Acute Exposure to the Food-Borne Pathogen Listeria monocytogenes Does Not Induce α-Synuclein Pathology in the Colonic ENS of Nonhuman Primates

Figures & data

Table 1 Information per Study Subject on Treatment, Fecal and Blood Shedding, Colitis Score, Clinical and Pathological Remarks.

Animal	Age (Years)	Sex	Weight (kg)	Treatment	Fecal	Blood	Clinical Remarks	Colitis Score	Pathological Description
MO1	9	F	3.9	Mock^b	Negative	Negative	Unremarkable	1	No inflammation noted. Tissue is unremarkable.
MO2	7	F	5.3	Mock^a	Negative	Negative	Unremarkable	1	No inflammation noted. Tissue is unremarkable.
MO3	10	F	8.8	Mock^b	Negative	Negative	Unremarkable	1.5	Sparse eosinophilic infiltration of the lamina propria.
LM1	10	F	4.7	LM	Negative	Negative	Animal reported for vomit 12 days after inoculation.	1	No inflammation noted. Tissue is unremarkable.
LM2	6	F	5.3	LM^a	Negative	Negative	Unremarkable	1	No inflammation noted. Tissue is unremarkable.
LM3	10	F	4.8	LM^c	Negative	Negative	Animal reported for diarrhea in cage pan 3 days after inoculation.	2	Dead cells present at the base of the lamina propria flanked by foamy macrophages, increased (relative to normal) lymphocytes and plasma cells.
LM4	7	F	5	LM	Negative	Negative	Unremarkable	2	Sparse to dense population of eosinophils in the lamina propria.
LM5	7	F	5.4	LM	Negative	Negative	Animal reported for diarrhea in cage pan 3 days after inoculation.	2	Pockets of dense and dead cells in the upper region of the lamina propria.
LM6	7	F	6.1	LM	Negative	Negative	Animal reported for leftover chow and inappetence 3 days after inoculation.	2	Pockets of dense cells and dead cells in the upper region of the lamina propria.
LM7	6	F	3.6	LM^a	Negative	Negative	Unremarkable	1.5	Sparse eosinophilic infiltration of the lamina propria and glandular separation indicative of an immune response.
LM8	11	F	6.7	LM^c	Negative	Negative	Unremarkable	2	Small clusters of foamy macrophages at the base of the lamina propria and pockets of dead cells in the mucosa.
LM9	7	F	4	LM^a	Negative	Negative	Unremarkable	3	Dense infiltration of eosinophils in the lamina propria and dense pockets of dead cells.
LM10	6	F	3.1	LM	Negative	Negative	Unremarkable	2	Areas of dense cells in the lamina propria and population of eosinophils across the mucosa.

Superscripts denote animals with one previous LM inoculation at either (a) 12–17 months or (b) 23–26 months prior to terminal inoculation/necropsy, while (c) denotes animals with two previous lm inoculations 36–48 months prior to their terminal inoculation/necropsy. Colitis scores are defined in the “Materials and Methods” section.

Table 2 Primary Antibodies Used for immunohistochemistry and Immunofluorescence

Antigen	Marker	Company	Species	Catalog No.	Lot No.	Dilution
HLA-DR	Antigen-presenting cell surface	Dako	Mouse	M0746	20071350	1:200
CD3	T cells (brightfield)	Dako	Mouse	M7254	20043703	1:100
CD3	T cells (fluorescence)	Abcam	Rat	AB11089	GR33823902	1:100
CD20	B cells	Thermo Fisher	Rabbit	RB9013-PO	9013P1705F	1:100
8-OHdG	Oxidative stress	Abcam	Mouse	AB48508	GR3250401	1:800
PGP 9.5	Pan-neuronal (brightfield)	Abcam	Rabbit	AB503067	GR45123-2	1:400
PGP 9.5	Pan-neuronal (fluorescence)	Invitrogen	Chicken	PA1-10011	VJ3103801	1:500
α-syn	Pre-synaptic protein	Abcam	Rabbit	AB138501	GR221666-8	1:400
p-α-syn	α-Syn phosphorylated	Wako	Mouse	015–25191	LKM6409	1:400

Abbreviations: HLA-DR, human leukocyte antigen DR; CD3, cluster of differentiation 3; CD20, cluster of differentiation 20; 8-OHdG, 8-hydroxy-2ʹ-deoxyguanonsine; PGP 9.5, protein gene product 9.5; α-syn, alpha-synuclein; p-α-syn, alpha synuclein phosphorylated at serine 129.

Table 3 Cecum Typhlitis Score per Study Subject

Animal	Typhlitis Score	Pathological Description
MO1	2	Mild scatterings of hemosiderin across entire cecum and a notable population of eosinophils.
MO2	1.5	Very small resident increase in inflammatory cells
MO3	2	Increased cell density
LM1	2	Nodular proliferation of a patch of eosinophils. Greater densities of eosinophils throughout the sample.
LM2	1.5	The tissue presents with inflammation but is overall minimal in its colitis score.
LM3	2	Elevated cell population. Dense areas across the lamina propria.
LM4	1	No inflammation noted. Tissue is unremarkable.
LM5	1	No inflammation noted. Tissue is unremarkable.
LM6	1	No inflammation noted. Tissue is unremarkable.
LM7	1.5	Marginally above baseline in areas of cell density.
LM8	1.5	Slightly increased cell density near the crypts in the lamina propria.
LM9	2	Cells in the lamina propria are denser. There is a notable population of eosinophils.
LM10	1	No inflammation noted. Tissue is unremarkable.

Figure 1 Representative HE-stained proximal colon sections images of cynomolgus macaques of no-colitis control (A, B); MO3) and moderate colitis (C, D; LM9) after oral LM inoculation. (A, C) at 4× magnification. (B, D) at 40× magnification. Note increased lymphoplasmacytic and eosinophilic inflammation in the lamina propria with glandular displacement in the colitis subject. Black arrow, plasma cell; circle, eosinophil; square, lymphocyte. Scale bar = 100µm for all panels.

Abbreviations: A, adipocytes; G, gland; LP, lamina propria; MP, myenteric plexus; S, serosal surface.

Figure 2 Images of proximal colon of cynomolgus macaques treated with mock (MO1) or LM (LM7) inoculations immunostained against markers of inflammation (A-C; HLA-DR, CD3, CD20) and oxidative stress (D; 8-OHdG) and their %AAT expression and OD expression (mean ± SEM). Scale bar = 25µm for all panels.

Figure 3 Images of proximal colon of cynomolgus macaques treated with mock (MO2) or LM (LM9) inoculations immunostained against pan-neuronal maker PGP9.5 (A), α-syn (B), p-α –syn (C, black arrows) and their %AAT expression and OD expression (mean ± SEM). Lewy body in human PD brain (D, black arrowhead). Scale bar = 25µm for all panels.

Figure 4 Double immunofluorescence images of proximal colon showing α-syn colocalization with pan-neuronal marker PGP9.5 in myenteric ganglia of no-colitis control (MO2) and colitis (LM9) cynomolgus macaques. Scale bar = 50µm for all panels.

Figure 5 Pearson correlations graphs comparing colitis score vs α-syn optical density (A), HLA-DR %AAT vs α-syn OD (B), colitis score vs α-syn %AAT (C), and HLA-DR %AAT vs α-syn %AAT (D). Each point represents average per subject; the blue points correspond to Mock- and red points to LM- inoculated subjects.

Figure 6 Triple immunofluorescence images of proximal colon sections showing α -syn-ir, HLA-DR-ir and CD3-ir in myenteric ganglia of no-colitis (MO2) and colitis (LM1) subjects. Scale bar = 50µm for all panels.