) the overall perception of the scion by the rootstock as non-self, or (4) a combination of the above.

Interactions between rootstocks and compost influence the active rhizosphere bacterial communities in citrus.

BACKGROUND: While the rootstock genotype (belowground part of a plant) can impact rhizosphere microbial communities, few studies have examined the relationships between rootstock genotype-based recruitment of active rhizosphere bacterial communities and the availability of root nutrients for plant uptake. Rootstocks are developed to provide resistance to disease or tolerance of abiotic stresses, and compost application is a common practice to also control biotic and abiotic stresses in crops. In this field study, we examined: (i) the effect of four citrus rootstocks and/or compost application on the abundance, diversity, composition, and predicted functionality of active rhizosphere bacterial communities, and (ii) the relationships between active rhizosphere bacterial communities and root nutrient concentrations, with identification of bacterial taxa significantly correlated with changes in root nutrients in the rhizosphere. RESULTS: The rootstock genotype determined differences in the diversity of active rhizosphere bacterial communities and also impacted how compost altered the abundance, diversity, composition, and predicted functions of these active communities. Variations in the active bacterial rhizobiome were strongly linked to root nutrient cycling, and these interactions were root-nutrient- and rootstock-specific. Direct positive relationships between enriched taxa in treated soils and specific root nutrients were detected, and potentially important taxa for root nutrient uptake were identified. Significant differences in specific predicted functions were related to soil nutrient cycling (carbon, nitrogen, and tryptophan metabolisms) in the active bacterial rhizobiome among rootstocks, particularly in soils treated with compost. CONCLUSIONS: This study illustrates that interactions between citrus rootstocks and compost can influence active rhizosphere bacterial communities, which impact root nutrient concentrations. In particular, the response of the rhizobiome bacterial abundance, diversity, and community composition to compost was determined by the rootstock. Specific bacterial taxa therefore appear to be driving changes in root nutrient concentrations in the active rhizobiome of different citrus rootstocks. Several potential functions of active bacterial rhizobiomes recruited by different citrus rootstocks did not appear to be redundant but rather rootstock-specific. Together, these findings have important agronomic implications as they indicate the potential for agricultural production systems to maximize benefits from rhizobiomes through the choice of selected rootstocks and the application of compost. Video Abstract.

Trunk Injection of Oxytetracycline for Huanglongbing Management in Mature Grapefruit and Sweet Orange Trees.

Huanglongbing (HLB) is a devastating bacterial disease associated with 'Candidatus Liberibacter asiaticus'. The location of the pathogen within the vasculature of the tree has left growers with limited options for the effective management of the disease. Trunk injection is a crop protection technique that applies therapeutics directly into the xylem of woody tree species and allows for their systemic uptake and transport, which may provide more effective management of vascular diseases such as HLB. In this study, mature 'Valencia' and 'Hamlin' sweet orange (Citrus sinensis) and 'Duncan' grapefruit (C. paradisi) trees were injected with oxytetracycline (OTC) in the spring and/or fall to evaluate the effects of injection timing and response to injection. In addition to seasonal evaluations of tree health and bacterial titer, preharvest fruit drop, yield, and fruit quality were measured at harvest to determine the effects of OTC injection. The benefits associated with injection included a reduction in fruit drop, an increase in fruit yield and fruit size, and improvements in juice quality. However, results varied due to the timing of injection and were not consistent across all three varieties. Residue analysis at different time points after injection suggests that trunk injection effectively delivers therapeutics to mature citrus trees. This study provides fundamental information on the short-term benefits associated with trunk injection of OTC for HLB management in citrus groves. The potential for use of trunk injection at the commercial scale and the possible risks are discussed.

SuperSour: A New Strategy for Breeding Superior Citrus Rootstocks.

Citrus crops have a long history of cultivation as grafted trees on selected rootstock cultivars, but all current rootstocks have significant limitations and traditional methods of rootstock breeding take at least 2-3 decades to develop and field test new rootstocks. Citrus production in the United States, and other parts of the world, is impaired by a wide range of biotic and abiotic problems, with especially severe damage caused by the disease huanglongbing (HLB) associated with Candidatus Liberibacter asiaticus. All major commercial citrus scion cultivars are damaged by HLB, but tree tolerance is significantly improved by some rootstocks. To overcome these challenges, the USDA citrus breeding program has implemented a multi-pronged strategy for rootstock breeding that expands the diversity of germplasm utilized in rootstock breeding, significantly increases the number of new hybrids evaluated concurrently, and greatly reduces the time from cross to potential cultivar release. We describe the key components and methodologies of this new strategy, termed "SuperSour," along with reference to the historical favorite rootstock sour orange (Citrus aurantium), and previous methods employed in citrus rootstock breeding. Rootstock propagation by cuttings and tissue culture is one key to the new strategy, and by avoiding the need for nucellar seeds, eliminates the 6- to 15-year delay in testing while waiting for new hybrids to fruit. In addition, avoiding selection of parents and progeny based on nucellar polyembryony vastly expands the potential genepool for use in rootstock improvement. Fifteen new field trials with more than 350 new hybrid rootstocks have been established under the SuperSour strategy in the last 8 years. Detailed multi-year performance data from the trials will be used to identify superior rootstocks for commercial release, and to map important traits and develop molecular markers for the next generation of rootstock development. Results from two of these multi-year replicated field trials with sweet orange scion are presented to illustrate performance of 97 new hybrid rootstocks relative to four commercial rootstocks. Through the first 7 years in the field with endemic HLB, many of the new SuperSour hybrid rootstocks exhibit greatly superior fruit yield, yield efficiency, canopy health, and fruit quality, as compared with the standard rootstocks included in the trials.

Identification of citrus immune regulators involved in defence against Huanglongbing using a new functional screening system.

Huanglongbing (HLB) is the most devastating citrus disease in the world. Almost all commercial citrus varieties are susceptible to the causal bacterium, Candidatus Liberibacter asiaticus (CLas), which is transmitted by the Asian citrus psyllid (ACP). Currently, there are no effective management strategies to control HLB. HLB-tolerant traits have been reported in some citrus relatives and citrus hybrids, which offer a direct pathway for discovering natural defence regulators to combat HLB. Through comparative analysis of small RNA profiles and target gene expression between an HLB-tolerant citrus hybrid (Poncirus trifoliata × Citrus reticulata) and a susceptible citrus variety, we identified a panel of candidate defence regulators for HLB-tolerance. These regulators display similar expression patterns in another HLB-tolerant citrus relative, with a distinct genetic and geographic background, the Sydney hybrid (Microcitrus virgata). Because the functional validation of candidate regulators in tree crops is always challenging, we developed a novel rapid functional screening method, using a C. Liberibacter solanacearum (CLso)/potato psyllid/Nicotiana benthamiana interaction system to mimic the natural transmission and infection circuit of the HLB complex. When combined with efficient virus-induced gene silencing in N. benthamiana, this innovative and cost-effective screening method allows for rapid identification and functional characterization of regulators involved in plant immune responses against HLB, such as the positive regulator BRCA1-Associated Protein, and the negative regulator Vascular Associated Death Protein.

Impact of Soil Microbial Amendments on Tomato Rhizosphere Microbiome and Plant Growth in Field Soil.

There is increased interest by the agricultural industry in microbial amendments that leverage natural beneficial interactions between plants and soil microbes to improve crop production. However, translating fundamental knowledge from laboratory experiments into efficient field application often has mixed results, and there is less clarity about the interaction between added microbes and the native microbial community, where microorganisms belonging to the same phylogenic clades often reside. In this study, four commercially available microbial amendments were examined in two greenhouse experiments using field soil to assess their impact on tomato plant growth and the native soil microbial communities. The amendments contained different formulations of plant growth-promoting bacteria (Lactobacilli, Rhizobia, etc.), yeasts, and mycorrhizal fungi. The application of the tested amendments in greenhouse conditions resulted in no significant impact on plant growth. A deeper statistical analysis detected variations in the microbial communities that accounted only for 0.25% of the total species, particularly in native taxa not related to the inoculated species and represented less than 1% of the total variance. This suggests that under commercial field conditions, additional confounding variables may play a role in the efficacy of soil microbial amendments. This study confirms the necessity of more in-depth validation requirements for the formulations of soil microbial amendments before delivery to the agricultural market in order to leverage their benefits for the producers, the consumers, and the environment.

Tracing Penicillin Movement in Citrus Plants Using Fluorescence-Labeled Penicillin.

Huánglóngbìng (HLB), citrus greening, is one of the most destructive diseases of citrus plants worldwide. In North America, HLB is caused by the phloem-limited bacterium Candidatus Liberibacter asiaticus and is transmitted by the Asian citrus psyllid, Diaphorina citri. No cure exists at present, and the use of antibiotics for the control of HLB has gained interest due to the significant losses to the citrus industry. Because of unsatisfactory results when using foliar applications of antibiotics, concerns were raised regarding the uptake and translocation of these materials within trees. We, therefore, investigated a method that allows us to study the movement of antibiotic materials in citrus plants. Herein, we utilized a fluorescence-labeled penicillin, BOCILLINTM FL-Penicillin (FL-penicillin), to study the uptake and translocation of penicillin in citrus plants. FL-penicillin was applied by puncture to the stem of young citrus seedlings and was traced by using fluorescence microscopy. After application, we detected FL-penicillin in the leaves and in the stem xylem and phloem tissues above and below the application site in both intact and partially bark-girdled citrus seedlings, indicating that it is easily taken up and transported through the plant vascular system. In addition, we detected FL-penicillin in the gut of D. citri, which were allowed to feed on the treated plants, suggesting translocation of this molecule into the vascular tissue. We propose that the use of fluorescent-labeled molecules could be an effective tool for understanding the uptake and translocation of antibiotics and other macromolecules in plants and insects.

Cooperative and distinct functions of MK2 and MK3 in the regulation of the macrophage transcriptional response to lipopolysaccharide.

The p38MAPK downstream targets MAPKAP kinases (MK) 2 and 3 are critical for the regulation of the macrophage response to LPS. The extents to which these two kinases act cooperatively and distinctly in regulating LPS-induced inflammatory cytokine expression are still unclear. To address this uncertainty, whole transcriptome analyses were performed using bone marrow-derived macrophages (BMDM) generated from MK2-/- or MK2/3-/- animals and their wild-type littermates. The results suggest that in BMDM, MK2 and MK3 not only cooperatively regulate the transcript expression of signaling intermediates, including IL-10, IL-19, CXCL2 and the IL-4 receptor (IL-4R)α subunit, they also exert distinct regulatory effects on the expression of specific transcripts. Based on the differential regulation of gene expression by MK2 and MK3, at least six regulatory patterns were identified. Importantly, we confirmed our previous finding, which showed that in the absence of MK2, MK3 negatively regulates IFN-ß. Moreover, this genome-wide analysis identified the regulation of Cr1A, NOD1 and Serpina3f as similar to that of IFN-ß. In the absence of MK2, MK3 also delayed the nuclear translocation of NFκB by delaying the ubiquitination and subsequent degradation of IκBß, reflecting the substantial plasticity of the response of BMDM to LPS.

Non-invasive Imaging and Modeling of Liver Regeneration After Partial Hepatectomy.

The liver has a unique regenerative capability upon injury or partial resection. The regeneration process comprises a complex interplay between parenchymal and non-parenchymal cells and is tightly regulated at different scales. Thus, we investigated liver regeneration using multi-scale methods by combining non-invasive imaging with immunohistochemical analyses. In this context, non-invasive imaging can provide quantitative data of processes involved in liver regeneration at organ and body scale. We quantitatively measured liver volume recovery after 70% partial hepatectomy (PHx) by micro computed tomography (µCT) and investigated changes in the density of CD68+ macrophages by fluorescence-mediated tomography (FMT) combined with µCT using a newly developed near-infrared fluorescent probe. In addition, angiogenesis and tissue-resident macrophages were analyzed by immunohistochemistry. Based on the results, a model describing liver regeneration and the interactions between different cell types was established. In vivo analysis of liver volume regeneration over 21 days after PHx by µCT imaging demonstrated that the liver volume rapidly increased after PHx reaching a maximum at day 14 and normalizing until day 21. An increase in CD68+ macrophage density in the liver was detected from day 4 to day 8 by combined FMT-µCT imaging, followed by a decline towards control levels between day 14 and day 21. Immunohistochemistry revealed the highest angiogenic activity at day 4 after PHx that continuously declined thereafter, whereas the density of tissue-resident CD169+ macrophages was not altered. The simulated time courses for volume recovery, angiogenesis and macrophage density reflect the experimental data describing liver regeneration after PHx at organ and tissue scale. In this context, our study highlights the importance of non-invasive imaging for acquiring quantitative organ scale data that enable modeling of liver regeneration.

IL-1ß and TNFα Differentially Influence NF-κB Activity and FasL-Induced Apoptosis in Primary Murine Hepatocytes During LPS-Induced Inflammation.

Macrophage-derived cytokines largely influence the behavior of hepatocytes during an inflammatory response. We previously reported that both TNFα and IL-1ß, which are released by macrophages upon LPS stimulation, affect Fas ligand (FasL)-induced apoptotic signaling. Whereas TNFα preincubation leads to elevated levels of caspase-3 activity and cell death, pretreatment with IL-1ß induces increased caspase-3 activity but keeps cells alive. We now report that IL-1ß and TNFα differentially influence NF-κB activity resulting in a differential upregulation of target genes, which may contribute to the distinct effects on cell viability. A reduced NF-κB activation model was established to further investigate the molecular mechanisms which determine the distinct cell fate decisions after IL-1ß and TNFα stimulation. To study this aspect in a more physiological setting, we used supernatants from LPS-stimulated bone marrow-derived macrophages (BMDMs). The treatment of hepatocytes with the BMDM supernatant, which contains both IL-1ß and TNFα, sensitized to FasL-induced caspase-3 activation and cell death. However, when TNFα action was blocked by neutralizing antibodies, cell viability after stimulation with the BMDM supernatant and FasL increased as compared to single FasL stimulation. This indicates the important role of TNFα in the sensitization of apoptosis in hepatocytes. These results give first insights into the complex interplay between macrophages and hepatocytes which may influence life/death decisions of hepatocytes during an inflammatory reaction of the liver in response to a bacterial infection.

Taurine transporter (TauT) deficiency impairs ammonia detoxification in mouse liver.

Hepatic ammonia handling was analyzed in taurine transporter (TauT) KO mice. Surprisingly, hyperammonemia was present at an age of 3 and 12 months despite normal tissue integrity. This was accompanied by cerebral RNA oxidation. As shown in liver perfusion experiments, glutamine production from ammonia was diminished in TauT KO mice, whereas urea production was not affected. In livers from 3-month-old TauT KO mice protein expression and activity of glutamine synthetase (GS) were unaffected, whereas the ammonia-transporting RhBG protein was down-regulated by about 50%. Double reciprocal plot analysis of glutamine synthesis versus perivenous ammonia concentration revealed that TauT KO had no effect on the capacity of glutamine formation in 3-month-old mice, but doubled the ammonia concentration required for half-maximal glutamine synthesis. Since hepatic RhBG expression is restricted to GS-expressing hepatocytes, the findings suggest that an impaired ammonia transport into these cells impairs glutamine synthesis. In livers from 12-, but not 3-month-old TauT KO mice, RhBG expression was not affected, surrogate markers for oxidative stress were strongly up-regulated, and GS activity was decreased by 40% due to an inactivating tyrosine nitration. This was also reflected by kinetic analyses in perfused liver, which showed a decreased glutamine synthesizing capacity by 43% and a largely unaffected ammonia concentration dependence. It is concluded that TauT deficiency triggers hyperammonemia through impaired hepatic glutamine synthesis due to an impaired ammonia transport via RhBG at 3 months and a tyrosine nitration-dependent inactivation of GS in 12-month-old TauT KO mice.

HCV modifies EGF signalling and upregulates production of CXCR2 ligands: Role in inflammation and antiviral immune response.

BACKGROUND & AIMS: To affect immune response and inflammation, the hepatitis C virus (HCV) substantially influences intercellular communication pathways that are decisive for immune cell recruitment. The present study investigates mechanisms by which HCV modulates chemokine-mediated intercellular communication from infected cells. METHODS: Chemokine expression was studied in HCVcc-infected cell lines or cell lines harbouring a subgenomic replicon, as well as in serum samples from patients. Expression or activity of mediators and signalling intermediates was manipulated using knockdown approaches or specific inhibitors. RESULTS: HCV enhances expression of CXCR2 ligands in its host cell via the induction of epidermal growth factor (EGF) production. Knockdown of EGF or of the p65 subunit of the NF-κB complex results in a substantial downregulation of HCV-induced CXCR2 ligand expression, supporting the involvement of an EGF-dependent mechanism as well as activation of NF-κB. Furthermore, HCV upregulates expression of CXCR2 ligands in response to EGF stimulation via downregulation of the T-cell protein tyrosine phosphatase (TC-PTP [PTPN2]), activation of NF-κB, and enhancement of EGF-inducible signal transduction via MEK1 (MAP2K1). This results in the production of a cytokine/chemokine pattern by the HCV-infected cell that can recruit neutrophils but not monocytes. CONCLUSIONS: These data reveal a novel EGF-dependent mechanism by which HCV influences chemokine-mediated intercellular communication. We propose that this mechanism contributes to modulation of the HCV-induced inflammation and the antiviral immune response. LAY SUMMARY: In most cases hepatitis C virus (HCV) results in chronic infection and persistent viral replication, taking decades until development of overt disease. To achieve such a course, the respective virus must have developed mechanisms to circumvent antiviral response, to modulate the inflammatory response and to utilise the infrastructure of its host with moderate effect on its viability. The present study provides novel data indicating that HCV induces epidermal growth factor production in its host cell, enhancing epidermal growth factor-inducible expression of chemokines that bind to the CXCR2 receptor and recruit neutrophile granulocytes. Importantly, chemokines are critical mediators determining the pattern of immune cells recruited to the site of injury and thereby the local inflammatory and immunological milieu. These data strongly suggest that HCV triggers mechanisms that enable the virus to influence the inflammatory and immunological processes of its host.

Genome resequencing and transcriptome profiling reveal structural diversity and expression patterns of constitutive disease resistance genes in Huanglongbing-tolerant Poncirus trifoliata and its hybrids.

Huanglongbing (HLB) is the most destructive bacterial disease of citrus worldwide. While most citrus varieties are susceptible to HLB, Poncirus trifoliata, a close relative of Citrus, and some of its hybrids with Citrus are tolerant to HLB. No specific HLB tolerance genes have been identified in P. trifoliata but recent studies have shown that constitutive disease resistance (CDR) genes were expressed at much higher levels in HLB-tolerant Poncirus hybrids and the expression of CDR genes was modulated by Candidatus Liberibacter asiaticus (CLas), the pathogen of HLB. The current study was undertaken to mine and characterize the CDR gene family in Citrus and Poncirus and to understand its association with HLB tolerance in Poncirus. We identified 17 CDR genes in two citrus genomes, deduced their structures, and investigated their phylogenetic relationships. We revealed that the expansion of the CDR family in Citrus seems to be due to segmental and tandem duplication events. Through genome resequencing and transcriptome sequencing, we identified eight CDR genes in the Poncirus genome (PtCDR1-PtCDR8). The number of SNPs was the highest in PtCDR2 and the lowest in PtCDR7. Most of the deletion and insertion events were observed in the UTR regions of Citrus and Poncirus CDR genes. PtCDR2 and PtCDR8 were in abundance in the leaf transcriptomes of two HLB-tolerant Poncirus genotypes and were also upregulated in HLB-tolerant, Poncirus hybrids as revealed by real-time PCR analysis. These two CDR genes seem to be good candidate genes for future studies of their role in citrus-CLas interactions.

Model Based Targeting of IL-6-Induced Inflammatory Responses in Cultured Primary Hepatocytes to Improve Application of the JAK Inhibitor Ruxolitinib.

IL-6 is a central mediator of the immediate induction of hepatic acute phase proteins (APP) in the liver during infection and after injury, but increased IL-6 activity has been associated with multiple pathological conditions. In hepatocytes, IL-6 activates JAK1-STAT3 signaling that induces the negative feedback regulator SOCS3 and expression of APPs. While different inhibitors of IL-6-induced JAK1-STAT3-signaling have been developed, understanding their precise impact on signaling dynamics requires a systems biology approach. Here we present a mathematical model of IL-6-induced JAK1-STAT3 signaling that quantitatively links physiological IL-6 concentrations to the dynamics of IL-6-induced signal transduction and expression of target genes in hepatocytes. The mathematical model consists of coupled ordinary differential equations (ODE) and the model parameters were estimated by a maximum likelihood approach, whereas identifiability of the dynamic model parameters was ensured by the Profile Likelihood. Using model simulations coupled with experimental validation we could optimize the long-term impact of the JAK-inhibitor Ruxolitinib, a therapeutic compound that is quickly metabolized. Model-predicted doses and timing of treatments helps to improve the reduction of inflammatory APP gene expression in primary mouse hepatocytes close to levels observed during regenerative conditions. The concept of improved efficacy of the inhibitor through multiple treatments at optimized time intervals was confirmed in primary human hepatocytes. Thus, combining quantitative data generation with mathematical modeling suggests that repetitive treatment with Ruxolitinib is required to effectively target excessive inflammatory responses without exceeding doses recommended by the clinical guidelines.

Comparative transcriptome analysis during early fruit development between three seedy citrus genotypes and their seedless mutants.

Identification of genes with differential transcript abundance (GDTA) in seedless mutants may enhance understanding of seedless citrus development. Transcriptome analysis was conducted at three time points during early fruit development (Phase 1) of three seedy citrus genotypes: Fallglo (Bower citrus hybrid (Citrus reticulata×C. reticulata×C. paradisi)×Temple (C. reticulata×C. sinensis)), grapefruit (C. paradisi), Pineapple sweet orange (C. sinensis), and their seedless mutants. Seed abortion in seedless mutants was observed at 26 days post anthesis (Time point 2). Affymetrix transcriptomic analysis revealed 359 to 1077 probe sets with differential transcript abundance in the comparison of seedless versus seedy fruits for each citrus genotypes and time points. The GDTA identified by 18 microarray probe sets were validated by qPCR. Hierarchical clustering analysis revealed a range of GDTA associated with development, hormone and protein metabolism, all of which may reflect genes associated with seedless fruit development. There were 14, 9 and 12 genes found exhibiting similar abundance ratios in all three seedless versus seedy genotype comparisons at time point 1, 2 and 3, respectively. Among those genes were genes coding for an aspartic protease and a cysteine protease, which may play important roles in seedless fruit development. New insights into seedless citrus fruit development may contribute to biotech approaches to create seedless cultivars.

IL-1ß-induced and p38MAPK-dependent activation of the mitogen-activated protein kinase-activated protein kinase 2 (MK2) in hepatocytes: Signal transduction with robust and concentration-independent signal amplification.

The IL-1ß induced activation of the p38MAPK/MAPK-activated protein kinase 2 (MK2) pathway in hepatocytes is important for control of the acute phase response and regulation of liver regeneration. Many aspects of the regulatory relevance of this pathway have been investigated in immune cells in the context of inflammation. However, very little is known about concentration-dependent activation kinetics and signal propagation in hepatocytes and the role of MK2. We established a mathematical model for IL-1ß-induced activation of the p38MAPK/MK2 pathway in hepatocytes that was calibrated to quantitative data on time- and IL-1ß concentration-dependent phosphorylation of p38MAPK and MK2 in primary mouse hepatocytes. This analysis showed that, in hepatocytes, signal transduction from IL-1ß via p38MAPK to MK2 is characterized by strong signal amplification. Quantification of p38MAPK and MK2 revealed that, in hepatocytes, at maximum, 11.3% of p38MAPK molecules and 36.5% of MK2 molecules are activated in response to IL-1ß. The mathematical model was experimentally validated by employing phosphatase inhibitors and the p38MAPK inhibitor SB203580. Model simulations predicted an IC50 of 1-1.2 µm for SB203580 in hepatocytes. In silico analyses and experimental validation demonstrated that the kinase activity of p38MAPK determines signal amplitude, whereas phosphatase activity affects both signal amplitude and duration. p38MAPK and MK2 concentrations and responsiveness toward IL-1ß were quantitatively compared between hepatocytes and macrophages. In macrophages, the absolute p38MAPK and MK2 concentration was significantly higher. Finally, in line with experimental observations, the mathematical model predicted a significantly higher half-maximal effective concentration for IL-1ß-induced pathway activation in macrophages compared with hepatocytes, underscoring the importance of cell type-specific differences in pathway regulation.

Quantitative and integrative analysis of paracrine hepatocyte activation by nonparenchymal cells upon lipopolysaccharide induction.

Gut-derived bacterial lipopolysaccharides (LPS) stimulate the secretion of tumour necrosis factor (TNF) from liver macrophages (MCs), liver sinusoidal endothelial cells (LSECs) and hepatic stellate cells (HSCs), which control the acute phase response in hepatocytes through activation of the NF-κB pathway. The individual and cooperative impact of nonparenchymal cells on this clinically relevant response has not been analysed in detail due to technical limitations. To gain an integrative view on this complex inter- and intracellular communication, we combined a multiscale mathematical model with quantitative, time-resolved experimental data of different primary murine liver cell types. We established a computational model for TNF-induced NF-κB signalling in hepatocytes, accurately describing dose-responsiveness for physiologically relevant cytokine concentrations. TNF secretion profiles were quantitatively measured for all nonparenchymal cell types upon LPS stimulation. This novel approach allowed the analysis of individual and collective paracrine TNF-mediated NF-κB induction in hepatocytes, revealing strongest effects of MCs and LSECs on hepatocellular NF-κB signalling. Simulations suggest that both cell types act together to maximize the NF-κB pathway response induced by low LPS concentrations (0.1 and 1 ng/mL). Higher LPS concentrations (≥ 5 ng/mL) induced sufficient TNF levels from MCs or LSECs to induce a strong and nonadjustable pathway response. Importantly, these simulations also revealed that the initial cytokine secretion (1-2 h after stimulation) rather than final TNF level (10 h after stimulation) defines the hepatocellular NF-κB response. This raises the question whether the current experimental standard of single high-dose cytokine administration is suitable to mimic in vivo cytokine exposure. DATABASE: The computational models described in this manuscript are available in the JWS database via the following link: https://jjj.bio.vu.nl/database/beuke.

Ammonia Attenuates LPS-Induced Upregulation of Pro-Inflammatory Cytokine mRNA in Co-Cultured Astrocytes and Microglia.

Hepatic encephalopathy (HE) is associated with cerebral microglia activation. Ammonia, a major toxin of HE, activates microglia in vitro but does not trigger pro-inflammatory cytokine synthesis. In the present study we analysed effects of ammonia on lipopolysaccharide (LPS)-induced upregulation of microglia activation and cytokine mRNA as well as on cytokine secretion in mono-cultured microglia and co-cultured astrocytes and microglia. In mono-cultured microglia LPS (100 ng/ml, 18 h) strongly elevated mRNA levels of the microglia activation marker CD14 and the pro-inflammatory cytokines IL-1α/ß, IL-6 and TNF-α. NH4Cl (5 mmol/l) had no effect on LPS-induced upregulation of CD14, IL-1α/ß and IL-6 mRNA but enhanced LPS-induced upregulation of TNF-α mRNA in mono-cultured microglia. In co-cultured astrocytes and microglia, however, LPS-induced upregulation of IL-1α/ß, TNF-α, IL-6, CD14 but not of IL-10, IL-12A/B or TGFß1-3 mRNA was attenuated by NH4Cl. LPS-induced upregulation of IL-1α/ß, IL-6 and TNF-α was also diminished by the TGR5-ligands allopregnanolone and taurolithocholic acid in mono-cultured microglia. NH4Cl also attenuated LPS-induced release of MCP-1, IL-6 and IL-10 in mono-cultured microglia. mRNA level of surrogate marker for microglia activation (CD14) and for the anti-inflammatory M2-type microglia (CD163, CXCL1, CXCL2) were also elevated in post mortem brain tissue taken from the fusiforme gyrus of patients with liver cirrhosis and HE. The findings suggest that ammonia attenuates LPS-induced microglia reactivity in an astrocyte-dependent way. One may speculate that these anti-inflammatory effects of ammonia may be triggered by neurosteroids derived from astrocytes and may account for absence of microglia reactivity in cerebral cortex of cirrhotic patients with HE.

Model-Based Characterization of Inflammatory Gene Expression Patterns of Activated Macrophages.

Macrophages are cells with remarkable plasticity. They integrate signals from their microenvironment leading to context-dependent polarization into classically (M1) or alternatively (M2) activated macrophages, representing two extremes of a broad spectrum of divergent phenotypes. Thereby, macrophages deliver protective and pro-regenerative signals towards injured tissue but, depending on the eliciting damage, may also be responsible for the generation and aggravation of tissue injury. Although incompletely understood, there is emerging evidence that macrophage polarization is critical for these antagonistic roles. To identify activation-specific expression patterns of chemokines and cytokines that may confer these distinct effects a systems biology approach was applied. A comprehensive literature-based Boolean model was developed to describe the M1 (LPS-activated) and M2 (IL-4/13-activated) polarization types. The model was validated using high-throughput transcript expression data from murine bone marrow derived macrophages. By dynamic modeling of gene expression, the chronology of pathway activation and autocrine signaling was estimated. Our results provide a deepened understanding of the physiological balance leading to M1/M2 activation, indicating the relevance of co-regulatory signals at the level of Akt1 or Akt2 that may be important for directing macrophage polarization.

Metabolic variations in different citrus rootstock cultivars associated with different responses to Huanglongbing.

Huanglongbing (HLB) is one of the most destructive bacterial diseases of citrus. No resistant cultivars have been identified, although tolerance has been observed in the genus Poncirus and some of its hybrids with Citrus that are commonly used as rootstocks. In this study we exploited this tolerance by comparing five different tolerant hybrids with a cultivar that shows pronounced HLB sensitivity to discern potential contributing metabolic factors. Whole leaves of infected and non-infected greenhouse-grown seedlings were extracted and subjected to untargeted GC-TOF MS based metabolomics. After BinBase data filtering, 342 (experiment 1) and 650 (experiment 2) unique metabolites were quantified, of which 122 and 195, respectively, were assigned by chemical structures. The number of metabolites found to be differently regulated in the infected state compared with the non-infected state varied between the cultivars and was largest (166) in the susceptible cultivar Cleopatra mandarin (Citrus reticulata) and lowest (3) in the tolerant cultivars US-897 (C. reticulata 'Cleopatra' × Poncirus trifoliata) and US-942 (C. reticulata 'Sunki' × P. trifoliata) from experiment 2. Tolerance to HLB did not appear to be associated with accumulation of higher amounts of protective metabolites in response to infection. Many metabolites were found in higher concentrations in the tolerant cultivars compared with susceptible Cleopatra mandarin and may play important roles in conferring tolerance to HLB. Lower availability of specific sugars necessary for survival of the pathogen may also be a contributing factor in the decreased disease severity observed for these cultivars.