Periodic Reporting for period 2 - immunogut (Unravelling the role of aging in post-stroke gut-brain axis signalling)
Période du rapport: 2022-09-01 au 2023-08-31
Aging is a major risk factor for stroke. Post-stroke inflammation greatly affects its severity and offers an extended therapeutic time window. Age-associated changes in gut permeability are likely to regulate intestinal immune response in the elderly, particularly in the setting of stroke, predisposing elderly stroke patients to infections and poor recovery. In this project, I show that in aged mice, stroke causes a prolonged and sustained gut barrier breach that provokes intestinal immune response, leads to translocation of microbial components across the epithelial barrier and results in exacerbated neurological damage. By delineating intestinal norepinephrine dynamics after stroke, and manipulating sympathetic signaling to the gut in young and aged mice, I determined that gut barrier breach fails to recover due to a secondary inflammatory response localized to the gut in aged stroke.
Objectives
Aim 1: Testing whether aging increases post-stroke gut permeability and bacterial translocation.
Here I observed significant increases in early bacterial seeding of organs after sham or stroke surgery in aged mice, indicating a breach of the gut barrier in aged mice. I found a significant and sustained increase in intestinal permeability in aged stroke compared to young.
Aim 2: Testing whether aging alters the frequencies and biological functions of intestinal innate immune subsets in response to loss of gut barrier integrity
I have showed that a proximal and important consequence of early bacterial translocation is a massive mobilization of local innate immune responses in the intestinal lamina propria. As the intestine harbors ~70% of the body’s Monocytes/Macrophages (Mo/M), and reactive intestinal Mo/M populations generate toxic cytokines, proteases, and ROS that further damage the intestinal epithelium, a conceptually new hypothesis is that this process leads to a feed-forward cycle of bacterial translocation lamina propria innate immune toxic responses further epithelial barrier breakdown.
Aim 3. Testing whether aging increases the translocation of microbial metabolites across the intestinal barrier to the systemic circulation after stroke
Although the breach in the intestinal barrier is self-limited in young stroke, this is not the case in aged stroke where gut barrier disruption begins earlier and lasts much longer (Fig. 2b). Functional outcomes are worse in aged compared to young stroke (Fig. 1b-d). Indeed, myeloid responses are a major driver of stroke severity. In aged stroke, a critical vulnerability arises from persistent intestinal permeability that is amplified by reactive intestinal innate immune responses. The concept that a dysregulated intestinal innate immune response, enhanced in aged stroke, will worsen stroke outcome is novel and has been investigated in Aims 2-3.
Objectives
Aim 1: Testing whether aging increases post-stroke gut permeability and bacterial translocation.
Here I observed significant increases in early bacterial seeding of organs after sham or stroke surgery in aged mice, indicating a breach of the gut barrier in aged mice. I found a significant and sustained increase in intestinal permeability in aged stroke compared to young.
Aim 2: Testing whether aging alters the frequencies and biological functions of intestinal innate immune subsets in response to loss of gut barrier integrity
I have showed that a proximal and important consequence of early bacterial translocation is a massive mobilization of local innate immune responses in the intestinal lamina propria. As the intestine harbors ~70% of the body’s Monocytes/Macrophages (Mo/M), and reactive intestinal Mo/M populations generate toxic cytokines, proteases, and ROS that further damage the intestinal epithelium, a conceptually new hypothesis is that this process leads to a feed-forward cycle of bacterial translocation lamina propria innate immune toxic responses further epithelial barrier breakdown.
Aim 3. Testing whether aging increases the translocation of microbial metabolites across the intestinal barrier to the systemic circulation after stroke
Although the breach in the intestinal barrier is self-limited in young stroke, this is not the case in aged stroke where gut barrier disruption begins earlier and lasts much longer (Fig. 2b). Functional outcomes are worse in aged compared to young stroke (Fig. 1b-d). Indeed, myeloid responses are a major driver of stroke severity. In aged stroke, a critical vulnerability arises from persistent intestinal permeability that is amplified by reactive intestinal innate immune responses. The concept that a dysregulated intestinal innate immune response, enhanced in aged stroke, will worsen stroke outcome is novel and has been investigated in Aims 2-3.
Aim 1: Testing whether aging increases post-stroke gut permeability and bacterial translocation
To induce stroke in aged mice, I have established in the lab the distal middle cerebral artery occlusion (dMCAo) model (Fig. 1a-c). I found that ischemic infarcts are significantly larger in aged (20-22 mo) than in young (3 mo) male C57bl mice (Fig. 1d)1. I have found a significant decrease in the levels of the epithelial tight-junction molecules Zonula Occludens-1 (ZO-1) (Fig. 1e-f), and E-cadherin (Fig. 1g) in the intestines of aged mice. interestingly.
WP2. Confirm loss of barrier integrity using FITC-dextran, measurements of LPS, and quantification of bacteria and 16S sequencing in distal organs.
I observed significant increases in early bacterial seeding of organs after sham surgery or dMCAo at 2h in aged mice indicating an earlier breach of the gut barrier in aged mice (Fig. 2a). In additional experiments, I assessed gut permeability using oral administration of FITC-dextran out to seven days after dMCAo. I found a significant and sustained increase in intestinal permeability in aged stroke compared to young (Fig. 2b). In a small series of stroke patients, there was a 22.2% incidence of secondary bacterial infection emanating from intestinal bacteria that correlated with increased mortality3. I found increased levels of serum LPS in aged stroked mice (Fig. 6c). Bacterial dissemination, indicated by colonies grown from organs’ extracts harvested at different time points post-stroke, showed significant increase in aged stroke (Fig. 2d), that positively correlated with the magnitude of gut barrier breach in these mice, but not in young stroke (Fig. 2e). These data suggest that in aged stroke, the intestinal response, both in terms of microbial translocation as well as the associated lamina propria immune response, may be a major negative determinant of stroke outcome.
Aim 2: Testing whether aging alters the frequencies and biological functions of intestinal innate immune subsets in response to loss of gut barrier integrity
I showed that in aged mice, almost all cellular population of the innate immune response are increased in both colon (Fig. 3a) and jejunum (Fig. 3b) of aged mice post-stroke. Additionally, I found that pro-inflammatory Mo/M (P2) increase in the jejunum after stroke.
Aim 3. Testing whether aging increases the translocation of microbial metabolites across the intestinal barrier to the systemic circulation after stroke
To induce stroke in aged mice, I have established in the lab the distal middle cerebral artery occlusion (dMCAo) model (Fig. 1a-c). I found that ischemic infarcts are significantly larger in aged (20-22 mo) than in young (3 mo) male C57bl mice (Fig. 1d)1. I have found a significant decrease in the levels of the epithelial tight-junction molecules Zonula Occludens-1 (ZO-1) (Fig. 1e-f), and E-cadherin (Fig. 1g) in the intestines of aged mice. interestingly.
WP2. Confirm loss of barrier integrity using FITC-dextran, measurements of LPS, and quantification of bacteria and 16S sequencing in distal organs.
I observed significant increases in early bacterial seeding of organs after sham surgery or dMCAo at 2h in aged mice indicating an earlier breach of the gut barrier in aged mice (Fig. 2a). In additional experiments, I assessed gut permeability using oral administration of FITC-dextran out to seven days after dMCAo. I found a significant and sustained increase in intestinal permeability in aged stroke compared to young (Fig. 2b). In a small series of stroke patients, there was a 22.2% incidence of secondary bacterial infection emanating from intestinal bacteria that correlated with increased mortality3. I found increased levels of serum LPS in aged stroked mice (Fig. 6c). Bacterial dissemination, indicated by colonies grown from organs’ extracts harvested at different time points post-stroke, showed significant increase in aged stroke (Fig. 2d), that positively correlated with the magnitude of gut barrier breach in these mice, but not in young stroke (Fig. 2e). These data suggest that in aged stroke, the intestinal response, both in terms of microbial translocation as well as the associated lamina propria immune response, may be a major negative determinant of stroke outcome.
Aim 2: Testing whether aging alters the frequencies and biological functions of intestinal innate immune subsets in response to loss of gut barrier integrity
I showed that in aged mice, almost all cellular population of the innate immune response are increased in both colon (Fig. 3a) and jejunum (Fig. 3b) of aged mice post-stroke. Additionally, I found that pro-inflammatory Mo/M (P2) increase in the jejunum after stroke.
Aim 3. Testing whether aging increases the translocation of microbial metabolites across the intestinal barrier to the systemic circulation after stroke
These findings suggest that peripheral inhibition of the pro-inflammatory PGE2-EP2 signalling pathway is sufficient to decrease intestinal inflammation and restore healthy gut functions in aging mice after stroke. I suggest that myeloid cell reprogramming from EP2 blockade in the periphery improves immune cell responses in the aged gut that further improves neurological recovery, and call for similar validation and exploration in human-focused studies.
These results are exciting, since they imply that reprogramming myeloid cells to rejuvenate immune responses in the aged gut, may improve post-stroke inflammation. This could be a compelling approach to treat many other age-associated diseases.
References
1 Doyle, K. P., Fathali, N., Siddiqui, M. R. & Buckwalter, M. S. Distal hypoxic stroke: a new mouse model of stroke with high throughput, low variability and a quantifiable functional deficit. J Neurosci Methods 207, 31-40 (2012).
2 Liu, Q. et al. Peripheral TREM1 responses to brain and intestinal immunogens amplify stroke severity. Nat Immunol 20, 1023-1034 (2019).
3 Stanley, D. et al. Translocation and dissemination of commensal bacteria in post-stroke infection. Nat Med 22, 1277-1284 (2016).
4 Minhas, P. S. et al. Restoring metabolism of myeloid cells reverses cognitive decline in ageing. Nature 590, 122-128 (2021).
5 Johansson, J. U. et al. Suppression of inflammation with conditional deletion of the prostaglandin E2 EP2 receptor in macrophages and brain microglia. J Neurosci 33, 16016-16032 (2013).
6 Johansson, J. U. et al. Prostaglandin signaling suppresses beneficial microglial function in Alzheimer's disease models. J Clin Invest 125, 350-364 (2015).
7 Liu, Q. et al. PGE2 signaling via the neuronal EP2 receptor increases injury in a model of cerebral ischemia. Proc Natl Acad Sci U S A 116, 10019-10024 (2019).
These results are exciting, since they imply that reprogramming myeloid cells to rejuvenate immune responses in the aged gut, may improve post-stroke inflammation. This could be a compelling approach to treat many other age-associated diseases.
References
1 Doyle, K. P., Fathali, N., Siddiqui, M. R. & Buckwalter, M. S. Distal hypoxic stroke: a new mouse model of stroke with high throughput, low variability and a quantifiable functional deficit. J Neurosci Methods 207, 31-40 (2012).
2 Liu, Q. et al. Peripheral TREM1 responses to brain and intestinal immunogens amplify stroke severity. Nat Immunol 20, 1023-1034 (2019).
3 Stanley, D. et al. Translocation and dissemination of commensal bacteria in post-stroke infection. Nat Med 22, 1277-1284 (2016).
4 Minhas, P. S. et al. Restoring metabolism of myeloid cells reverses cognitive decline in ageing. Nature 590, 122-128 (2021).
5 Johansson, J. U. et al. Suppression of inflammation with conditional deletion of the prostaglandin E2 EP2 receptor in macrophages and brain microglia. J Neurosci 33, 16016-16032 (2013).
6 Johansson, J. U. et al. Prostaglandin signaling suppresses beneficial microglial function in Alzheimer's disease models. J Clin Invest 125, 350-364 (2015).
7 Liu, Q. et al. PGE2 signaling via the neuronal EP2 receptor increases injury in a model of cerebral ischemia. Proc Natl Acad Sci U S A 116, 10019-10024 (2019).