The critical role of JNK and p38 MAPKs for TLR4 induced microgliamediated

vailable online at www.pelagiaresearchlibrary.com European Journal of Experimental iology, 15, 5(8):3- ISSN: 8 915 OEN (US): EJEU The critical role of JNK and p38 MPKs for TLR induced microgliamediated neurotoxicity Sagar Gaikwad, ivyesh Patel, Naveen. R. and Reena grawal-rajput Laboratory of Immunology, epartment of Human Health and iseases, School of iological Sciences and iotechnology, Indian Institute of dvanced Research, Gandhinagar, Gujarat, India STRT Identifying signal transduction pathways and understanding their role in microglia-mediated neuroinflammation and neurotoxicity may provide clinical benefits in neurodegenerative diseases. Microglia activation and inflammation is the first line of defense mechanism by the host to remove pathogen/injurious stimuli and initiate the tissue healing process. Inflammation should be tightly regulated. However, dysregulation leads to bystander tissue damage in most neurodegenerative diseases. Evidence suggests that activated microglia excessively secretes chronic neurotoxic factors, including TNF-α, IL1-β, NO and reactive oxygen species (ROS), driving progressive neurotoxicity. The present study provides the molecular mechanisms involved in TLR mediated excessive inflammatory responses by microglia. We used stimulated V murine microglia cells, as an in vitro model system to examine TLR induced microglia-mediated chronic neuroinflammation. Here we demonstrate that β- amyloid and enhanced TLR expression on microglia. stimulation significantly enhanced inflammatory cytokines by microglia in time and dose dependent manner at mrn and protein levels. Subsequently, phosphorylation of PI3 kinase was observed but it is partially involved in inflammation regulation. In addition, TLR activation markedly induces phosphorylation of JNK 1/, p38 and ERK1/ MPKs. Our study revealed that JNK 1/ and p38 MPK are crucial players during TLR mediated inflammation and neurotoxicity. In addition, combined inhibition of p38 and JNK synergistically increases neuroprotection against β induced neurotoxicity. In conclusion, our findings suggest that TLR activation by and β-amyloid significantly increases inflammation while p38 MPK and JNK 1/ inhibition provides neuroprotection through negative regulation of NF-κ. Thus p38 MPK and JNK 1/ are good therapeutic targets in inflammatory neurodegenerative diseases like lzheimer s disease. Key words: Microglia, neuroinflammation, TLR, lzheimer s disease, JNK 1/, p38 MPKs. INTROUTION tight control of innate immunity is essential because morbidity from brain infection / injury can be caused directly by the acute chronic neuroinflammation, as well as by a disproportionate immune response. Recent studies described a direct role of microglial toll-like receptor (TLR) not only in pathogen/injury clearance but also in exacerbating neurodegeneration [1-]. However, detailed molecular mechanisms of regulation of excessive inflammatory responses and neurotoxicity by microglia are poorly understood. Microglia, the resident macrophage-like cells, functions as the basic immune defense system of the brain [3]. Evidence suggests that activation of innate immunity in the NS triggers neurodegeneration through a toll-like receptor -dependent pathway []. TLR induced microglia activation is a characteristic feature of most neurodegenerative diseases including lzheimer s disease, Multiple sclerosis, Parkinson s disease, stroke, ischemia and amyotrophic lateral sclerosis as well as posttraumatic 3

Gaikwad S. et al Euro. J. Exp. io., 15, 5(8):3- brain injury [-7]. Neurotoxicity induced by β-amyloid or in NS depends on the presence and activation of microglial TLR [1-, 8-1]. Toll-like receptors (TLRs) recognize pathogen/danger-associated molecular patterns (PMPs/MPs) and induce innate immune responses that are essential for host defense against infection or injury [11]. mong the TLR family, TLR functions as a receptor for the endotoxin lipopolysaccharide () [1], which binds -binding protein (LP) and the 1. This complex in turn binds to TLR and initiates an intracellular signaling pathway including activation of p38, JNK [13] and ERK MPKs that regulates gene expression through NF-κ activation [1]. TLR functions through an accessory protein (M-) [1]. TLR activation results in increased secretion of cytokines which dictates the fate of brain pathology. MPKs are serine/threonine protein kinases, plays important role in a variety of cellular functions like cell proliferation, differentiation and apoptosis [15-1]. There are three main members of the MPK family, c-jun N- terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38 MPKs exert different biological functions. ctivation of ERK1/ is involved in proliferation and survival while JNK and p38 MPK are associated with apoptosis [15]. Several studies have shown that induces neuroinflammation via a mechanism mediated by the JNK pathway and induces apoptosis in brain [15, 17]. Microglia are rapidly activated upon tissue damage, can efficiently clear apoptotic cells [18], and can promote neuro-repair through the production of growth factors [3]. The spectrum of activated microglia phenotypes is diverse and generally beneficial. However, when microglia activation becomes exaggerated or dysregulated, the response becomes neurotoxic. Therefore, it is of critical importance to elucidate the mechanisms that are specifically involved in the dysregulated response of microglia which contribute to neuronal damage. In the current study, we explored whether there is a causative link between microglial MPK signaling and microglia-dependent neuronal damage. Our study demonstrates that TLR activation by and β-amyloid significantly increases inflammation whereas p38 MPK and JNK 1/ inhibition provides neuroprotection. ollectively, p38 MPK and JNK 1/ are good therapeutic targets in neurodegenerative diseases. MTERILS N METHOS.1 ell culture and treatment V microglial cells are suitable in vitro model system for examining inflammation in primary microglia or animal brain [19]. V microglia cells (kind gift from Prof. nirban asu) were maintained at 37 under an atmosphere of 5% O in EMEM supplemented with 5% fetal bovine serum (FS), 1 U/ml penicillin and 1 mg/ml streptomycin. For experiments, V cells were pretreated with ultrapure -Rs-TLR inhibitor (Invivogen), P1835-ERK1/ inhibitor, SP15-JNK1/ inhibitor, S19-p38 inhibitor and LY9-PI3K inhibitor (ll 1µM concentration) for 1 hr followed by stimulation with lipopolysaccharide ((1µg/ml) from Escherichia coli, serotype 55:5) or fibrillated β amyloid (5 µm) for hrs. Preparation of fibrillated amyloid β (β): briefly, acetyl-β (Sigma-ldrich) was dissolved in sterile PS (1 µm) and stored at until use. β peptides were aggregated by incubation at 37 for days and then 5 µm fibrillated β was used to treat V cells. ll Inhibitors and are from Sigma ldrich and cell culture related reagents were purchased from Invitrogen unless otherwise mentioned.. Semiquantitative RT-PR Relative mrn levels of TLR, IL-1β, IL-, TNF-α, inos and OX in microglia were determined by RT-PR as described earlier []..3 Western blotting V microglial cells were plated in well plates at a density of 1 1 cells/well and differentially treated as indicated. ell lysates were prepared in sodium dodecyl sulphate (SS)-containing sample buffer and 3µg total protein from each sample were separated by 1% SS-polyacrylamide gel electrophoresis (SS-PGE) followed by transfer to PVF membranes and blocking, then blots were probed with the primary antibodies: rabbit anti-tlr (1:5); rabbit anti-phospho p38 (1:1); rabbit anti-phospho JNK 1/ (1:5): rabbit anti-phospho ERK 1/ (1:1) and mouse anti-β-ctin (1:1) all from Invitrogen. 35

Gaikwad S. et al Euro. J. Exp. io., 15, 5(8):3-. Nitric oxide (NO) measurement The nitrite (a stable oxidative end product of NO) production in the culture medium was measured using the Griess reagent (Sigma ldrich) as per manufacturer s instructions. riefly, culture supernatants (phenol red-free) was mixed with Griess reagents (1:1 ratio) in a 9 well plate for 15 min. bsorbance was measured at 5 nm with microplate reader (iorad). Sodium nitrite was used as a standard and each experiment was performed in triplicate..5 ROS measurement Microglial cells were treated as indicated for hrs. Then, cells were washed with PS and incubated with 5 μm ell ROX deep red (Invitrogen) in dark for 3 min at 37. fter washing, intracellular ROS levels were measured by mean fluorescence intensity with a multimode plate reader (Molecular evises) at /55 nm (excitation / emission).. Neuronal cell survival assay ifferentiated neuroa cells with neuronal morphology used as alternative source of neurons. The neuronal cells were cultured with differentially treated microglia conditioned medium for 8 hrs to examine microglia mediated neurotoxicity. The neuronal cell death was determined with the MTT assay (Sigma) as per manufacturer s instructions..7 ELIS The production TNF-α in the culture supernatants was measured with sandwich ELIS kits (eiosciences) according to the manufacturer's instructions..8 Statistical analysis The data expressed as ± SEM and statistical significance was analysed using one way analysis of variance (NOV) followed by Tukey s multiple comparison test. p value less than.5 (p<.5) was considered to be statistically significant. RESULTS 3.1 β amyloid and induced TLR mediated neuroinflammation Microglia are the resident innate immune cells of the brain, provides first line of defense through inflammatory response and removes infectious materials/debris/or plaques by phagocytosis to provide protection during NS pathologies. TLR is reported to be upregulated in brains of patients with [1, -1]. We investigated effect of β amyloid (β) on TLR expression in V microglia cell. We observed that V microglia stimulated with fibrillar β significantly enhanced TLR mrn levels (Figure 1) accompanied with increased production of inflammatory mediators, NO and TNF-α (Figure 1-1) and the effect was TLR dependent as evidenced by inhibition of TLR (-Rs, a potent TLR antagonist) significantly decreased β-induced production of NO and TNF-α (Figure 1-1). The observations suggest that β induced TLR mediated inflammation in V microglia. For further studies we used, a potent TLR ligand, to study TLR induced microglia mediate immune responses. Resting microglial cells have highly ramified processes which are very motile. This motility allows microglia to screen the neuronal parenchyma for danger signals, pathogens, metabolic products and deteriorated tissues. onversion of ramified to amoeboid morphology, increased size of microglia as well as increased expression of several microglia activation markers, are essential features of the inflammatory response of the brain to various insults []. Therefore, we initially confirmed that, in our experimental paradigm, treatment resulted in an intense microglia activation and inflammation as evidenced by increased amoeboid morphology (Figure 1), increased expression of microglia activation markers like TLR, IL-1β, IL-, TNF-α, inos, and OX- (Figure 1E) accompanied with increased production of NO and TNF-α (Figure 1F-1G). ollectively, the data suggests that β amyloid and induced microglia activation and neuroinflammation through TLR signalling. 3. TLR stimulation enhanced neuroinflammation in dose dependent manner s mentioned above, β induced TLR mediated microglia activation and neuroinflammation. We investigated the effect of TLR ligand concentration ( 1-1 ng/ml) on microglia mediated inflammation. We observed that dose dependently enhanced expression of TLR mrn and protein levels (Figure -) accompanied with subsequent increase in pro-inflammatory cytokines IL-1β, IL- and TNF-α mrn levels (Figure ). In addition, significantly enhanced production of NO and ROS in a dose dependent manner (Figure -E). The data indicates that TLR activation and subsequent inflammatory response depends on concentration of ligand () and the inflammatory response is directly proportional to dose of the TLR ligand. Thus the possible reason for exacerbated neuroinflammation in patient s brain could be due to increased TLR activation resulting from higher accumulation of β. 3

TNF- TNF-α (pg/ml) (pg/ml) NO production Nitrite (µm) ( M) TNF- TNF-α (pg/ml) NO production ( M) Nitrite (µm) Gaikwad S. et al Euro. J. Exp. io., 15, 5(8):3-1. 3.9 ensitometry TLR Ramified morphology moeboid morphology β-amyloid 8 Untreated (1µg/ml) 3 1 b-amyloid (b) yloid ( ) TLR inhibitor+(b) β-amyloid TLR inhibitor+ β-amyloid bitor+( ) E F 5 G IL- IL-1β TNF-α OX- inos TLR 1 8 1µg/ml 15 1 5 1 µg/ml Figure 1. β amyloid and induced TLR mediated neuroinflammation. V microglial cells were stimulated with 5µM fibrillar β and TLR mrn expression was assayed by RT-PR (after hrs) (), secreted NO and TNF-α in culture supernatant were analysed by Griess reaction and ELIS (after hrs), respectively ( and ). V microglial cells were stimulated with 1µg/ml and ramified or amoeboid morphology was observed under inverted phase-contrast microscope (Olympus) at x magnification (), mrn levels TLR, proinflammatory cytokines (IL-1β, IL-, and TNF-α) and inflammatory mediators inos and OX was assayed by RT-PR (after hrs) (E), secreted NO and TNF-α in culture supernatant were analysed by Griess reaction and ELIS (after hrs), respectively (F and G).( sterisk indicates p<.1) 3.3 PI3 kinase partially regulates TLR induced inflammation in V microglia The phosphatidylinositol 3-kinase (PI3K) signaling plays a key role in regulation of inflammation [3]. We examined the TLR induced activation of PI3K in microglia cells. We observed that stimulation significantly increases activation of PI3K (Figure 3-3). ctivation of PI3K was peaked at 3 min and decreased after min (Figure 3-3). To examine the role of PI3K in TLR induced inflammation by microglia, we used LY9, a selective PI3K inhibitor. Microglial cells were pretreated with PI3K inhibitor for 1 hr followed by stimulation for hrs and mrn levels of proinflammatory were determined. PI3K inhibition does not affect expression of IL-, TNF-α, inos and ox (Figure 3), whereas inhibition of TLR confirms the inhibition of induced inflammation (Figure 3). Similarly, TNF-α production was partially affected by PI3K inhibition (Figure 3). The results suggest that inhibition of PI3K partially inhibits TLR induced inflammation in V microglia. 3. TLR stimulation enhanced inflammation and ROS production through activation of p38 and JNK 1/ MPKs ctivation of MPK signaling plays an important role in mediating microglia activation []. In addition, JNK and p38 MPKs are also involved in neuronal death [5]. TLR upregulation is also associated with MPKs activation and neuronal cell apoptosis [-7]. We therefore investigated whether MPKs activation are important for TLR induced microglia-mediated inflammation and neuronal cell death. We first investigated the activation of MPK pathways in our experimental paradigm. V microglia cells were treated with or without for 5- minutes and MPKs were detected by Western blotting using specific antibodies for the phosphorylated (activated) forms of P-JNK, P-p38 and P-ERK1/. stimulation significantly enhanced phosphorylation of p38, JNK, and ERK1/ as compared to untreated V microglia (Figure -). ctivation of p38, JNK, and ERK1/, was peaked at 3 min, which declined after min (Figure -). It should 37

Relative ROS production Relative ROS production NO Nitrite production (µm) ( M) Gaikwad S. et al Euro. J. Exp. io., 15, 5(8):3- be noted that did not activated MPKs in neuronal cells (data not shown). In contrast, neuronal MPKs (p38, JNK and ERK 1/) were reported to be induced by soluble, diffusible factors released from activated microglia [5]. These observations suggest that MPKs activation in neurons is induced by microglia secreted inflammatory mediators. The TNF-α, ROS and inos-derived NO has been suggested to play an important role in inducing neuronal cell death, and their blocking may provide neuroprotection. To address the relationship between MPK signaling and inflammation, we used selective inhibitors for the MPK pathways: ERK1/ inhibitor (P1835), JNK1/ inhibitor (SP15), and p38 inhibitor (S19. Microglial cells were pretreated with MPK inhibitors for 1 hr followed by stimulation for hrs and inflammatory mediators were determined. Interestingly, JNK and p38 inhibition significantly decreased production of TNF-α, NO and ROS (Figure -F), while the ERK1/ and PI3K inhibition did not show inhibitory effect on inflammation (Figure -F). The results suggest that JNK and p38 activation play indispensable role for TLR induced inflammation and oxidative stress which may enhance neurotoxicity during pathophysiology. TLR 1 8 1 1 1 (ng/ml) TLR IL-1β IL- TNF-α 1 1 1 (ng/ml) 1 1 1 (ng/ml) 9 k k E 3..5. 1.5 1..5. 1 1 1 1 (ng/ml) ng/ml 1 1 ns.1.1.3 1 1.5 g/ml Figure. TLR stimulation enhanced neuroinflammation in dose dependent manner. V microglial cells were stimulated different concentrations (1-1 ng/ml); TLR mrn expression was assayed by RT-PR (after hrs) () and TLR protein levels were analysed by Western blot (). Expression of proinflammatory cytokines (IL-1β, IL- and TNF-α) mrn levels were assayed by RT-PR (after hrs) (), production of NO and ROS were measured by Griess reagent and ell-rox deep red reagent, respectively ( and F). sterisks indicates p<.1, indicates p<.1; indicates p<.5 38

TNF- production (pg/ml) +LY 5 +LY 1 +LY+RS Relative fold p-pi3k (Fold change vs control) Gaikwad S. et al Euro. J. Exp. io., 15, 5(8):3- p-pi3k (85 k) p-pi3k (55 k) ( k) 15 3 (min) (1 µg/ml) p-pi3k (p55) p-pi3k (p85) 15 3 (min) (1 µg/ml) 5 IL-1β IL- TNF-α inos 15 1 5 OX- +LY +LY+RS Figure 3. PI3 kinase partially regulates TLR induced inflammation in V microglia. V microglial cells were stimulated with for different time point (15- min) and phosphorylation of PI3K was assayed by Western blot () and relative fold changes were done by densitometry analysis using Image J (). V microglial cells were pretreated with PI3K inhibitor (LY) for 1 hrs followed by stimulation, mrn levels of inflammatory mediators was analysed by RT-PR (after hrs) () and secretion of TNF-α was measured by ELIS (after hrs) (). sterisks indicates p<.1, indicates p<.1; indicates p<.5 and indicate p<.5 vs treated group/ 3.5 Inhibition of p38 and JNK ameliorates β induced Microglia-mediated Neurotoxicity through negative regulation of NF-κ onsequent to above observations, we examined the possible role of the MPK signaling in TLR induced microglia-mediated neurotoxicity. To examine the functional importance of TLR on neuronal cell survival during neuron-microglia coculture. We used the -Rs, a potent TLR signaling inhibitor, to investigate the role of TLR in β-mediated neurotoxicity in neuron-microglia culture. activated microglia are well known to trigger neuronal cell death through secretion of neurotoxic products and therefore used as a positive control for detection of microglia-mediated neuronal toxicity. Exposure of differentiated neuroa cells to supernatant from β activated microglia showed significantly increased neuronal cell death however β induced neuronal cell death significantly reversed by TLR inhibition as examined by MTT assay (Figure 5). These results indicate that TLR signaling is major contributor in β induced neuronal insults. Microglial cells were pretreated with MPK inhibitors for 1 hr followed by β stimulation for hrs and the conditioned media (M) were collected. Neuronal cells were treated with or without M for 8 hrs and neuronal cell survival were assayed. The inhibition of JNK and p38 significantly ameliorated neurons from TLR induced microglia-mediated neuronal death, while inhibition of ERK1/ did not show a neuroprotection (Figure 5). In addition, combined inhibition of JNK and p38 synergistically enhanced neuroprotection (Figure 5). NF-κ is well described for its important role in NS pathologies, so we investigated the effect of JNK and p38 inhibition of NF-κ. We observed that β enhanced NF-κ activation through TLR (Figure 5 and 5) as evidenced by decreased NF-κ by TLR inhibition. Interestingly, JNK and p38 inhibition also results in decreased NF-κ while ERK inhibition did not limited NF-κ (Figure 5 and 5). ollectively, the 39

NO production ( M) TNF- TNF-α production (pg/ml) (pg/ml) Relative ROS production p-p38 (% vs control) p-erk 1/ (% vs control) p-jnk (% vs control) Gaikwad S. et al Euro. J. Exp. io., 15, 5(8):3- data suggests that inhibition of JNK and p38 provides neuroprotection through negative regulation of NF-κ activation and combined inhibition of JNK/p38 MPK synergistically potentiates the neuroprotection against microglia-mediated neurotoxicity. 35 3 5 15 ns ns 1 5 5 15 3 p-p38 5 15 3 (min) (1 µg/ml) 1 8 15 1 1 8 ns 5 15 3 E 5 p-erk 1/ 5 15 3 (min) (1 µg/ml) 5 3 1 F 5 S P SP LY 1 g/ml S P SP LY 1 g/ml 5 15 3 p-jnk 5 15 3 (min) (1 µg/ml) S P SP LY Figure. TLR stimulation enhanced inflammation and ROS production through activation of p38 and JNK 1/ MPKs. V microglial cells were stimulated with for different time point (15- min) and phosphorylation of p38, ERK1/ and JNK was assayed by Western blot (,, and ). V microglial cells were pretreated with MPKs inhibitors for 1 hrs followed by stimulation for hrs and production of NO, TNF-α and ROS were measured (, and E). sterisk indicates p<.1 vs untreated control, indicates p<.1 vs treated group ISUSSION In the present study, we demonstrated (i) β and induced TLR mediated microglia activation and neuroinflammation, as evidenced by the amoeboid morphology and up-regulation of TLR, IL-1β, IL-, TNF-α, inos, and OX-, (ii) PI3K and MPK are markedly activated in microglia after TLR stimulation with and (iii) inhibition of p38 and JNK provide neuroprotection against TLR induced microglia-mediated neuronal cell death. We for the first time report that combined inhibition of JNK and p38 MPK synergistically enhanced neuroprotection during TLR induced microglia mediated neuroinflammation and neurotoxicity. Several evidences suggest that MPK are rapidly activated in microglia /macrophages with various activating stimuli. In microglia cultures, stimulation induces activation of p38, JNK, and ERK 1/ [8-3]. onsistent with the studies, we also report activation of p38, JNK, and ERK1/ in activated V microglia. Importantly, Xie and coworkers shown that MPK signaling pathways were activated in neurons that had been in co-culture with microglia stimulated with and IFN-γ, but not in neurons treated with and IFN-γ [5]. These evidences suggest that microglia secreted factors like TNF-α, NO and ROS activates p38, JNK1/ and ERK 1/ and may be potentiates neuronal loss in aberrant neuroinflammatory conditions. Several studies have reported that MPKs (p38, JNK and ERK 1/) are involved in neuronal cell death. stimulated microglia shows activation of MPKs, play key roles in increased inflammatory mediators like inos, IL-1β, or TNF-α [31-33]; thus, there might be link between activation of MPKs and increased neuroinflammation. The molecular mechanisms by which MPK pathways contribute to microglia-mediated neuroinflammation was elucidated using selective inhibitors. Inflammatory mediators like TNF-α and NO produced from β-amyloid or activated microglia enhanced neuronal cell death [3]. However, our findings suggest inhibition of JNK and p38 significantly reduced NO, TNF-α and ROS levels (Figure -F), in contrast inhibition of ERK 1/ and PI3K did not blocked TLR induced microglia-mediated inflammation. These results suggest that the JNK and p38 pathways are important regulators of TLR induced microglia mediated inflammation. Furthermore, we investigated the role of

RS S P SP RS S P SP Relative P-p5 P-p5 NF- NF-κ (rbitrary units) S P SP LY S+SP % ell Survival % Neuronal survival % Neuronal survival % Neuronal survival Gaikwad S. et al Euro. J. Exp. io., 15, 5(8):3- the MPKs in microglia mediated neuronal cell death. Our observation that inhibition of p38 and JNK provides neuroprotection against activated microglia-mediated neurotoxicity (Figure 5) suggests the critical role of JNK and p38 MPKs signaling during the neurotoxicity. In addition, inhibition ERK1/ did not protects neuronal cell death suggests that activation of ERK1/ is not a problem in TLR induced microglia-mediated neuroinflammation and subsequent neurotoxicity. 15 ontrol TLR inhibitor 1 1 75 ns 5 5 ns 5 β (5 µm) 1 g/ml β (5 µm) (5 k) ( k) P-p5 NF-κ β (1 (5 g/ml) µm) Figure 5. Inhibition of p38 and JNK ameliorates β induced microglia-mediated neurotoxicity. ifferentiated neuroa cells were exposed to supernatants from β or activated microglia either untreated or treated with TLR inhibitor. Neuronal cell viability was examined by MTT assay (). V microglial cells were pretreated with or without MPKs inhibitors for 1 hr followed by activation with β for hrs and the conditioned media were collected. ifferentiated neuroa cells were exposed to conditioned media for 8 hrs and neuronal cell survival was analysed by MTT assay (). ifferentiated neuroa cells were exposed to supernatants from β or activated microglia either untreated or treated with TLR inhibitor. ifferentiated neuroa cells were exposed to supernatants from differentially treated microglia for 1 hr and NF-κ activation was assayed by Western blot (), relative P-NF-κ (p5) was assayed by densitometric analysed using ImageJ (=p<.1, =p<.1, =p<.1 vs β treated group and ns=non significant). ONLUSION Our findings suggest that combined inhibition of JNK1/ and p38 MPK signaling could be a potential therapeutic strategy against brain pathologies like lzheimer s disease where TLR induced microglia-mediated neuroinflammation is implicated in disease progression. cknowledgements We thank Prof. nirban asu (National rain Research entre, India) for kind gift of V microglial cell line. The study was funded by epartment of Science and Technology (SR/SI/59/11(G)) and epartment of iotechnology (T/PR897/ME/9/1/1) (both the grants were obtained from Government of India). 1

Gaikwad S. et al Euro. J. Exp. io., 15, 5(8):3- REFERENES [1] Trotta, T., et al., J Neuroimmunol, 1. 8(1-): p. 1-1. [] Gaikwad, S. and R. grawal-rajput, Lipopolysaccharide from Rhodobacter sphaeroides ttenuates Microglia- Mediated Inflammation and Phagocytosis and irects Regulatory T ell Response, Int J Inflam, 15. OI: 1.1155/15/313. [3] Kreutzberg, G.W., Trends Neurosci, 199. 19(8): p. 31-8. [] Lehnardt, S., et al., Proc Natl cad Sci U S, 3. 1(1): p. 851-9. [5] McGeer, P.L., et al., Neurology, 1988. 38(8): p. 185-91. [] ickson,.w., et al., Microglia and cytokines in neurological disease, with special reference to IS and lzheimer's disease. Glia, 1993. 7(1): p. 75-83. [7] Trapp,.., et al., J Neuroimmunol, 1999. 98(1): p. 9-5. [8] Tang, S.., et al., Exp Neurol, 8. 13(1): p. 11-1. [9] He, Y.,. Zhou, and W. Jiang, Neural Regen Res, 13. 8(9): p. 7-53. [1] Tang, S.., et al., Pivotal role for neuronal Toll-like receptors in ischemic brain injury and functional deficits. Proc Natl cad Sci U S, 7. 1(3): p. 13798-83. [11] Takeda, K. and S. kira, Int Immunol, 5. 17(1): p. 1-1. [1] Hoshino, K., et al., J Immunol, 1999. 1(7): p. 379-5. [13] Wright, S.., J Exp Med, 1999. 189(): p. 5-9. [1] Shimazu, R., et al., J Exp Med, 1999. 189(11): p. 1777-8. [15] Kim, E.K. and E.J. hoi, rch Toxicol, 15. 89(): p. 87-8. [1] Kaminska,., et al., nat Rec (Hoboken), 9. 9(1): p. 19-13. [17] Wang, L.W., et al., J Neuroinflammation, 1. 9: p. 175. [18] Gehrmann, J., et al., Neuropathol ppl Neurobiol, 1995. 1(): p. 77-89. [19] Henn,., et al., LTEX, 9. (): p. 83-9. [] Walter, S., et al.,. ell Physiol iochem, 7. (): p. 97-5. [1] Jin, J.J., et al., J Neuroinflammation, 8. 5: p. 3. [] Nimmerjahn,., F. Kirchhoff, and F. Helmchen, Science, 5. 38(57): p. 131-8. [3] ahill,.m., J.T. Rogers, and W.. Walker, J Signal Transduct, 1. 1: p. 3587. [] Kim, S.H.,.J. Smith, and L.J. Van Eldik, Neurobiol ging,. 5(): p. 31-9. [5] Xie, Z.,.J. Smith, and L.J. Van Eldik, Glia,. 5(): p. 17-9. [] Fernandez-Lizarbe, S., M. Pascual, and. Guerri, J Immunol, 9. 183(7): p. 733-. [7] Katome, T., [Inhibition of stress-responsive signaling pathway prevents neural cell death following optic nerve injury]. Nihon Ganka Gakkai Zasshi, 1. 118(11): p. 97-15. [8] Rao, K.M., T. Meighan, and L. owman, J Toxicol Environ Health,. 5(1): p. 757-8. [9] Pyo, H., et al., Neuroreport, 1998. 9(5): p. 871-. [3] Hambleton, J., et al., Proc Natl cad Sci U S, 199. 93(7): p. 77-8. [31] Liu, J. and. Lin, ell Res, 5. 15(1): p. 3-. [3] Poddar, R. and S. Paul, J Neurochem, 13. 1(): p. 558-7. [33] Morrison, R.S., et al., dv Exp Med iol,. 513: p. 1-8. [3] Xie, Z., et al., J Neurosci,. (9): p. 38-9.