Hypothalamic TLR2 triggers sickness behavior via a microglia-neuronal axis
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1 Hypothalamic TLR triggers sickness behavior via a microglia-neuronal axis Sungho Jin, *, Jae Geun Kim,, *, Jeong Woo Park, Marco Koch,, Tamas L. Horvath and Byung Ju Lee Department of Biological Sciences, University of Ulsan, Ulsan, 68-79, Republic of Korea; Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Incheon, 6-77, Republic of Korea, Program in Integrative Cell Signaling and Neurobiology of Metabolism, Section of Comparative Medicine, Yale University School of Medicine, New Haven, CT 65, USA; Institute of Anatomy, University of Leipzig, Leipzig, Germany. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to T.L.H. or B.J.L. ( tamas.horvath@yale.edu; bjlee@ulsan.ac.kr) Supplementary Information Supplementary Figure Legends Supplementary Figure S. CSK-induced activation of microglia was absent in the hypothalamic arcuate nucleus (Arc) of TLR KO mice. Representative images show immunosignals of Iba- in the hypothalamic arcuate nucleus (Arc). Intracerebroventricular injection of CSK () induced activation of microglia in the Arc of wild-type (WT) mice, but not in that of TLR KO mice. = control. Scale bar = µm ( µm for higher magnification view in inset). Supplementary Figure S. Effect of CSK on the morphological changes of microglia in the hypothalamus, hippocampus, cortex and circumventricular organs (CVOs). (A,B) Representative images showing immunosignals of Iba--positive microglia in the mouse hippocampus and cortex reveal that -induced central stimulation of TLR signaling did not cause morphological change of microglia in these brain regions. (C,D) Representative images show immunosignals of Iba--positive microglia in the CVOs, including area postrema (AP) (C) and organum vasculosum of the lamina terminalis (OVLT) (D). Scale bar = µm ( µm for higher magnification view in inset). = vehicle treated control group. = CSK treated group.
2 Supplementary Figure S. Microglia play a role in CSK-induced anorexia and body weight loss. (A) Representative images ( 6 mice analyzed) show immunosignals of Iba- in the hypothalamic Arc. Scale bar = µm. = vehicle treated control group. = CSK treated group. PBS-lip = liposome-encapsulated PBS treated group. Clo-lip = liposome-encapsulated clodronate treated group. (B) Clo-lip significantly reduced the number of Iba--positive microglia in the Arc (, n = sections/6 mice;, n = sections/6 mice; Clo-lip, 6 sections/ mice; Clo-lip +, n = 8 sections/ mice; P <. versus mice injected with PBS-lip by unpaired two-tailed Student's t-tests;, not significant). (C,D) -induced anorexia (C) and weight loss (D) were significantly attenuated by administration of Clo-lip prior to icv injection. (, n = mice;, n = ; Clo-lip, n = ; Clolip +, n = ; *P <.5, **P <., P <. by two-way ANOVA). All data are presented as mean ± s.e.m. Supplementary Figure S. TLR-induced changes in mrna expression of genes involved in inflammatory processes in the hypothalamus. (A) Real-time qpcr analysis revealed CSK ()- induced changes in mrna expression of TLR, IL-β and TNF-α in the hypothalamus and cortex (n = 6 values/ mice; **P <., P <. by two-way ANOVA;, not significant). (B,C) To further confirm the effect of TLR activation on hypothalamic inflammation, real-time qpcr was performed using mrna samples from the hypothalami of TLR KO (B, n = 9 values/ mice; P <. by twoway ANOVA;, not significant) and MyD88 KO (C, n = 6 values/ mice; P <. by two-way ANOVA;, not significant) mice h after icv administration of. The -induced increases in mrna expression of genes associated with inflammatory processes were almost completely suppressed in the TLR and MyD88 KO mice. = control. WT = wild-type mice. Supplementary Figure S5. Central activation of TLR increases the number of c-fos signals in the hypothalamic arcuate nucleus (Arc). To determine the effect of CSK () treatment on neuronal activity of the Arc, immunohistochemical observation of c-fos activity was performed using sections from mice after icv treatment with, in comparison with control (). Representative images (A) and calculated data (B) reveal that the number of c-fos-positive cells in the Arc increased by h after icv administration of (, n = 8 sections/ mice;, n = 6 sections/ mice; P <. by unpaired two-tailed Student's t-tests). All data are presented as mean ± s.e.m. Scale bar = µm.
3 Supplementary Figure S6. Simplified diagram showing TLR-induced microglial activation that stimulates POMC neuronal activity and induces anorexia. TLR signaling contributes to microglial activation and induces inflammatory reactions via NF-κB and COX pathways in the hypothalamic Arc. The activated microglia, characterized by change in their morphology, regulate synaptic input organization onto the POMC neurons through increased contact area onto the surface of POMC soma. Consequently, inhibitory GABAergic inputs decrease and excitatory glutamatergic inputs increase on the surface of POMC soma contacted by microglia. These synaptic changes result in activation of the POMC neurons, which causes anorexia via α-msh binding to melanocortin (MC) receptors on the targets of POMC neurons such as neurons in the paraventricular nucleus (PVN).
4 Supplementary Figure S. CSK-induced activation of microglia was absent in the hypothalamic arcuate nucleus (Arc) of TLR KO mice WT- WT- TLR KO-
5 Supplementary Figure S. Effect of CSK on the morphological changes of microglia in the hypothalamus, hippocampus, cortex and circumventricular organs (CVOs) A Hypothalamus Arc Hippocampus Cortex B C AP D OVLT
6 Supplementary Figure S. Microglia play a role in CSK-induced anorexia and body weight loss A Iba- Clo-lip PBS-lip B # of microglia cells in Arc 8 6 Clo-lip Clo-lip + C h food intake (g) * ** Clo-lip Clo-lip + D Body weight change (g) * ** Clo-lip Clo-lip +
7 Supplementary Figure S. TLR-induced changes in mrna expression of genes involved in inflammatory processes in the hypothalamus A Hypothalamus TLR 5 IL-β TNF-α 5 5 TLR 5 ** IL-β Cortex TNF-α B TLR 8 6 WT WT TLR KO TLR KO MyD88 COX- TNF-α 8 6 WT WT TLR KO TLR KO IL-β 6 5 mpges- 8 6 C TLR 5 5 WT WT MyD88 KO MyD88 KO ** MyD88 COX TNF-α WT WT MyD88 KO MyD88 KO IL-β mpges- 5
8 Supplementary Figure S5. Central activation of TLR increases the number of c-fos signals in the hypothalamic arcuate nucleus (Arc) 6 5 am c-fos P L c-fos 7 C T B Number of c-fos -positive cells in Arc A
9 Supplementary Figure S6. Simplified diagram showing TLR-induced microglial activation that stimulates POMC neuronal activity and induces anorexia Decreased food intake Neurons in PVN α-msh MCR Ligand TLR/ MyD88 NF-κB/COX Resting microglia Activated microglia POMC neurons Glutamatergic Synaptic input GABAergic Synaptic input
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