Shaista Naqvi

PGE 2 Induces Macrophage IL-10 Production and a Regulatory-like Phenotype via a Protein Kinase A-SIK-CRTC3 Pathway

The polarization of macrophages into a regulatory-like phenotype and the production of IL-10 plays an important role in the resolution of inflammation. We show in this study that PGE2, in combination with LPS, is able to promote an anti-inflammatory phenotype in macrophages characterized by high expression of IL-10 and the regulatory markers SPHK1 and LIGHT via a protein kinase A–dependent pathway. Both TLR agonists and PGE2 promote the phosphorylation of the transcription factor CREB on Ser133. However, although CREB regulates IL-10 transcription, the mutation of Ser133 to Ala in the endogenous CREB gene did not prevent the ability of PGE2 to promote IL-10 transcription. Instead, we demonstrate that protein kinase A regulates the phosphorylation of salt-inducible kinase 2 on Ser343, inhibiting its ability to phosphorylate CREB-regulated transcription coactivator 3 in cells. This in turn allows CREB-regulated transcription coactivator 3 to translocate to the nucleus where it serves as a coactivator with the transcription factor CREB to induce IL-10 transcription. In line with this, we find that either genetic or pharmacological inhibition of salt-inducible kinases mimics the effect of PGE2 on IL-10 production.

Dectin-1 Regulates IL-10 Production via a MSK1/2 and CREB Dependent Pathway and Promotes the Induction of Regulatory Macrophage Markers

In response to infection by fungal pathogens, the innate immune system recognises specific fungal pathogen associated molecular patterns (PAMPs) via pattern recognition receptors including the C-type lectin dectin-1 and members of the Toll Like Receptor (TLR) family. Stimulation of these receptors leads to the induction of both pro- and anti-inflammatory cytokines. The protein kinases MSK1 and 2 are known to be important in limiting inflammatory cytokine production by macrophages in response to the TLR4 agonist LPS. In this study we show that MSKs are also activated in macrophages by the fungal derived ligand zymosan, as well as the dectin-1 specific agonists curdlan and depleted zymosan, via the ERK1/2 and p38α MAPK pathways. Furthermore, we show that MSKs regulate dectin-1 induced IL-10 production, and that this regulation is dependent on the ability of MSKs to phosphorylate the transcription factor CREB. IL-10 secreted in response to zymosan was able to promote STAT3 phosphorylation via an autocrine feedback loop. Consistent with the decreased IL-10 secretion in MSK1/2 knockout macrophages, these cells also had decreased STAT3 tyrosine phosphorylation relative to wild type controls after stimulation with zymosan. We further show that the reduction in IL-10 production in the MSK1/2 macrophages results in increased secretion of IL-12p40 in response to zymosan relative to wild type controls. The production of high levels of IL-10 but low levels of IL-12 has previously been associated with an M2b or ‘regulatory’ macrophage phenotype, which was initially described in macrophages stimulated with a combination of immune complexes and LPS. We found that zymosan, via dectin-1 activation, also leads to the expression of SphK1 and LIGHT, markers of a regulatory like phenotype in mouse macrophages. The expression of these makers was further reinforced by the high level of IL-10 secreted in response to zymosan stimulation.

CREB phosphorylation at Ser133 regulates transcription via distinct mechanisms downstream of cAMP and MAPK signalling

CREB (cAMP-response-element-binding protein) is an important transcription factor for the activation of a number of immediate early genes. CREB is phosphorylated on Ser133 by PKA (protein kinase A), promoting the recruitment of the co-activator proteins CBP (CREB-binding protein) and p300; this has been proposed to increase the transcription of CREB-dependent genes. CREB is also phosphorylated on Ser133 by MSK1/2 (mitogen- and stress-activated kinase 1/2) in cells in response to the activation of MAPK (mitogen-activated protein kinase) signalling; however, the relevance of this to gene transcription has been controversial. To resolve this problem, we created a mouse with a Ser133 to alanine residue mutation in the endogenous Creb gene. Unlike the total CREB knockout, which is perinatally lethal, these mice were viable, but born at less than the expected Mendelian frequency on a C57Bl/6 background. Using embryonic fibroblasts from the S133A-knockin mice we show in the present study that Ser133 phosphorylation downstream of PKA is required for CBP/p300 recruitment. The requirement of Ser133 phosphorylation for the PKA-mediated induction of CREB-dependent genes was, however, promoter-specific. Furthermore, we show that in cells the phosphorylation of CREB on Ser133 by MSKs does not promote strong recruitment of CBP or p300. Despite this, MSK-mediated CREB phosphorylation is critical for the induction of CREB-dependent genes downstream of MAPK signalling.

A Phytophthora infestans RXLR effector targets plant PP1c isoforms that promote late blight disease

Plant pathogens deliver effectors to alter host processes. Knowledge of how effectors target and manipulate host proteins is critical to understand crop disease. Here, we show that in planta expression of the RXLR effector Pi04314 enhances leaf colonization by Phytophthora infestans via activity in the host nucleus and attenuates induction of jasmonic and salicylic acid-responsive genes. Pi04314 interacts with three host protein phosphatase 1 catalytic (PP1c) isoforms, causing their re-localization from the nucleolus to the nucleoplasm. Re-localization of PP1c-1 also occurs during infection and is dependent on an R/KVxF motif in the effector. Silencing the PP1c isoforms or overexpression of a phosphatase-dead PP1c-1 mutant attenuates infection, demonstrating that host PP1c activity is required for disease. Moreover, expression of PP1c–1mut abolishes enhanced leaf colonization mediated by in planta Pi04314 expression. We argue that PP1c isoforms are susceptibility factors forming holoenzymes with Pi04314 to promote late blight disease.

MSK1 and MSK2 inhibit lipopolysaccharide-induced prostaglandin production via an interleukin-10 feedback loop.

ABSTRACT Prostaglandin production is catalyzed by cyclooxygenase 2 (cox-2). We demonstrate here that MSK1 and MSK2 (MSK1/2) can exert control on the induction of cox-2 mRNA by Toll-like receptor (TLR) agonists. In the initial phase of cox-2 induction, MSK1/2 knockout macrophages confirmed a role for MSK in the positive regulation of transcription. However, at later time points both lipopolysaccharide (LPS)-induced prostaglandin and cox-2 protein levels were increased in MSK1/2 knockout. Further analysis found that while MSKs promoted cox-2 mRNA transcription, following longer LPS stimulation MSKs also promoted degradation of cox-2 mRNA. This was found to be the result of an interleukin 10 (IL-10) feedback mechanism, with endogenously produced IL-10 promoting cox-2 degradation. The ability of IL-10 to do this was dependent on the mRNA binding protein TTP through a p38/MK2-mediated mechanism. As MSKs regulate IL-10 production in response to LPS, MSK1/2 knockout results in reduced IL-10 secretion and therefore reduced feedback from IL-10 on cox-2 mRNA stability. Following LPS stimulation, this increased mRNA stability correlated to an elevated induction of both of cox-2 protein and prostaglandin secretion in MSK1/2 knockout macrophages relative to that in wild-type cells. This was not restricted to isolated macrophages, as a similar effect of MSK1/2 knockout was seen on plasma prostaglandin E2 (PGE2) levels following intraperitoneal injection of LPS.

Potato NPH3/RPT2-Like Protein StNRL1, Targeted by a Phytophthora infestans RXLR Effector, Is a Susceptibility Factor

An effector from the potato late blight pathogen targets a host protein that negatively regulates immunity. Plant pathogens deliver effectors to manipulate host processes. We know little about how fungal and oomycete effectors target host proteins to promote susceptibility, yet such knowledge is vital to understand crop disease. We show that either transient expression in Nicotiana benthamiana, or stable transgenic expression in potato (Solanum tuberosum), of the Phytophthora infestans RXLR effector Pi02860 enhances leaf colonization by the pathogen. Expression of Pi02860 also attenuates cell death triggered by the P. infestans microbe-associated molecular pattern INF1, indicating that the effector suppresses pattern-triggered immunity. However, the effector does not attenuate cell death triggered by Cf4/Avr4 coexpression, showing that it does not suppress all cell death activated by cell surface receptors. Pi02860 interacts in yeast two-hybrid assays with potato NPH3/RPT2-LIKE1 (NRL1), a predicted CULLIN3-associated ubiquitin E3 ligase. Interaction of Pi02860 in planta was confirmed by coimmunoprecipitation and bimolecular fluorescence complementation assays. Virus-induced gene silencing of NRL1 in N. benthamiana resulted in reduced P. infestans colonization and accelerated INF1-mediated cell death, indicating that this host protein acts as a negative regulator of immunity. Moreover, whereas NRL1 virus-induced gene silencing had no effect on the ability of the P. infestans effector Avr3a to suppress INF1-mediated cell death, such suppression by Pi02860 was significantly attenuated, indicating that this activity of Pi02860 is mediated by NRL1. Transient overexpression of NRL1 resulted in the suppression of INF1-mediated cell death and enhanced P. infestans leaf colonization, demonstrating that NRL1 acts as a susceptibility factor to promote late blight disease.

RXLR Effector AVR2 Up-Regulates a Brassinosteroid-Responsive bHLH Transcription Factor to Suppress Immunity

PiAVR2 exploits cross talk between BR signaling and innate immunity in Solanum species, via NbCHL1, representing a novel, indirect mode of innate immune suppression by a filamentous pathogen effector. An emerging area in plant research focuses on antagonism between regulatory systems governing growth and immunity. Such cross talk represents a point of vulnerability for pathogens to exploit. AVR2, an RXLR effector secreted by the potato blight pathogen Phytophthora infestans, interacts with potato BSL1, a putative phosphatase implicated in growth-promoting brassinosteroid (BR) hormone signaling. Transgenic potato (Solanum tuberosum) plants expressing the effector exhibit transcriptional and phenotypic hallmarks of overactive BR signaling and show enhanced susceptibility to P. infestans. Microarray analysis was used to identify a set of BR-responsive marker genes in potato, all of which are constitutively expressed to BR-induced levels in AVR2 transgenic lines. One of these genes was a bHLH transcription factor, designated StCHL1, homologous to AtCIB1 and AtHBI1, which are known to facilitate antagonism between BR and immune responses. Transient expression of either AVR2 or CHL1 enhanced leaf colonization by P. infestans and compromised immune cell death activated by perception of the elicitin Infestin1 (INF1). Knockdown of CHL1 transcript using Virus-Induced Gene Silencing (VIGS) reduced colonization of P. infestans on Nicotiana benthamiana. Moreover, the ability of AVR2 to suppress INF1-triggered cell death was attenuated in NbCHL1-silenced plants, indicating that NbCHL1 was important for this effector activity. Thus, AVR2 exploits cross talk between BR signaling and innate immunity in Solanum species, representing a novel, indirect mode of innate immune suppression by a filamentous pathogen effector.

Oomycetes Seek Help from the Plant：Phytophthora infestans Effectors Target Host Susceptibility Factors

Plants have a sophisticated immune system to defend against a wide range of invaders, including insects, nematodes, bacteria, oomycetes, fungi, and viruses. Microbes may manipulate or suppress immunity by delivering effector proteins, either to the inside or outside of plant cells. Much attention has been focused on identifying the targets of effector proteins in the host and on characterizing how effector activities suppress immunity. The best studied effector proteins are bacterial type III effectors (T3Es), many of which target positive regulators of immunity in order to inhibit their activity (Deslandes and Rivas, 2012).

Transgenic Multivitamin Biofortified Corn: Science, Regulation, and Politics

Micronutrient deficiency is a major global challenge because at any one time up to 50 % of the world’s population may suffer from diseases caused by a chronic insufficient supply of vitamins and minerals, and this largely reflects the lack of access to a diverse diet [1]. In developed countries, micronutrient deficiency is addressed by encouraging the consumption of fresh fruits and vegetables, along with supplementation and fortification programs to enhance the nutritional value of staple foods [2]. In contrast, the populations of developing countries typically subsist on a monotonous diet of milled cereal grains such as rice or maize, which are poor sources of vitamins and minerals. Strategies that have been proposed to overcome micronutrient deficiencies in developing countries include supplementation, fortification, and the implementation of conventional breeding and genetic engineering programs to generate nutrient-rich varieties of staple crops. Unfortunately, the first two strategies have been largely unsuccessful because of the insufficient funding, poor governance, and dysfunctional distribution network in developing country settings [3]. Biofortification programs based on conventional breeding have enjoyed only marginal success because of the limited available genetic diversity and the time required to develop crops with enhanced nutritional properties as well as desirable agronomic characteristics. It is also impossible to conceive of a conventional breeding strategy that would ever produce “nutritionally complete” cereals [2]. More promising results have been obtained by engineering the metabolic pathways leading to provitamin A, vitamin B9, and vitamin C (β-carotene, folate, and ascorbate) in the same transgenic corn line [4]. Genetic engineering therefore has immense potential to improve the nutritional properties of staple crops and contribute to better health, although a number of technical, economical, regulatory, and sociopolitical constraints remain to be addressed.

Beta Interferon Production Is Regulated by p38 Mitogen-Activated Protein Kinase in Macrophages via both MSK1/2- and Tristetraprolin-Dependent Pathways

ABSTRACT Autocrine or paracrine signaling by beta interferon (IFN-β) is essential for many of the responses of macrophages to pathogen-associated molecular patterns. This feedback loop contributes to pathological responses to infectious agents and is therefore tightly regulated. We demonstrate here that macrophage expression of IFN-β is negatively regulated by mitogen- and stress-activated kinases 1 and 2 (MSK1/2). Lipopolysaccharide (LPS)-induced expression of IFN-β was elevated in both MSK1/2 knockout mice and macrophages. Although MSK1 and -2 promote the expression of the anti-inflammatory cytokine interleukin 10, it did not strongly contribute to the ability of MSKs to regulate IFN-β expression. Instead, MSK1 and -2 inhibit IFN-β expression via the induction of dual-specificity phosphatase 1 (DUSP1), which dephosphorylates and inactivates the mitogen-activated protein kinases p38 and Jun N-terminal protein kinase (JNK). Prolonged LPS-induced activation of p38 and JNK, phosphorylation of downstream transcription factors, and overexpression of IFN-β mRNA and protein were similar in MSK1/2 and DUSP1 knockout macrophages. Two distinct mechanisms were implicated in the overexpression of IFN-β: first, JNK-mediated activation of c-jun, which binds to the IFN-β promoter, and second, p38-mediated inactivation of the mRNA-destabilizing factor tristetraprolin, which we show is able to target the IFN-β mRNA.