L’activation des réponses immunitaires des plantes repose sur la reconnaissance de motifs moléculaires associés aux pathogènes (aussi appelés PAMP) par des récepteurs de l’immunité, également nommés PRR (pattern recognition receptors). La chitine, principal composant de la paroi des champignons, est un PAMP bien caractérisé qui induit des réponses de défense aussi bien chez les mammifères que chez les plantes.La première partie de cette étude met en évidence que deux chito-oligosaccharides, la chitine et le chitosan, agissent comme des PAMP chez la vigne (Vitis vinifera) puisqu’ils induisent des évènements précoces de signalisation, l’expression de gènes de défense et une résistance contre des agents pathogènes. Ces résultats suggèrent que des systèmes de perception existent chez la vigne. Une analyse phylogénétique a permis d’identifier trois récepteurs kinases à domaine LysM (LysM-RK ou LYK) chez V. vinifera (VvLYK1-1, -2, -3) appartenant au même clade que le récepteur à la chitine chez Arabidopsis et nommé AtCERK1 (Arabidopsis thaliana Chitin Elicitor Receptor Kinase 1). Leur analyse fonctionnelle a été réalisée par complémentation du mutant d’Arabidopsis Atcerk1, affecté dans la perception de la chitine. Nos résultats montrent que VvLYK1-1 et VvLYK1-2, mais pas VvLYK1-3, complémentent fonctionnellement le mutant Atcerk1 en restaurant l’activation des MAPK (Mitogen-Activated Protein Kinases) et l’expression de gènes de défense induits par les chito-oligosaccharides. De plus, l’expression de VvLYK1-1 chez Atcerk1 restaure la résistance basale à l’agent de l’oïdium de la vigne (Erysiphe necator).La seconde partie du projet s’est focalisée sur les éliciteurs oligosaccharidiques de type « damage-associated molecular patterns (DAMP) ». Ces molécules endogènes peuvent provenir de la dégradation de la paroi lors d’une attaque et sont capables d’activer les réponses immunitaires de la plante. Les DAMP les mieux caractérisés actuellement sont les oligogalacturonates (OG), des fragments de pectine qui induisent des réponses immunitaires chez de nombreuses espèces végétales dont l’activation de MAPK, la production d’H2O2, l’expression de gènes de défense et le dépôt de callose. Nous avons montré dans cette étude que les xyloglucanes (Xh), des fragments d’hémicellulose pariétale purifiés, induisaient l’activation de MAPK et l’expression de gènes de défense chez la vigne et Arabidopsis, afin d’induire une résistance contre le champignon nécrotrophe Botrytis cinerea. Les Xh induisent également la production de resvératrol, une phytoalexine majoritaire chez la vigne, et un dépôt de callose chez Arabidopsis. Par une approche génétique, nous avons identifié certains composants de la signalisation induite par les Xh chez Arabidopsis. L’utilisation de mutants suggère que la résistance induite par les Xh contre B. cinerea est dépendante des voies de la camalexine, de l’acide salicylique, de l’acide jasmonique et de l’éthylène chez Arabidopsis. De manière globale, nos résultats mettent en lumière que les xyloglucanes peuvent être considérés comme de nouveaux éliciteurs de l’immunité chez la vigne et Arabidopsis. / Activation of the plant immune responses requires recognition of common pathogen-associated molecular pattern (PAMP) by their cognate pattern recognition receptors (PRR). Chitin, a major component of fungal cell walls, is a well-known PAMP that triggers defense responses in several mammal and plant species.In the first part of this study, we show that two chitooligosaccharides, chitin and chitosan, act as PAMPs in grapevine (Vitis vinifera) as they elicit immune signaling events, defense gene expression, and resistance against pathogens. These two PAMPs are active in grapevine suggesting that at least one perception system exists. Phylogenetic analysis clearly distinguished three V. vinifera LysM Receptor Kinases (VvLYK1-1, -2, -3) located in the same clade as the Arabidopsis Chitin Elicitor Receptor Kinase 1 (AtCERK1), which mediates chitin-induced immune responses. Their functional characterization was achieved by complementation assays in the Atcerk1 mutant, impaired in chitin perception. Our results provide evidence that VvLYK1-1 and VvLYK1-2, but not VvLYK1-3, functionally complement the loss of AtCERK1 function by restoring chitooligosaccharide-induced MAPK activation and immune gene expression. Moreover, expression of VvLYK1-1 in Atcerk1 restored penetration resistance to the non-adapted grapevine powdery mildew (Erysiphe necator).The second part of this study focused on damaged-associated molecular patterns (DAMP), endogenous molecules that can be released from the plant cell wall during an attack and activate the plant innate immunity. Until now, the best characterized DAMPs are oligogalacturonides (OG) coming from pectin fragments that induce innate immune responses in various plant species, including MAPK activation, H2O2 production, defense gene expression and callose deposition. In this study, we showed that purified xyloglucans (Xh), derived from the plant cell wall hemicellulose, elicit MAPK activation and immune gene expression in grapevine (V. vinifera) and Arabidopsis to trigger induced resistance against the necrotrophic fungus Botrytis cinerea. Xh also elicit the production of resveratrol, the main grapevine phytoalexin, and callose deposition in Arabidopsis. Using a genetic approach, we identified some signaling components of Xh-induced immunity. The use of Arabidopsis mutants suggests that Xh-induced resistance against B. cinerea is dependent on the camalexin, salicylate, jasmonate and ethylene pathways. Taken together, our data highlight that Xh can be considered as new elicitors of grapevine and Arabidopsis immunity.

Entre os PRRs (receptores de reconhecimento padrão), NOD-like receptors (NLRs), tal como NOD2, são responsáveis pela detecção intracelular de muramil dipeptídeo (MDP); padrão molecular associado a patógeno (PAMP), encontrado no peptidoglicano (PGN) de praticamente todas bactérias GRAM positiva e negativa. Após o reconhecimento e estimulação por MDP, NOD2 recruta diretamente a serina-treonina quinase RIPK2, uma proteína adaptadora importante na ativação de NF?B mediada por NOD2. A expressão de NOD2 foi descrita em macrófagos e em outras células. Além disso, trabalhos anteriores indicaram que PRRs desempenham papel crucial na ativação de células gliais da medula espinal, na indução e manutenção da dor inflamatória crônica e dor neuropática. No presente estudo, avaliamos o papel de NOD2 na modulação da sensibilidade à dor, focando sua importância na ativação de células da glia da medula espinal, bem como a sua via de sinalização (RIPK2) e liberação de citocinas pró-nociceptivas, como o fator de necrose tumoral alfa (TNF-?), interleucina-6 (IL-6) e interleucina-1 beta (IL-1?). Os resultados demonstram que camundongos selvagens tratados com MDP, apresentaram diminuição no limiar nociceptivo mecânico (pico entre 3 e 5 horas) comparado com o grupo controle (veículo), retornando ao basal após 48 horas. Além disso, camundongos NOD2-/- , RIPK2-/- , TNFR1/2-/- e IL-6 -/- tratados com MDP não diferiram o limiar nociceptivo mecânico, comparado com seus respectivos grupos controle (veículo). Entretanto, camundongos TNFR1- /- , CCR2-/- , TLR4-/- , MyD88-/- e TRIF-/- tratados com MDP, apresentaram diminuição no limiar nociceptivo mecânico similar aos camundongos selvagens tratados com MDP. Adicionalmente, o pré-tratamento de camundongos selvagens com IL-1ra, propentofilina, minociclina, fluorocitrato e SB 203580 inibiu o desenvolvimento da hipersensibilidade mecânica induzida por MDP. Estes dados sugerem que a ativação do sensor intracellular NOD2 esta presente em células da glia da medula espinal e estimula a ativação das vias de sinalização RIPK2 e p38 MAPK com subsequente produção de IL-1?, IL-6 e TNF?, por uma via de sinalização independente de TLR4, MyD88 e TRIF. Finalmente, estes mecanismos contribuem para o processo de hipersensibilidade mecânica durante a neuropatia periférica e representam uma nova abordagem para elucidar os mecanismos envolvidos na fisiopatologia da dor crônica. / Among PRRs (pattern recognition receptors), NOD-like receptors (NLRs), such as NOD2 are responsible by intracellular detection of muramyl dipeptide (MDP); pathogen-associated molecular pattern (PAMP) found in the peptidoglycan (PGN) from virtually all gram positive and gram negative bacteria. Upon recognition and stimulation by MDP, NOD2 recruits directly the receptor-interacting serine/threonine-protein kinase 2 (RIPK2), an adaptor protein important in the NOD2-mediated NF?B activation. The expression of NOD2 has been described in macrophages and other cells. Moreover, previous work has indicated that PRRs play a crucial role in the activation of spinal cord glial cells, in the induction and maintenance of chronic inflammatory and neuropathic pain. In the present study, we aimed to evaluate the role of NOD2 in the modulation of pain sensitivity, focusing on its importance in the activation of spinal cord glial cells, as well as its signaling pathway (RIPK2) and release of pro-nociceptive cytokines, such as tumour necrosis factor-alpha (TNF-?), interleukin-6 (IL-6) and interleukin-1beta (IL-1?). The results demonstrate that WT mice treated with MDP showed a decrease in mechanical nociceptive threshold (peak 3 to 5 hours) compared with the control group (vehicle), returning to the base line after 48 hours. Furthermore, NOD2-/- , RIPK2-/- , TNFR1/2-/- and IL-6 -/- mice treated with MDP did not differ the mechanical nociceptive threshold compared with their respective control groups (vehicle). However, TNFR1-/- , CCR2-/- , TLR4-/- , MyD88-/- and TRIF-/- mice treated MDP, showed a decrease in mechanical nociceptive threshold similar to WT mice treated with MDP. In addition, the pretreatment of WT mice with IL-1ra, propentofylline, minocycline, fluorocitrate and SB 203580 inhibited the development of mechanical hypersensitivity induced by MDP. These data suggest that activation of the intracellular sensor NOD2 present in spinal cord glial cells stimulates the activation of RIPK2 and p38 MAPK signaling pathways and subsequent production of IL-1?, IL-6 and TNF?, in a TLR4-, MyD88- and TRIF-independent signaling pathway. Finally, these mechanisms contribute to the process of mechanical hypersensitivity during peripheral neuropathy and represent a novel approach for elucidating the mechanisms underlying pathophysiology of chronic pain.

Mycobacteria are multifaceted pathogens capable of causing both acute disease as well as an asymptomatic latent infection. Host immune responses during mycobacterial infection involve potent cell effector functions including that of CD4+, CD8+ and γδT cells, macrophages and dendritic cells (DCs). Further, the critical regulators of protective immunity to mycobacterial infection include IFN-γ, IL-12, IL-23, TNF-α, lymphotoxins, CD40, nitric oxide and reactive oxygen species. However, the success of mycobacterial infection often relies in its ability to evade immune surveillance mechanisms mediated by sentinels of host immunity by modulating host signal transduction pathways and expression of immunoregulatory molecules. Therefore, the key to control mycobacterial growth and limit pathogenesis lies in the understanding the interactions between Mycobacterium and primary responders like macrophages and DCs. In this scenario, the role of pattern recognition receptors (PPRs) in orchestrating host immune responses assumes central importance. The cell surface receptors play crucial role in influencing overall immune responses. Of the PRRs, the Toll-like receptors (TLRs) form key immune surveillance mechanisms in recognition as well as control of mycobacterial infection. Among them, TLR2 is the primary interacting receptor on antigen presenting cells that recognize the invading mycobacteria. Mycobacterial cell wall constituents such as LAM, LM, PIM and 19-kDa protein have been shown to activate TLR2 signaling leading to proinflammatory responses. Recent reports have suggested that PE_PGRS antigens of M. tuberculosis interact with TLR2. For example, RV0754, Rv0978c, RV1917c have been implicated in modulation of human DCs. The 19-kDa lipoprotein, LpqH (Rv3763) and LprG (Rv1411c) utilize TLR2 signaling to inhibit macrophage responsiveness to IFN-γ triggered MHC class II expression and mycobacterial antigen presentation. Interestingly, recognition and amplification of pathogenic-specific signaling events play important roles in not only discriminating the invading microbes, but also in regulating explicit immune responses. In this context, integration of key signaling centers, which modulate host immunity to pathogenic mycobacterial infections, remains unexplored. In accordance to above observations, signal transduction pathways downstream to TLRs play a critical role in modulation of battery of host cells genes in terms of expression and production of immune modulatory cytokines and chemokines, recruitment of cellular machineries to site of infections etc. This suggests the decisive role for TLRs in modulation of host cell fate decisions. However, during the ensuing immunity to invading pathogens, beside TLR signaling pathways, various other signaling molecules are thought to execute specific functions in divergent cellular contexts. Recent studies from our laboratory have clearly demarcated a novel cross talk of TLR2-NOTCH1 and TLR2-Wnt signaling pathways during mycobacterial infections. The current study primary focuses on the broad range of cross talk of TLR2 and Sonic hedgehog (SHH) signaling pathways and its functional significance. The present investigation demonstrates that M. bovis BCG, a vaccine strain, triggers a robust activation of SHH signaling in macrophages compared to infection with diverse Gram-positive or Gram-negative microbes. This observation was further evidenced by the heightened SHH signaling signatures during in vivo scenario in cells /tissues from pulmonary tuberculosis (TB) individuals as well as tuberculous meningitis (TBM) patients. Furthermore, we show that the sustained TNF-α secretion by macrophages upon infection with M. bovis BCG is a critical necessity for SHH activation. Significantly, perturbation studies implicate a vital role for M. bovis BCG stimulated TLR2/PI3K/PKC/MAPK/NF-κB axis to induce TNF-α, that contributes to enhance SHH signaling. The TNF-α driven SHH signaling downregulates M. bovis BCG induced TLR2 signaling events leading to modulation of battery of genes that regulate various functions of macrophages genes like Vegf-a, Socs-3, Cox-2, Mmp-9 and M1/M2 genes. Importantly, utilizing whole-genome microRNA (miRNA) profiling, roles for specific miRNAs were identified as the molecular regulators that bring about the negative-feedback loop comprising TLR2-SHH signaling events. Thus, the current study illustrates how SHH signaling tightly regulates the kinetics and strengths of M. bovis BCG specific TLR2 responses, emphasizing a novel role for SHH signaling in host immune responses to mycobacterial infections. As described, variety of host factors contributes for ensuing effective host defenses and modulation of host cell fate decisions. Interestingly, avirulent pathogenic mycobacteria, including the vaccine strain M. bovis BCG, unlike virulent M. tuberculosis, cause extensive apoptosis of infected macrophages, which suggests a significant contribution of the apoptosis process to the initiation and subsequent amplification of innate as well as adaptive immune responses. Among various cues that could lead to apoptosis of host cells, the initiation of the apoptotic machinery by posttranscriptional mechanisms assumes significant importance. Among posttranscriptional control mechanisms, miRNAs are suggested to regulate several biological processes including immune responses. Various effectors of host immunity are known to be regulated by several miRNAs, and a prominent one among them, miRNA-155 (miR-155), often exhibits crucial roles during innate or adaptive immune responses. In this perspective, we identified a novel role of miR-155 during M. bovis BCG induced apoptosis of macrophages. The genetic and signaling perturbations data suggested that miR-155 regulates PKA signaling by directly targeting a negative regulator of PKA, protein kinase inhibitor alpha (PKI-α). Enhanced activation of PKA signaling resulted in induced expression of the apoptotic genes as well as Caspase-3 cleavage and Cytochrome c translocation. Thus, augmented PKA signaling by M. bovis BCG-driven miR-155 dictates cell fate decisions of infected macrophages, emphasizing a novel role for miR-155 in host immunity to mycobacterial infections. In perspective of these studies, important directives are often comprised of sequential and coordinated activation of TLR and NLR-driven signal transduction pathways, thus exhibiting foremost influence in determining the overall strength of the innate immune responses. As described, TLR2 exhibits dominant role in sensing various agonists including pathogen-associated molecular patterns (PAMPs) of microbes at the cell surface and generally considered as major effectuator of proinflammatory responses. Interestingly, NLRs like NOD1 or NOD2 often act in contrary, thus regulating anti-inflammatory responses as well as polarization of T cells towards skewed Th2 phenotype. This presents an interesting conundrum to functionality of DCs or macrophages in terms of effector functions during rapidly evolving immunological processes including effects originating from immunosuppressive effectors such as CTLA-4 or TGF-. DCs like macrophages are important sentinels of innate immunity, possesses array of PRRs that include TLRs and NOD-like receptors (NLRs). Signaling events associated with innate sensors like TLRs and NLRs often act as regulatory circuits that modulate the overall functions of DCs in terms of maturation process, cytokine or chemokine production, receptor expression, migration to secondary lymphoid organs for antigen presentation for effectuating Th polarization. TLR2, while acting as sensors for extracellular cues or endocytic network, drives signaling events in response to recognition of PAMPs including mycobacterial antigens like ESAT-6, PE_PGRS antigens, while NOD1 and NOD2 operate as cytosolic sensors initiating signaling pathways upon recognition of diaminopimelic acid (DAP) and muramyl dipeptide (MDP), components of bacterial peptidoglycan. Thus, TLRs or NOD receptors could trigger similar or contrasting immune responses by cooperative or non-cooperative sensing, consequently exhibiting immense complexity during combinatorial triggering of host DCs-PRR repertoire. In view of these observations, our current investigation comprehensively demonstrated that maturation process of human DCs were cooperatively regulated by signaling cascades initiated by engagements of TLR2, NOD1 and NOD2 receptors. Importantly, combined triggering of TLR2 and NOD receptors abolished the TGF-β or CTLA-4-mediated impairment of human DCs maturation, which required critical participation of NOTCH1-PI3K signaling cohorts. Thus, our data delineated the novel insights in modulation of macrophages and DCs effector functions by mycobacterial TLR2 or NOD agonists and broaden our understanding on the signal dynamics and integration of multiple signals from PRRs during mycobacterial infections. Altogether, our findings establish the understanding of conceptual frame work in fine tuning of TLR2 responses by SHH signaling as well as potential co-operativity among TLRs and NODs to modulate NOTCH1 dependent DCs maturation. Importantly, our study provides mechanistic and functional insights into various molecular regulators of macrophage cell fate decisions like miR-31. miR-150 and miR-155, which can fuel the search for attractive and effective drug targets and novel therapeutics to combat diseases of the hour like tuberculosis.