TLR2 immunotherapy suppresses neuroinflammation, tau spread, and memory loss in rTg4510 mice

In Alzheimer ’ s disease, chronic neuroinflammation is accompanied by amyloid and tau pathologies. Especially, aberrant microglial activation is known to precede the regional tau pathology development


Introduction
Neuroinflammation in the central nervous system occurs by activation of glial cells, mainly microglia and astrocytes.Together with amyloid and tau pathologies, neuroinflammation is one of the hallmarks of Alzheimer's disease (AD) (Wilson et al., 2023).In physiological conditions, microglia retain their resting state and become timely activated to maintain brain homeostasis.In pathological conditions, however, dysfunctional microglia show chronic activation, induce neuroinflammation by secretion of proinflammatory cytokines and thereby form neurotoxic environment.Notably, recent studies showed that the number of activated microglia and the levels of proinflammatory cytokines are significantly lower in non-demented individuals with AD neuropathology compared to demented AD patients (Perez-Nievas et al., 2013;Barroeta-Espar et al., 2019;Gefen et al., 2019).When compared inflammatory cytokine profiles of AD patients, patients with higher proinflammatory cytokine levels at early stages of AD developed more severe neuropathology at late stages of AD (Sudduth et al., 2013).In terms of tau spread, progressive spatial activation of microglia followed by tau pathology was observed in the brains of P301S tau transgenic mice and AD patients (Yoshiyama et al., 2007;Pascoal et al., 2021).
Microglia express diverse activating and inhibitory immunoreceptors that modulate their functions (Wang and Colonna, 2019).Tolllike receptors (TLRs) are important immunoreceptors residing on microglia that regulate microglial activation (Fiebich et al., 2018).Located on the cell membrane or in endosomes, TLRs recognize pathogenic ligands through leucine-rich extracellular domain and conduct inflammation via downstream signaling pathway (Squillace and Salvemini, 2022).Among them, TLR2 has been actively studied in the context of AD pathology.Previous studies reported that TLR2 interacts with amyloid β (Aβ) to trigger neuroinflammation and blocking TLR2 reduces Aβ accumulation in APP/PS1 mice (Jana et al., 2008;Liu et al., 2012;McDonald et al., 2016).Also, elevated levels of TLR2 were reported in the brains of P301L mutant tau transgenic mice and human patients with AD (Bonham et al., 2018;Momtazmanesh et al., 2020).Interestingly, a recent paper showed that tau fibrils induce microglial inflammation via TLR2/MyD88/NF-κB pathway, and blocking the TLR2-MyD88 interaction alleviates tau pathology in PS19 tau transgenic mice (Dutta et al., 2023).However, it remains unknown how TLR2 contributes to tau spread and whether tau spread can be blocked by immunotherapy.
In this study, we found that TLR2 deficiency reduces memory loss and microglial activation in rTg4510 tau transgenic mice.Microglial TLR2 interacts with oligomeric tau upon activation to induce inflammatory responses.Neuronal tau uptake was significantly reduced when neurons were co-cultured with Tlr2 knockout microglia or after intracranial injection of tau into the hippocampus of Tlr2 knockout mice.Moreover, treatment with anti-TLR2 monoclonal antibody Tomaralimab significantly reduces tau spread and cognitive impairment in rTg4510 mice.These results suggest that TLR2 activation in microglia aggravates tau pathogenesis in AD.

Mice
The mice used in the study: human P301L tau responder line (The Jackson Laboratory #015815), tetracycline-controlled transactivator line (The Jackson Laboratory #016198), Tlr2 knockout mice (The Jackson Laboratory #004650).Mice were maintained in a specific pathogen-free animal facility.All experimental procedures were approved by Seoul National University Animal Care and Use Committee and performed according to the Korean Ministry of Food and Drug Safety.Mice were assigned randomly to experimental groups.

Behavioral tests
2.2.1 Y-maze test − mice were placed in the end of one arm of Ymaze (32.5 cm length × 15 cm height) and allowed to move freely for 7 min.An entry into the arm was counted when the whole-body including tail is placed inside the arm.The percentage of spontaneous alternation was estimated as the ratio of the number of alternations to the number of total entries.2.2.2 Novel object recognition test − mice were habituated in the empty chamber (30 cm length × 30 cm width × 25 cm height) to move freely for 7 min at 24 h intervals for 2 days.During the test period, two objects were placed in the chamber and one of the objects were changed every day for 3 days (novel object), while the other kept unchanged.The object exploration was defined as mouse sniffing or touching the object.The discrimination index was estimated as the ratio of the time of exploring a novel object to the total time of exploring two objects.2.2.3 Passive avoidance test − an apparatus with a light and dark compartments (20 × 20 × 20 cm) separated by a sliding door was used for the test.Mice were placed in the closed light compartment and allowed to move freely for 1 min.After opening the door, the latency time for the mice entering the dark compartment was measured with a 5 min cut-off.An electric shock (0.25 mA, 2 s) was delivered by the floor grids in the dark compartment for conditioning, and the test was performed after 24 h.

Recombinant tau preparation
Human 0N4R tau was subcloned into pET-6xHis vector, expressed in BL21-DE3, and purified using Ni-NTA agarose (Qiagen).Monomeric tau was incubated with DyLight 488/594 NHS Ester (Thermo Scientific) for fluorescence labeling.For oligomerization, 24 μM monomeric tau in PBS containing 5 mM dithiothreitol and 6 μM heparin was incubated for 1 h (low-molecular-weight) or 1.5 h (high-molecular-weight) at room temperature.Tau proteins were subjected to native polyacrylamide gel electrophoresis (PAGE) to confirm its molecular sizes.

Western blot
Cells were lysed in RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors.Samples were subjected to SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membrane.After blocking, the membrane was incubated overnight at 4 • C with primary antibodies.After washed, the membrane was incubated with anti-mouse IgG HRP (Santa Cruz Biotechnology #sc-516102, 1:5000) or anti-rabbit IgG HRP (Cell Signaling Technology #7074, 1:5000) for 1 h.After washed, the membrane was analyzed with a chemiluminescent imaging system.

TLR2 reporter assay
HEK-Blue Null1 and HEK-Blue hTLR2 cells (5.4 × 10 4 cells per well) were seeded into the 96-well plate containing reagents in a volume of 180 μl per well and incubated in HEK-Blue Detection medium (Inviv-oGen) at 37 • C for 16 h.After 20 h, secreted embryonic alkaline phosphatase (SEAP) levels were determined using a spectrophotometer at 620 nm.

Real-time quantitative reverse transcription PCR
Cells were incubated with or without oligomeric tau (200 nM) for 24 h or 48 h, in the presence or absence of Tomaralimab (100 nM or 500 nM).Cells were harvested and RNA extraction was performed by using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer's instructions.Complementary DNA was synthesized by using QuantiTect reverse transcription kit (Qiagen) and amplified using a SYBR Green PCR master mix (Applied Biosystems).

Tau spread assay in transwell system
SH-SY5Y cells were differentiated by incubation with 10 μM retinoic acid (Sigma-Aldrich) for 7 to 10 days.Differentiated SH-SY5Y cells were transduced with adenoviruses carrying tau four-repeat domains (Adeno-Tau4R, donor) or TLR2 (Adeno-TLR2, recipient) with a multiplicity of infection (MOI) of 500 or 100, respectively.Cells were then subcultured into the insert (donor) or bottom (recipient) wells of transwell plate (Corning).After allowing cells to adhere for 4 h, recipient cells were treated with Tomaralimab for 3 days.The amount of Tau4R in the recipient cells was analyzed by western blotting.

Tau spread assay in vivo
The brains of three 6-month-old rTg4510 mice were homogenized in DPBS (10 % w/v) and centrifuged at 3,000 xg at 4 • C for 5 min.The supernatant was aliquoted and kept at − 80 • C until use.The brain lysates (2.5 μl) were injected into the right hippocampus (stereotaxic coordinates: anteroposterior = -1.9mm, mediolateral = -1.4mm, dorsoventral = -1.5 mm from bregma) of 4-to 5-month-old rTg4510 mice with a speed of 1 μl/ml.Tomaralimab administration (30 mg/kg, intravenous) was initiated from the day of the lysate injection and continued for 8 weeks at intervals of once a week.Numbers of AT8positive cells in the CA3 region of the left hippocampus were counted.

Statistics
All statistical analyses were performed using GraphPad Prism 10 Software.Normality of data distribution was analyzed using the Shapiro-Wilk test.If the data did not meet the assumptions of the parametric tests, nonparametric tests were used.Unpaired t-test was performed when comparing two independent groups.Paired t-test was performed when analyzing passive avoidance test data.One-way ANOVA test, Kruskal-Wallis test, and two-way ANOVA test were performed when comparing multiple groups.P values were obtained from two-tailed test.No statistical test was used to predetermine sample sizes.

TLR2 deficiency alleviates cognitive functions and tau pathology
To examine whether gene expression of TLR families show any correlation with diagnosis of AD, we used Agora Gene Comparison Tool (https://agora.adknowledgeportal.org) to obtain RNA sequencing (RNA-seq) data from Religious Orders Study and Memory and Aging Project (ROSMAP), Mayo RNAseq (MAYO), and Mount Sinai Brain Bank (MSBB) human cohort studies.The differential RNA expression levels of each receptor vary across the brain regions, while the expression of TLR2 specifically showed a significant increase in the parahippocampal gyrus (Fig. 1A).The brain region of the parahippocampal gyrus includes entorhinal cortex, which is important for memory functions and shows atrophy in early stages of AD (Rodrigue and Raz, 2004;Echávarri et al., 2011;Suh et al., 2011).Also, early tau pathology develops in the entorhinal cortex in AD, and pathologic tau further spreads to the hippocampus and the neocortex as disease progresses (Polydoro et al., 2014;Kaufman et al., 2018;Berron et al., 2021;Delpech et al., 2021).We therefore hypothesized that elevated TLR2 expression in the parahippocampal gyrus may promote tau pathology progression in AD.
To address whether TLR2 expression is correlated with tau pathology and memory functions, we crossed wild-type (WT) and Tlr2 knockout (Tlr2 KO) mice to either rTg4510 mice, which express a human P301L mutant tau under the control of the Ca 2+ -calmodulin kinase II (CaMKII) promoter, or its control tetracycline-controlled transactivator (tTA) mice without P301L tau responder (Ramsden et al., 2005;SantaCruz et al., 2005).At 6 months of age, rTg4510/WT mice showed cognitive deficits in the Y-maze test (Fig. 1B), novel object recognition test (Fig. 1C) and passive avoidance test (Fig. 1D) compared to control tTA/ WT and tTA/Tlr2 KO mice.However, the cognitive functions of rTg4510/Tlr2 KO mice were comparable to those of control tTA/WT and tTA/Tlr2 KO mice (Fig. 1B-D).Total entries in the Y-maze test showed no difference between experimental groups (Fig. 1B).
When immunostained with AT8 antibody which detects pathological and phosphorylated tau, AT8-positive tau was significantly reduced by Tlr2 deficiency in the frontal cortex and hippocampal dentate gyrus of rTg4510 mice, and slightly reduced in the entorhinal cortex (Fig. 1E-F).Recent single-nucleus RNA-seq study of entorhinal cortex revealed an increased TLR2 expression in microglia (Grubman et al., 2019).In the entorhinal cortex, TLR2 expression was highly elevated in the microglia but not much in the astrocytes of rTg4510/WT mice compared to control tTA/WT mice (Supplementary Fig. 1).Interestingly, among all microglia, those engulfing AT8-positive tau showed an elevated expression of TLR2 (Supplementary Fig. 1A).Iba1-positive microglial activation was significantly reduced in the frontal cortex and entorhinal cortex by Tlr2 KO in rTg4510 mice, while it showed no difference in the hippocampus (Fig. 1E, G).Compared to control tTA/WT and tTA/Tlr2 KO mice, loss of NeuN-positive neurons in the cortex was significant in rTg4510/WT mice, but this neuronal loss was partly alleviated in rTg4510/Tlr2 KO mice (Supplementary Fig. 2).These results indicate that TLR2 expression in microglia is correlated with tau pathology development and memory loss.

TLR2 activation increases binding of oligomeric tau in microglia
As TLR2 was reported as a receptor that binds to diverse pathogenic  , 2008;de Oliviera Nascimento et al., 2012;Liu et al., 2012), we addressed whether TLR2 binds to tau.We prepared monomeric and oligomeric human recombinant tau proteins (Fig. 2A), and performed co-immunoprecipitation assay after addition of tau proteins to cell extracts overexpressing GFP-tagged TLR2 (TLR2-GFP).TLR2 bound to both monomeric and oligomeric tau proteins, but preferably bound to oligomeric tau than monomeric tau (Fig. 2B-C).To determine a region of TLR2 responsible for tau binding, we generated TLR2 mutant lacking the ligand binding domain (ΔLBD), mutants that inhibit heterodimerization of TLR2 with TLR1 or TLR6 (ΔDimer and D327W/F349L) (Jin et al., 2007;Kang et al., 2009), and mutant that inhibits TLR2-mediated NF-κB signaling (P681H) (Underhill et al., 1999) (Fig. 2D).The ligand binding domain of TLR2 partially overlaps with dimerization domain (Fig. 2D).From the co-immunoprecipitation assay, TLR2 ΔDimer and D327W/F349L mutants showed little binding affinity for oligomeric tau compared to ΔLBD and P681H mutants (Fig. 3E), indicating a possibility that dimerization of TLR2 is important for tau binding.Since TLR2 forms heterodimer with TLR1 and TLR6, we further examined which TLR2 heterodimer binds to oligomeric tau.We assessed tau uptake in BV2 cells, which show high expression levels of TLR1 and TLR6.To induce the formation of TLR2/TLR1 and TLR2/TLR6 heterodimers, BV2 cells were treated tau oligomers with TLR2 agonists Pam3CSK4 and Pam2CSK4, respectively (Brandt et al., 2013;Kang et al., 2009).Treatment with Pam3CSK4, but not Pam2CSK4, increased tau uptake into BV2 cells in a dose-dependent manner, suggesting that oligomeric tau binds to TLR2/TLR1 heterodimer (Supplementary Fig. 3).Interestingly, Pam3CSK4 treatment increased TLR2 expression levels and specifically enhanced colocalization of DyLight 488-oligomeric tau and TLR2 in BV2 cells (Fig. 2F-G).These results indicate that TLR2 heterodimerization increases binding of oligomeric tau as a pathogenic ligand.

Tau activates TLR2 to induce inflammatory responses in monocyte and microglial cells
To determine whether tau binding to TLR2 induces inflammatory responses, we used HEK-Blue hTLR2 cells, a human TLR2/NF-κB-SEAP reporter HEK293 cells which can detect TLR2 activation with ligand binding.Indeed, treatment of oligomeric tau increased TLR2 activation in HEK-Blue hTLR2 cells compared to control HEK-Blue Null1 cells (Fig. 3A).In THP-1 human monocyte cells which show high levels of TLR2 expression, oligomeric tau treatment increased levels of TLR2, MyD88, and NLRP3 proteins compared to untreated cells, which are players of inflammatory responses (Fig. 3B-E).Furthermore, oligomeric tau treatment drastically increased mRNA levels of proinflammatory cytokines TNF-α, IL-1β, IL-6, and IL-8 compared to untreated THP-1 cells (Fig. 3F-I).Such increase of proinflammatory cytokines was also observed in BV2 cells (Supplementary Fig. 4) and mouse primary microglia (Fig. 3J-L).These results indicate that oligomeric tau activates TLR2 and promotes inflammatory responses in both monocyte and microglial cells.

Microglial TLR2 activation promotes neuronal tau uptake
Accumulating evidence has demonstrated that tau spreads between neurons via synaptic connections (Clavaguera et al., 2009;de Calignon et al., 2012;Wu et al., 2013) and microglia contribute to tau spread by the mechanisms including secretion of tau-containing exosomes and proinflammatory cytokines (Asai et al., 2015;Wang et al., 2022).We therefore hypothesized that microglial TLR2 activation might promote tau spread between neurons.Since TLR2 was reported to be expressed with low levels in neurons and high levels in microglia (Tang et al., 2007;Dzamko et al., 2017), we first examined whether TLR2 in neurons plays a role in tau uptake.When primary cultured mouse cortical neurons were treated with oligomeric tau, tau uptake into Tlr2 KO neurons showed no significant difference compared to that of WT neurons (Fig. 4A-B).Next, we examined the effect of microglial TLR2 on neuronal tau uptake in the neuron-microglia co-culture system.Compared to single-cultured WT neurons, co-culture with WT microglia significantly increased tau uptake into WT neurons (Fig. 4A-B).On the contrary, co-culture with Tlr2 KO microglia did not show such increase of tau uptake into WT neurons (Fig. 4A-B).Interestingly, tau uptake in Tlr2 KO neurons showed no difference between single-culture and coculture with WT or Tlr2 KO microglia (Fig. 4A-B).Thus, TLR2 in both neurons and microglia are necessary for efficient tau uptake into neurons.
To address a role of TLR2 in neuronal tau uptake in vivo, we injected oligomeric tau into the hippocampus of WT and Tlr2 KO mice.After tau injection, Iba1-positive microglia highly colocalized with human oligomeric tau in the hippocampus of WT mice (Fig. 4C-D).In the hippocampus of Tlr2 KO mice, however, Iba1-positive microglia showed significantly less association with human oligomeric tau compared to WT mice (Fig. 4C-D).Furthermore, tau uptake into MAP2-positive neurons in the hippocampus was significantly reduced in Tlr2 KO mice compared to WT mice (Fig. 4E-F).These results suggest that TLR2 in neurons mediates tau uptake and TLR2 in microglia promotes neuronal tau uptake via enhancing the inflammatory responses.(A) TLR2 is activated by tau treatment.HEK-Blue Null1 or HEK-Blue hTLR2 cells were treated 200 nM oligomeric tau (oTau) in the presence or absence of Tomaralimab (100 nM or 500 nM) for 20 h.The level of secreted embryonic alkaline phosphatase (SEAP) was quantified with the optical density at 620 nm.Kruskal-Wallis with Dunn's test, n = 3. (B-E) Tau activates NLRP3 inflammasome via TLR2.THP-1 cells were co-treated with 200 nM oTau in the presence or absence of Tomaralimab (100 nM or 500 nM) for 24 or 48 h (B).The relative levels of TLR2 (C), MyD88 (D), and NLRP3 (E) were normalized to β-actin.One-way ANOVA with Tukey test, n = 5. (F-I) Tau-induced TLR2 activation increases production of proinflammatory cytokines.THP-1 cells were treated with 200 nM oTau in the presence or absence of Tomaralimab (100 nM or 500 nM) for 24 or 48 h.The mRNA levels of proinflammatory cytokines TNF-α (F), IL-1β (G), IL-6 (H), and IL-8 (I) were measured.One-way ANOVA with Tukey test, n = 4. (J-L) Tau-induced inflammation is attenuated by Tomaralimab in microglia.Mouse primary microglia were treated with 200 nM oTau in the presence or absence of Tomaralimab (100 nM or 500 nM) for 24 h.The mRNA levels of proinflammatory cytokines TNF-α (J), IL-1β (K), and IL-6 (L) were measured.One-way ANOVA with Tukey test, n = 3-5.Data are represented as mean ± SEM.

Blocking TLR2 function alleviates tau pathology progression
Next, we examined tau spread in differentiated SH-SY5Y human neuroblastoma cells using transwell culture system.We transduced differentiated SH-SY5Y cells with adenovirus expressing tau four-repeat domains (Adeno-Tau4R) or TLR2 (Adeno-TLR2).Cells were then subcultured into the insert well (Adeno-Tau4R-transduced donor cells) and bottom well (Adeno-TLR2-transduced recipient cells), and the amount of Tau4R in the recipient cells were analyzed after 3 days.In recipient cells, tau spread was increased by TLR2 transduction and Tomaralimab treatment reduced tau spread dose-dependently, to the levels comparable to the control recipient cells without TLR2 transduction (Fig. 5A-B).Similar to the increase of TLR2 levels after treatment of THP-1 cells with tau oligomers (Fig. 3B-C), Tau4R transduction into donor cells increased endogenous TLR2 level in the recipient cells after tau spread occurred in the transwell assays (Fig. 5A-B).
To assess the effect of Tomaralimab on tau spread in vivo, we injected the brain lysate of 6-month-old rTg4510 mice into the right hippocampus of 4-to 5-month-old rTg4510 mice.Since Tomaralimab exhibited a half-life of 8 ± 2 days in pigs (Arslan et al., 2012), we injected mice with Tomaralimab once a week (Fig. 5C).When analyzed the left hippocampus of rTg4510 mice after 8 weeks, number of AT8-positive neurons increased in tau-seeded rTg4510 mice compared to non-seeded rTg4510 mice, indicating tau spread to the contralateral hemisphere after tau seeding (Fig. 5D-E).On the other hand, intravenous injection of Tomaralimab significantly prevented the increase of AT8-positive neurons in the left hippocampus of rTg4510 mice, suggesting that Tomaralimab inhibits tau spread in vivo (Fig. 5D-E).
To address the effect of TLR2 immunotherapy on cognitive deficits in AD model mice, we intravenously injected Tomaralimab into 4-monthold rTg4510 or control non-carrier WT mice for 15 weeks and examined cognitive functions (Fig. 5F).Likewise, rTg4510 mice treated with vehicle showed cognitive deficits in the novel object recognition test, while rTg4510 mice injected with 30 mg/kg Tomaralimab showed improved cognitive functions (Fig. 5G).When analyzed the brains with immunohistochemistry, development of AT8-positive tau pathology in rTg4510 mice was significantly prevented with Tomaralimab treatment compared to vehicle treatment (Fig. 5H-I).Taken together, these results suggest that TLR2 mediates tau pathology progression by promoting tau spread and inflammatory responses, and blocking its function delays cognitive impairment in AD.

Discussion
By exploring RNA-seq data of human AD cohort studies, we found that TLR2 expression levels are significantly elevated in the parahippocampal gyrus of AD patients.A recent study reported that TLR2 interacts with tau fibrils to induce neuroinflammation in microglia (Dutta et al., 2023).Notably, PS19 tau transgenic mice treated with an engineered peptide that specifically blocks the TLR2-MyD88 interaction showed reduced neuroinflammation and tau deposition (Rangasamy et al., 2018;Dutta et al., 2023).On the other hand, our study highlighted that blocking TLR2 using a monoclonal antibody Tomaralimab reduces neuroinflammation and tau spread in rTg4510 mice, suggesting that TLR2 immunotherapy may alleviate tau pathology in AD.In addition to TLR2, the expression levels of other TLRs show significant changes in AD brains with different extents and regional specificities, which indicate their roles in AD pathogenesis.Indeed, TLR4 was reported to mediate microglial activation following Aβ accumulation and to induce memory loss after Aβ injection into the mouse cerebral ventricle (Udan et al., 2008;Reed-Geaghan et al., 2009;Balducci et al., 2017).Thus, the additional mechanisms how these TLRs aggravate or alleviate AD together needs to be further investigated.
Previous studies have shown that inflammatory cytokines in the brain worsen AD pathology in mouse models.For example, viral expression of IL-4 and IL-10 in the hippocampus of TgCRND8 and Tg2576 APP transgenic mice increases Aβ deposition and memory loss, respectively (Chakrabarty et al., 2012(Chakrabarty et al., , 2015)).Sustained IL-1β production also exacerbates tau pathology in 3xTg-AD mice (Ghosh et al., 2013).In this study, we found that oligomeric tau activates TLR2 and increases the production of proinflammatory cytokines in monocytes and microglial cells, which may build-up deleterious environment for neurons and promote tau spread.Recently, TLR2 was detected in extracellular vesicles as well as in plasma membranes, and cryo-electron tomography imaging visualized extracellular vesicles containing tau filaments in AD patient brains (Jeppesen et al., 2019;Fowler et al., 2023).Since microglia secrete vesicle-enveloped tau which might then be taken into neurons (Asai et al., 2015;Ruan et al., 2020), whether TLR2 functions in vesicle-mediated tau spread is a remaining question.
While TLRs are known as key receptors of innate immune responses, they are also expressed in T cells and function in adaptive immune responses (Kabelitz, 2007).Recent studies demonstrated that the number of peripheral CD8 + T cells increases in AD patients and shows a correlation with worsen memory functions, and CD8 + T cells in blood vessels infiltrate into AD brain to induce neuronal death (Gate et al., 2020;Unger et al., 2020;Chen et al., 2023).In addition, peripheral immune challenges induced by prenatal exposure to polyriboinosinicpolyribocytidilic acid (Poly I:C) induced AD-like neuropathology in WT mice and intravenous injection of IL-21 into 5XFAD mice enhanced Aβ deposition in the brain (Krstic et al., 2012;Agrawal et al., 2022).Increasing evidence indicate that dysfunction of peripheral immune responses has deleterious impact on AD pathology.Therefore, it would be also interesting to investigate whether peripheral TLR2-mediated immune responses affect the development of AD pathology, and evaluate the efficacy of Tomaralimab in the regulation of both peripheral and central immune responses.
In conclusion, we suggest that TLR2 functions as a tau receptor in neurons, microglia, and immune cells for promoting tau spread via inducing inflammatory responses, leading to memory impairments in AD.Our work demonstrate that blocking TLR2 with Tomaralimab rescues tau pathogenesis, thus providing a new therapeutic opportunity for tau pathology in AD.Fig. 5. Blocking TLR2 using Tomaralimab alleviates tau pathology progression.(A-B) Blocking TLR2 reduces tau spread in transwell culture system.Differentiated SH-SY5Y cells were transduced with adenovirus expressing tau four-repeat domains (Adeno-Tau4R, MOI 500, tau donor cells) or adenovirus expressing TLR2 (Adeno-TLR2, MOI 100, tau recipient cells).Cells were then subcultured into the insert (donor) or bottom (recipient) wells of transwell plate.Recipient cells in the bottom wells were incubated with or without Tomaralimab for 3 days.The relative levels of Tau4R in the recipient cells were examined (A) and normalized to β-actin (B).One-way ANOVA, n = 3. (C-E) Tomaralimab reduces tau spread in the hippocampus of rTg4510 mice.The right hippocampus of 4-to 5-month-old rTg4510 mice were intracranially injected with brain lysates of 6-month-old rTg4510 mice (10 % w/v in PBS, 2.5 μl) and intravenously treated with vehicle or Tomaralimab (30 mg/kg, once weekly).After 8 weeks, tau spread in the hippocampus was analyzed using immunohistochemistry (IHC) (C).Immunohistochemistry of AT8 and NeuN in the hippocampus (D).Arrows indicate the injection site.SML, stratum lacunosum moleculare; DG, dentate gyrus.The numbers of AT8-positive neurons in the left hippocampus were estimated (E).One-way ANOVA, n = 8 per group.(F-I) Intravenous injection of Tomaralimab ameliorates cognitive impairment in rTg4510 mice.The 4-month-old mice were intravenously treated with vehicle or Tomaralimab (10 or 30 mg/kg, once weekly) (F).After 14 weeks, mice were analyzed with the novel object recognition test (NOR).One-way ANOVA, n = 7-11 per group (G).After 15 weeks, tau pathology was analyzed using immunohistochemistry (IHC).
Immunohistochemistry of AT8 and NeuN in the hippocampus (H).Scale bar, 100 μm.The numbers of AT8-positive neurons were estimated (I).One-way ANOVA, n = 9-11 per group.Data are represented as mean ± SEM.

Fig. 3 .
Fig.3.Tau treatment induces TLR2 activation and inflammatory responses in monocyte and microglial cells.(A) TLR2 is activated by tau treatment.HEK-Blue Null1 or HEK-Blue hTLR2 cells were treated 200 nM oligomeric tau (oTau) in the presence or absence of Tomaralimab (100 nM or 500 nM) for 20 h.The level of secreted embryonic alkaline phosphatase (SEAP) was quantified with the optical density at 620 nm.Kruskal-Wallis with Dunn's test, n = 3. (B-E) Tau activates NLRP3 inflammasome via TLR2.THP-1 cells were co-treated with 200 nM oTau in the presence or absence of Tomaralimab (100 nM or 500 nM) for 24 or 48 h (B).The relative levels of TLR2 (C), MyD88 (D), and NLRP3 (E) were normalized to β-actin.One-way ANOVA with Tukey test, n = 5. (F-I) Tau-induced TLR2 activation increases production of proinflammatory cytokines.THP-1 cells were treated with 200 nM oTau in the presence or absence of Tomaralimab (100 nM or 500 nM) for 24 or 48 h.The mRNA levels of proinflammatory cytokines TNF-α (F), IL-1β (G), IL-6 (H), and IL-8 (I) were measured.One-way ANOVA with Tukey test, n = 4. (J-L) Tau-induced inflammation is attenuated by Tomaralimab in microglia.Mouse primary microglia were treated with 200 nM oTau in the presence or absence of Tomaralimab (100 nM or 500 nM) for 24 h.The mRNA levels of proinflammatory cytokines TNF-α (J), IL-1β (K), and IL-6 (L) were measured.One-way ANOVA with Tukey test, n = 3-5.Data are represented as mean ± SEM.