Behavioral implications of the dynamic regulation of microglia: the influence of a day versus a lifetime

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Silas Gilbert
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1 Behavioral implications of the dynamic regulation of microglia: the influence of a day versus a lifetime Laura K. Fonken Postdoctoral Fellow University of Colorado March 20, 2017

About me: Research Timeline 2008 2009 2010 2011 2012 2013 2014 2015 2016 SYR OSU OSU CU BS, Biology and Psychology Syracuse University Funding Research Assistant Ohio State

2 About me: Research Timeline SYR OSU OSU CU BS, Biology and Psychology Syracuse University Funding Research Assistant Ohio State University * OSU fellowship PhD, Neuroscience, Ohio State University AHA fellowship * * Postdoctoral Research Fellow University of Colorado * NIH NRSA fellowship NARSAD grant

3 About me: Research Timeline SYR OSU OSU Projects CU Early life experience on development Circadian disruption and mood 1 st author co-author Seasonal immune regulation Circadian disruption and obesity Air pollution and inflammation Circadian disruption and immune mir-155 and inflammatory disorders Circadian regulation of neuroinflammation Neuroinflammation and aging Review papers Stress induced neuroinflammation

4 Nervous Immune Endocrine MENTAL HEALTH

5 Immune to brain signaling Diffusion & active transport Legend Cytokine Microglia Macrophage Peripheral infection or injury Vagus nerve afferents

6 Microglia are the primary innate immune cell in the brain Legend MHCII TLR RAGE Cytokine Detections of peripheral immune signal

7 Immune to brain signaling Diffusion & active transport Legend Cytokine Microglia Macrophage Peripheral infection or injury Vagus nerve afferents Cytokine synthesis Neural changes Sickness behaviors

8 Sickness and depression Lethargy Anhedonia Social withdrawal Sleep disturbances Pain sensitivity Psychomotor agitation/slowing

9 A DAY A LIFETIME

10 Dynamic regulation of microglia phenotype 1. Circadian regulation of microglia 2. Diminished circadian rhythms with age 3. Age-associated accumulation of danger signals 4. Future directions

13 H1N1 antibody titer was 4X greater for individuals vaccinated in the morning compared to the afternoon. Long et al, Vaccine, 2016

14 Are there circadian differences in sickness behavior following infection? Saline or lipopolysaccharide ZT 12 Zeitgeber Time (ZT) Inactive phase ZT 18 ZT6 12 ZT 0 Active phase 4 ZT16 ZT

15 Sickness and depression Lethargy Anhedonia Social withdrawal Sleep disturbances Pain sensitivity Psychomotor agitation/slowing

16 Juvenile social investigation

Behavioral sickness response is suppressed during the active phase Time investigating (s) 150 100

17 Behavioral sickness response is suppressed during the active phase Time investigating (s) * * Baseline 3h 8h 24h Saline - ZT6 LPS - ZT6 Saline - ZT16 LPS - ZT16 Fonken et al, BBI, 2015

18 Behavioral sickness response is suppressed during the active phase % sucrose intake ZT6 - Saline ZT16 - Saline ZT6 - LPS ZT16 - LPS 0 Baseline Injection Recovery

19 Are there circadian differences in hippocampal cytokine production following infection? Saline or lipopolysaccharide Inactive phase ZT6 Active phase ZT16

20 The hippocampal inflammatory response is reduced in the active phase IL-1β gene expression IL-1b protein Relative mrna Inactive Active * pg/100ug tissue * 0 Saline LPS 0.0 Saline LPS Fonken et al, BBI, 2015

21 Suprachiasmatic nucleus (SCN) the master circadian pacemaker atop a hierarchy of oscillators SCN Peripheral Clocks

22 CLOCK BMAL1 CRYs PERs PERs CRYs CRYs PERs CRYs CK1ε/δ PERs CRYs Nucleus Cytoplasm

23 > 10% of the mammalian transcriptome is under circadian control

24 Do microglia rhythmically express circadian clock genes? Zeitgeber Time (ZT) ZT 18 ZT ZT 6 ZT 0 Percoll gradient myelin microglia 1X PBS 40% P 70% P

25 Clock genes are rhythmically expressed in microglia Relative mrna expression Per Zeitgeber time Per Zeitgeber time Fonken et al, BBI, 2015

Clock genes are rhythmically expressed in microglia Relative mrna expression 8 6 4 2 0 Rev-erb

26 Clock genes are rhythmically expressed in microglia Relative mrna expression Rev-erb Zeitgeber time BMAL Zeitgeber time Fonken et al, BBI, 2015

27 > 10% of the mammalian transcriptome is under circadian control

28 Microglia exhibit diurnal variation in basal cytokine expression Relative mrna expression IL-1b Zeitgeber time TNFa 0 6 Z Fonken et al, BBI, 2015

29 Do differences in basal cytokines indicate a difference in reactivity?

30 Microglia exhibit diurnal differences in response to an ex vivo LPS challenge IL1b mrna expression ZT0 ZT6 ZT12 ZT18 0ng LPS 10ng LPS 100ng LPS Fonken et al, BBI, 2015

31 + - inflammatory response

32 Aging Neuroimmune changes Circadian disruption + - inflammatory response Hypothesis: Aging disrupts diurnal rhythms in microglia, which may contributes to heightened neuroinflammatory responses throughout the day

33 Dynamic regulation of microglia phenotype 1. Circadian regulation of microglia 2. Diminished circadian rhythms with age 3. Age-associated accumulation of danger signals 4. Future directions

34 Fisher 344 Brown Norway Rats

35 Aging disrupts rhythmic expression of Per genes in microglia Relative mrna expression Per1 Young ~ Aged Zeitgeber time Per2 Young ~ Aged ~ Zeitgeber time Fonken et al, Neurobiol Aging, 2016

36 IL-1β is rhythmically expressed in young but not aged microglia 3 2 IL-1b Young ~ Aged Zeitgeber time Fonken et al, Neurobiol Aging, 2016

37 Diurnal differences in inflammatory potential are not apparent in aged microglia IL-1b mrna expression Young Aged Relative mrna expression ZT6 ZT ZT6 ZT18 0ng/ml LPS 10ng/ml LPS 100ng/ml LPS Fonken et al, Neurobiol Aging, 2016

38 Young Aged inflammatory response inflammatory response

39 Dynamic regulation of microglia phenotype 1. Circadian regulation of microglia 2. Diminished circadian rhythms with age 3. Age-associated accumulation of danger signals 4. Future directions

40 Aged rats have prolonged hippocampal cytokine expression following E. coli Relative mrna expression * * * IL1β IL18 TNF-α IL10 Young - saline Young - E. coli Aged - saline Aged - E. coli 4 days IP Vehicle or E. coli Fonken et al, J Neurosci, 2016

41 Immune to brain signaling Diffusion & active transport Legend Cytokine Microglia Macrophage Peripheral infection or injury Vagus nerve afferents Cytokine synthesis Neural changes Behavioral changes

(% baseline) 150 100 50 0 * * Base 8 h 24 h 4 day *

42 Aged rats show prolonged reductions in social exploration following infection Social investigation (% baseline) * * Base 8 h 24 h 4 day * Young - Saline Young - E. coli Aged - Saline Aged - E. coli

43 Aged rats have reduced sucrose preference following infection 100 * Young - Saline Sucrose preference (% total liquid intake) * Old - Saline Young - E. coli Old - E. coli 0 D0 D1 D4

44 Aged rats show prolonged cognitive impairments following infection Context A IP Vehicle or E. coli Shock 3 days 4 days Context B

45 Aged rats show prolonged cognitive impairments following infection Context A Vehicle E. coli Freezing % * 0 Young Old Context A IP Vehicle or E. coli 3 days 4 days Context B

Legend MHCII TLR RAGE Cytokine Priming of the neuroinflammatory response Surveillant Activated Young Inflammatory challenge + cytokines + duration

46 Legend MHCII TLR RAGE Cytokine Priming of the neuroinflammatory response Surveillant Activated Young Inflammatory challenge + cytokines + duration of activation + behavioral sickness response Primed Hyper-activated Aged +++ cytokines +++ duration of activation +++ behavioral sickness response

47 HMGB1 is a danger signal that may Healthy cell prime microglia Passive release Necrotic cell H H H Tissue damage H H H H H

48 HMGB1 is a danger signal that may prime microglia Healthy cell Active release H H H Inflammation Stress H Legend TLR RAGE HMGB1 H H H H

49 HMGB1 is elevated in the hippocampus and CSF of aged rats Hippocampal HMGB1 protein (hmgb1:b actin) HMGB1 B-actin Hippocampus Cerebrospinal fluid 1.0 * Young * Old CSF HMGB1 protein (pg/ml) Young Old Fonken et al, J Neurosci, 2016

50 HMGB1 mrna is elevated in old nondiseased human brain Relative HMGB1 mrna expression Adult (18-55) Old (56-75) * HMGB1 mrna r = 0.50 p = Age Fonken et al, unpublished observation

51 Nuclear co-localization of HMGB1 is reduced in microglia Fonken et al, J Neurosci, 2016

52 HMGB1+ microglia (% total CD11b+) * * Young Old 20 CA1 DG

53 Structure of HMGB1 N A box B box acid tail C

54 Does blockade of HMGB1 prevent microglia priming in the aged brain? ICM Vehicle or Box A 24 h Plate cells with LPS for 3h

55 IL-1β expression is reduced in microglia from aged rats pre-treated with Box A IL-1β * Young - Control Young - BoxA Aged - Control Aged - BoxA LPS (ng/ml) Fonken et al, J Neurosci, 2016

Legend MHCII TLR RAGE HMGB1 H Box-A Cytokine Aged Aged + Box-A H Reducing age-associated priming of H H H Box-A De-sensitized H the neuroinflammatory response Primed H

56 Legend MHCII TLR RAGE HMGB1 H Box-A Cytokine Aged Aged + Box-A H Reducing age-associated priming of H H H Box-A De-sensitized H the neuroinflammatory response Primed H Inflammatory challenge Hyper-activated H H H Activated H H H +++ cytokines +++ activation time +++ sickness behavior + cytokines + activation time + sickness behavior H H H H

57 Does blockade of HMGB1 prevent heightened immune responses in aged hippocampus? ICM Vehicle or Box A 24 h IP E. coli Collect tissue 4day

58 Pre-treating aged rats with Box A prevents the exaggerated inflammatory response IL-1β protein pg/100ug Hippocampal IL-1β 4 days post-e. coli Young * Aged Vehicle Box A Fonken et al, J Neurosci, 2016

59 Does blockade of HMGB1 prevent behavioral impairments in aged rats post-e. coli? ICM Vehicle or Box A 24 h Context A IP Vehicle or E. coli Shock 3 days 4 days Context B

60 Box A prevents age-associated cognitive impairments following immune challenge Freezing % Conditioned context * Saline - Vehicle Saline - BoxA E. coli - Vehicle E. coli - BoxA 0 Young Old Context A ICM Vehicle or Box A IP Vehicle or E. coli 3 days 24 h 4 days Context B Fonken et al, J Neurosci, 2016

61 A DAY A LIFETIME

62 Future directions Establish the neural circuitry mediating affective components of the behavioral sickness response Determine whether there are sex-differences in neuroinflammatory responses to stress Explore the development of circadian rhythms in microglia and establish whether environmental lighting influences microglia in early life

63 Overarching hypothesis The central nucleus of the amygdala is a key early neuroinflammation sensor that initiates and propagates the sickness response.

64 Aim 1: Is central amygdala activation critical for the behavioral sickness response? Saline (0 mg) 1 mg CNO 3 mg CNO Firing Rate (z) CNO time (min) IP CNO injection in a Gi DREADDexpressing neuronal population reduces neuronal firing (courtesy of M. Saddoris). Firing Rate (z) CNO 30 t

65 Aim 2: Interrogate amygdala circuits based on immune activation history

DREADD CeA NTS Pathway specific activation of PL

66 Aim 3: Functional mapping and projectionspecific manipulation of amygdala inputs Cav-Cre DIO-Gi DREADD CeA NTS Pathway specific activation of PL neurons using Gq DREADD (courtesy of M. Baratta).

Thank you! University of Colorado Dr. Steven Maier Dr.

Mike Weber Maier/Watkins lab Ohio State University Dr.

67 Thank you! University of Colorado Dr. Steven Maier Dr. Linda Watkins Dr. Michael Baratta Dr. Ruth Barrientos Heather D Angelo Rachel Daut Dr. Matthew Frank Dr. Andrew Gaudet Meagan Kitt Dr. Mike Weber Maier/Watkins lab Ohio State University Dr. Randy Nelson Dr. Courtney DeVries Nelson/DeVries lab Dr. Jonathan Godbout Dr. Diana Norden

68 Questions?

The Role of the Immune System in Translating Early-Life Experience to Later-Life Brain and Behavior

The Role of the Immune System in Translating Early-Life Experience to Later-Life Brain and Behavior STACI D. BILBO DEPARTMENT OF PSYCHOLOGY AND NEUROSCIENCE DUKE UNIVERSITY Thanks to! Dr. Mark Hutchinson