All mice detailed in the following experiments were of the strain C57BL/6. Male only or female only mice were used in different experiment. Mice (8–12 weeks old) were housed in groups of four to six, under a 12-h/12-h light-dark cycle at 19–23°C, with water and normal chow diet (RM1, SDS, United Kingdom) ad libitum. All mice were maintained at the Biomedical Research Facility, Southampton General Hospital, Southampton, United Kingdom.

Brain homogenate (10% w/v) from normal mice (NBH) or from terminal ME7 prion mice (ME7) was directly injected into the dorsal hippocampi of mice using a stereotaxic frame. Female C57BL/6 mice (10–12 weeks) were anesthetized with a ketamine/rompun mixture (85 mg/kg and 13 mg/kg), and the incision area was shaved with hair clippers (Wella, United Kingdom) and sterilized with 70% alcohol. Lacri-lube (Allergan, United Kingdom) was applied to each eye to prevent drying that comes from the removal of the blink reflex under anesthesia. The mouse was fitted to a stereotactic frame (Kopf Instruments, United States) using 45° atraumatic ear bars and an incisor bar (Kopf Instruments, United States). A sagittal incision was made exposing the skull, allowing for the location of bregma and burr holes were drilled bilaterally into the skull using a dentist’s drill, taking care not to damage the dura mater, at the appropriate coordinates: 2.0 mm posterior to bregma and ± 1.7 mm lateral to the midline. A 10 μl syringe with a 26s-gauge needle (Hamilton, United Kingdom) was inserted into the brain to a depth of 1.6 mm, and 1 μl of brain homogenate was injected at a flow rate of 1 μl/min. The needle remained in place for 2 min to allow for bolus diffusion, before being slowly removed. The incision was closed with sutures before the mice were placed in a heated chamber to recover. After recovery, mouse appearance was regularly checked over the following week, and weights were measured weekly thereafter, to ensure animal welfare was maintained.

Plastic cylinders, 20 cm long and 6.8 cm in diameter, were filled with 190 grams of normal chow food pellets (RM1, SDS) and placed in the cages. Mice were habituated once with a full tube in a group cage and then twice in individual cages with a full burrowing tube to establish baseline before weekly testing. For testing, mice were placed individually in cages overnight and the remaining pellets at the end of each session were weighed, with the amount displaced (“burrowed”) recorded. The mice were returned to their home cage after testing. Relative weight of pellets burrowed across successive weeks was compared to initial baseline and presented as percentage of baseline.

An attenuated strain of Salmonella typhimurium, S. typhimurium SL3261, has been used previously to model low-grade systemic inflammation and shows changes in both peripheral and central cytokine expression (Peters et al., 2010; Puntener et al., 2012). ME7 prion mice were given an intraperitoneal (i.p.) injection of 1 × 10⁶ colony forming units (cfu) S. typhimurium SL3261 or 200μl non-pyrogenic saline (Kent Pharmaceuticals, United Kingdom) at either 8- or 12-weeks post-inoculation (wpi). NBH-injected control mice were given a single i.p. injection of 200 μl non-pyrogenic saline or S. typhimurium (ME7 + SL3261). Mice were transcardially perfused with 0.9% saline containing heparin sodium (5 U/ml; CP Pharmaceuticals, United Kingdom), 4 weeks after exposure to S. typhimurium SL3261 (12 wpi or 16 wpi). Tissue was collected for immunohistochemistry and biochemical analysis of microglial activation and synaptic marker gene expression.

Immunohistochemistry was carried out on 10 μm-thick fresh frozen tissue sections cut from embedded brains using a Leica CM3050 cryostat. Tissue sections were air dried, fixed in cold ethanol, quenched with 1% hydrogen peroxide (Sigma-Aldrich, United Kingdom) in phosphate-buffered saline (PBS) and blocked with 2% (w/v) bovine serum albumin (Thermo Fisher Scientific, United Kingdom), 10% (v/v) normal rabbit serum (Sigma-Aldrich, United Kingdom) in PBS at room temperature (RT) for 30 min. Primary antibodies for rat anti-mouse major histocompatibility complex class II (MHCII; 14-5321-82, Thermo Fisher Scientific, United Kingdom) and rat anti-mouse CD11b (MA5-16258, Thermo Fisher Scientific, United Kingdom) were diluted 1:500 in PBS and incubated overnight at 4 degrees. Following incubation with the primary antibody, sections were washed and incubated with biotinylated rabbit anti-rat IgG antibody (BA-4000; Vector Laboratories, United Kingdom) for 60 min at RT. Sections were washed and incubated with Vectastain ABC kit (PK-4000; Vector Laboratories, United Kingdom) for 30 min at RT and staining developed with 0.05% (v/v) diaminobenzidine (DAB; Sigma Aldrich, United Kingdom) in phosphate buffer with 0.015% (v/v) hydrogen peroxide. Sections were counterstained with Harris hematoxylin (Sigma-Aldrich, United Kingdom) and differentiated in acid alcohol, dehydrated in increasing ethanol concentrations and xylene before mounting with DPX (Thermo Fisher Scientific, United Kingdom).

Images were captured using a Leica DM5000B microscope and a DFC300 FX camera at 2.5 × optical zoom with LAS X software (Leica Microsystems). All brightfield images were captured using identical exposure limit, gain and saturation. DAB staining was separated from other channels with the Color Deconvolution plugin using FIJI, built on ImageJ v2 (Ruifrok and Johnston, 2001; Rueden et al., 2017). Image threshold limit for each marker was set to a level removing background noise and was kept constant for all images. Images were converted to a binary image and percentage area of staining was quantified: the fold increase in area covered was calculated relative to ME7 + saline animals at the respective time point. At least every 4th section was taken for analysis to reduce the likelihood of counting a cell twice and area used to avoid over-valuing cell fragments.

Tissue samples enriched for the hippocampus and thalamus were isolated following transcardial perfusion and snap-frozen in liquid nitrogen. For mRNA analysis, samples were homogenized in 1 ml TRIzol reagent (Thermo Fisher Scientific, United Kingdom) using a motor-driven pellet pestle. After a 5-min incubation at RT, 200 μl 1-bromo-3-chloropropane (Sigma Aldrich, United Kingdom) was added, the mixture shaken vigorously for 15 s and incubated for 3 min at RT followed by centrifugation at 12,000 × g for 15 min at 4 degrees. 300 μl of the resultant RNA-containing aqueous phase was added to 500 μl RNase-free isopropanol (Sigma Aldrich, United Kingdom) and the samples incubated at room temperature for 10 min following a vigorous mixing. Samples were centrifuged at 12,000 × g for 10 min at 4 degrees before excess isopropanol removed and the pellet washed with 1 ml RNase-free ethanol (70% v/v). Samples were centrifuged at 7,000 × g for 10 min at 4 degrees before ethanol removed and samples air-dried at RT for 15 min. RNA was eluted in 20 μl. DNase/RNase-free water (Life Technologies, United Kingdom) before being placed at 60 degrees for 10 min. Samples were spun down before contaminants were removed using RNeasy MinElute Cleanup Kit (Qiagen, United Kingdom) as per manufacturers’ instructions.

Isolated RNA was quantified using a NanoDrop ND-1000 and all samples with an A₂₆₀:A₂₈₀ ratio of ≥ 1.8 were included. Four hundred nanogram of sample RNA was retrotranscribed using the TaqMan Reverse Transcription Reagents kit (Thermo Fisher Scientific, United Kingdom) following the manufacturer’s instructions for cDNA synthesis with random hexamers. qPCR analysis was performed in duplicate using 1x iTaq Universal SYBR Green Supermix (Bio-Rad, United Kingdom), as previously described (Ibbett et al., 2019). Primers, detailed in Table 1, were designed using Primer-BLAST (NCBI) with one primer of each pair recognizing an exon-exon boundary to prevent amplification of genomic DNA (Supplementary Table 1). GAPDH was used as a reference gene and relative expression of target genes calculated using the following equation based on the threshold cycle (Ct) values: 2^{(GAPDH Ct–target gene Ct)}. Fold change in expression between treatment groups was calculated relative to target gene expression in ME7 + saline mice.

Isolated RNA from ME7 + saline and ME7 + SL3261 brain samples taken at 12 wpi was pooled (n = 5 per treatment group) and analysis of synaptic plasticity genes was conducted in using an RT² Profiler PCR Array Mouse Synaptic Plasticity (330231; QIAGEN, United Kingdom). Sample preparation and loading was carried out as detailed in the manufacturer’s instructions. Analysis was completed using the provided analysis software.

Prism software (v8.4.3; Graphpad, United States) was used to perform statistical analyses and graphically present data as mean ± SD, along with individual data points. All data were subject to testing for normal and lognormal distribution using the Shapiro-Wilk test. If data was normally distributed, data were then analyzed with the appropriate parametric test and post hoc analysis depending on experimental design and number of independent variables, as described in the figure legend. If data failed to meet normal distribution but passed lognormality test, data were log transformed and analyzed with a parametric test. If normality was not met with log transformation, data were analyzed using the appropriate non-parametric equivalent statistical test as detailed in the figure legends.

To investigate the effect of a real, live bacterial infection at the early stage of disease, ME7 prion mice were infected with 1 × 10⁶ cfu S. typhimurium SL3261 (ME7 + SL3261) or saline and phenotypic changes to microglia were measured 4 weeks later (12-wpi). Microglia in the hippocampus and thalamus showed significant changes in expression of CD11b and MHCII after S. typhimurium infection (Figure 1). CD11b expression was significantly increased compared to saline-injected ME7 prion mice [t(8) = 2.683, p = 0.0139] (Figures 1A,C,E). Increased expression of MHCII is evident on cells that resemble microglia, but also on cells that resemble blood vessels, following infection with S. typhimurium [t(7) = 5.397, p = 0.0005] (Figures 1B,D,F). The increased expression of these immune activation markers indicates an exaggerated neuroinflammatory response.

Next, we investigated the effects of S. typhimurium infection in ME7 prion mice at mid-stage of disease. Analysis of the immune activation markers CD11b and MHCII, analyzed 4 weeks later (16-wpi), showed changes in expression levels (Figure 2). Increased CD11b expression on microglia [U(6, 15) = 0, p = 0.05] was observed in the hippocampus and thalamus after S. typhimurium infection (Figures 2A,C,E). Although MHCII expression appeared increased (Figures 2B,D,F), expression was not statistically different 4 weeks after S. typhimurium infection compared to saline-injected mice [U(8, 13) = 2, p = 0.20].

To determine if microglia underwent a functional change following systemic infection with S. typhimurium, we measured the expression of the proinflammatory cytokines, IL-1β and TNF-α (Figure 3). Expression levels of IL-1β [t(8) = 0.8024, p = 0.469] and TNF-α [t(8) = 1.464, p = 0.0907] were similar in early ME7 prion mice (12-wpi) challenged with S. typhimurium compared to saline injection (Figures 3A,B). Analysis of mice infected at mid-stage disease, showed significant increases in expression of cytokines following systemic infection with S. typhimurium [IL-1β [t(7) = 2.090, p = 0.0375] and TNF-α [t(7) = 2.126, p = 0.0355] (Figures 3C,D). This increase in proinflammatory cytokine production following infection with S. typhimurium suggests exaggerated activation of microglia because of systemic bacterial infection. These changes in neuroinflammation are only observed when mice are infected at the mid-stage of disease, when loss of synapses and underlying neuropathology is already present.

In the ME7 mouse model, microglial activation is observed from 8 weeks (Betmouni et al., 1996), while the first changes to neuronal networks, including synaptic loss, are detected from 12-wpi (Gray et al., 2009; Hilton et al., 2013). To investigate the effect of a systemic bacterial challenge on synapses in the hippocampus and thalamus, we employed use of a PCR profile array specifically targeted at changes in genes associated with synaptic plasticity. Results were compared to expression with mice injected with normal brain homogenate (NBH) (Figure 4). This array was composed of 84 genes central to synaptic plasticity, including genes associated with structure of the post-synaptic density and those involved in long-term potentiation (LTP) and long-term depression (LTD). Most genes associated with synaptic plasticity show downregulation in ME7 prion compared to NBH-injected mice (Figure 4A and Supplementary Table 2). We identified 5 genes (Cebpd, Egr2, Mmp9, Nfkbib, Timp1) that are significantly upregulated in ME7 prion compared to NBH-injected mice (Figure 4A and Supplementary Table 2). These genes are associated with early response in synaptic plasticity and extracellular matrix structure, and the expression levels were not further modified by systemic infection. Genes associated with the post-synaptic density, including Arc, Dlg4 and Synpo, showed decreased expression in ME7 prion mice, but no further decrease following systemic infection (Supplementary Table 2). Analysis of activity-related neuronal receptors which are involved in development of LTP and LTD, such as Grm1 and Grin2a, showed a decrease in response to ME7 prion disease (Supplementary Table 2) and were significantly lower following systemic exposure to S. typhimurium.

To confirm and extend the analysis of genes associated with activity-related neuronal receptors, we measured mRNA levels of Grm1 and Grin2a in ME7 prion mice infected with S. typhimurium at 12-wpi or 16-wpi. Analysis of mRNA transcript levels confirm a decrease in expression at early stage of disease (12-wpi) for Grm1 [F(2, 12) = 7.306; p = 0.0084] and Grin2a [F(2, 12) = 4.964; p = 0.0269], with post hoc analysis highlighting significantly lower expression 4 weeks after S. typhimurium infection compared to NBH control mice (Figures 5B,C). Grin2a expression was not modified when mice were infected at mid-stage disease (16-wpi) [F(2, 10) = 0.8174, p = 0.4690], whilst Grm1 expression is significantly reduced after infection at both early and midstage of disease [F(2, 10) = 5.843, p = 0.0209] (Figures 5E,F). To confirm changes to neurons, we analyzed expression of a pre-synaptic marker, synaptophysin (Syp; Figures 5A,D), as neuropathology in ME7 prion mice is known to preferentially affect the pre-synapse (Gray et al., 2009; Siskova et al., 2009; Hilton et al., 2013). Synaptophysin expression is reduced following systemic challenge with S. typhimurium at mid stage disease (16-wpi) [F(2, 10) = 4.929, p = 0.0324], but no changes were observed following infection at early stage disease (12-wpi) [F(2, 12) = 0.9821, p = 0.4027].

Finally, to investigate if changes in activity-related neuronal receptors are linked to altered behavior, we measured burrowing activity, a behavior that is decreased from 8 weeks in ME7 prion disease (Chouhan et al., 2017). Burrowing was measured on a weekly basis and showed a decrease as the disease progressed (Figure 6). ME7 prion mice, exposed to systemic bacterial infection from 8-wpi significantly alter the rate of burrowing deficit compared to saline-treated mice, measured 4 weeks later (p < 0.05). Mice inoculated with normal brain homogenate (NBH) did not show any changes to burrowing activity when exposed to saline or S. typhimurium.

We demonstrate that ME7 prion mice show increased neuroinflammation and reduced expression of selected synaptic markers, following systemic infection with S. typhimurium when compared to ME7 prion mice treated with saline. These observations confirm that a low-grade systemic bacterial infection can exaggerate neuropathology in the ME7 prion model of neurodegeneration, however, the extent and nature of the changes are dependent on the stage of disease. These results are in accordance with our previous study where we show that systemic infection of naïve mice with S. typhimurium results in a prolonged cerebrovascular and neuroinflammatory response (Puntener et al., 2012). Neuropathology in ME7 prion model is characterized by early deposition of PrP^Sc, microgliosis, loss of synaptophysin density within the molecular layers of the hippocampus and subsequent neuronal loss (Cunningham et al., 2003; Hilton et al., 2013). Increased expression of microglial activation markers and related morphological changes can be detected in ME7 mice from 8-wpi by assessing CD11b expression levels (Betmouni et al., 1996; Gomez-Nicola et al., 2013). Here we show that systemic infection with S. typhimurium results in a 1.6-fold increase when mice are infected at 8 wpi and analyzed 4 weeks later (Figure 1). A greater increase in CD11b expression was observed as disease progresses, with a 3.1-fold increase in expression over saline-treated ME7 prion mice, when mice are exposed to infected at 12-wpi and analyzed at 16-wpi (Figure 2). Systemic infection at the early stage of disease, induced a sixfold higher expression of MHCII, while infection at mid stage disease resulted in a twofold increase in expression. This difference in expression may be explained by the relative high expression of MHCII associated with the vasculature. Systemic infection with S. typhimurium results in the activation of endothelial cells which contributes to the crosstalk between the peripheral and central immune responses, with increased expression of cytokines and chemokines (van Sorge et al., 2011). Therefore, in addition to the effect on microglia, the activation of the cerebral vasculature may contribute to the enhanced neuropathology and neuroinflammation following infection with S. typhimurium. Vascular activation and cerebrovascular dysfunction may precede onset of neurodegeneration (Bell and Zlokovic, 2009; Sweeney et al., 2018), but if observations in the ME7 prion model translate to other neurodegenerative diseases requires further study. Our data suggest that systemic infection may enhance cerebrovascular dysfunction and neuroinflammation at the early stage of ME7 prion disease. Further functional studies, for example disruption of the blood brain barrier and/or infiltration of immune cells are required to identify underlying mechanisms. We cannot exclude that enhanced neuropathology is due to altered prion levels in the brain, and future studies will be required to investigate if microbial infections influence prion replication or seeding activity.

Previous studies have shown that microglia are primed by ME7 prion-driven neuropathology and respond with a greater potency to inflammatory stimuli compared to naïve microglia (Cunningham et al., 2009; Murray et al., 2012; Griffin et al., 2013; Skelly et al., 2019). Activation of primed microglia with central or systemic LPS results in exaggerated and prolonged cellular activation and production of the pro-inflammatory cytokines IL-1β and TNF-α (Cunningham et al., 2005b,2009). In this study we investigated if a real, live bacterial infection induces similar functional changes to microglia. Infection at early stage did not induce a significant change in cytokines production and IL-1β and TNF-α expression levels measured in hippocampal/thalamic were comparable to ME7 mice treated with saline. However, systemic infection of ME7 prion mice at mid-stage resulted in a significant increase in both IL-1β and TNF-α mRNA transcripts measured 4 weeks later (Figure 3). Microglial priming can occur because of misfolded protein deposition and/or the loss of synaptic density in the hippocampus. The latter is a neuropathological hallmark which has not yet taken place at 8-wpi. Microglia in the healthy brain contribute to homeostasis more effectively than primed microglia (Lim et al., 2015; Neher and Cunningham, 2019). Thus, it is possible that ME7 prion mice that were infected at early stage show a temporal profile of cytokine production similar to a naïve mouse. In naïve mice central cytokine expression peaks at 21 days following systemic bacterial challenge (Puntener et al., 2012), and measurement after 4 weeks may not capture this response. Microglial numbers in the hippocampus and thalamus increase in ME7 prion mice from 12-wpi (Gomez-Nicola et al., 2013). The differences in cytokine production in ME7 prion mice challenged at early and mid-stage disease could therefore also be explained by an increase in microglia numbers. Together, these data suggest that the exaggerated effects of systemic inflammation on cytokine production in the ME7 prion model are only present at the mid-stage of disease when higher number of microglia are present, along with concurrent protein deposition and synaptic pathology (Cunningham et al., 2005a,2009; Gray et al., 2009; Hilton et al., 2013).

Synaptic density in the stratum radiatum of the hippocampal CA1 is reduced in ME7 prion mice from 12 to 13 wpi, coinciding with behavioral deficits and motor function (Jeffrey et al., 2000; Hilton et al., 2013). Synaptic dysfunction and/or loss in the ME7 prion model has been described predominantly in the pre-synaptic compartment, mostly by measuring synaptophysin expression levels, although alterations to the post-synaptic density are also observed (Gray et al., 2009; Siskova et al., 2009, 2010). To investigate the effect of a bacterial infection on synaptic markers we analyzed the gene expression of a range of molecules linked to synaptic plasticity (Supplementary Table 2). We found reduced expression of gene transcription related to synaptic plasticity in ME7 prion mice, including LTP, LTD, postsynaptic density, neuronal receptors (in particular glutamate receptors) and other structural and plasticity genes (Table 2 and additionally Supplementary Table 2). Importantly, we found further reduction of these selected molecules in ME7 mice exposed to a bacterial infection. Loss of the perineuronal net is evident in ME7 prion mice at 16-wpi (Franklin et al., 2008; McRae and Porter, 2012). Given that profile array data shows a decrease in Ncam1, GABAR α5 subunit and AMPAR subunit expression, along with increased Mmp9 expression (Supplementary Table 2), it could be postulated that these are all indicative of early perineuronal net breakdown in ME7 prion mice after systemic bacterial challenge at 8-wpi. Loss of the perineuronal net may therefore detrimentally affect extra-synapse glutamate receptor expression (e.g., Grm1) as opposed to post-synaptic receptors (e.g., Grin2a). Together, these data do suggest that synaptic expression of glutamate receptors is altered in ME7 prion mice and further reduced following exposure to systemic bacterial infection.

To further investigate if these changes to synaptic markers depend on the time during disease progression, we measured expression of selected genes in mice infected at different stages of disease (Figure 5). We found an expected decrease in Syp expression levels in ME7 mice at 12 wpi, which was further decreased when mice were exposed to a systemic bacterial infection. No changes were observed in mice infected at early stage, suggesting that the bacterial infection per se does not impact Syp expression, but in the presence of an ongoing neurodegenerative disease it enhances synaptic degeneration. Previous studies showed no change in expression of glutamate receptors (NMDARs) in ME7 prion mice, when compared to control mice (Gray et al., 2009). We confirmed these observations by analyzing mRNA transcript levels for the N2a NMDAR subunit (Grin2a). We further show that systemic bacterial infection results in a significant reduction of Grin2a expression, especially at the early stage of ME7 prion disease. This response is associated with reduced levels of mGluR1 (Grm1). Both mGluR1 and the N2a NMDR are expressed at the post-synaptic compartment of neurons and the induction of persistent long-term potentiation (LTP) is dependent on activation of these receptors (van Dam et al., 2004; Ferraguti and Shigemoto, 2006; Cheyne and Montgomery, 2008; Mukherjee and Manahan-Vaughan, 2013). Our data suggest that systemic bacterial infection exaggerates the loss of glutamate receptor expression, which may explain reduced neuronal function and earlier onset and/or progression of neuropathology as observed with changes in burrowing activity. Interestingly, these changes to behavior and glutamate receptors occurred in the absence of enhanced IL-1β and TNF-α production.

A number of previous studies used the bacterial mimetic LPS to address the effect of systemic inflammation on AD-like neuropathology. These experiments have yielded conflicting results that seem to depend on dose, timing, and frequency of LPS administration (Wendeln et al., 2018; Tejera et al., 2019). The effect of a live systemic bacterial infection or bacterial sepsis has also been studied, using experimental models of neurodegeneration. For example, oral application of live Porphyromonas gingivalis significantly enhances amyloid and tau pathology, neuroinflammation and cognitive impairment in aged wild type mice and APP/PS1 transgenic mice (Ding et al., 2018; Ilievski et al., 2018; Dominy et al., 2019) and a real live Bordetella pertussis respiratory infection results in increased neuroinflammation and Aβ₄₀ load (McManus et al., 2014). Interestingly, recurrent, acute systemic infections with Streptococcus pneumoniae do not alter the onset or course of the disease when tested in various experimental models of neurodegeneration (Ebert et al., 2010), suggesting that enhanced neurodegeneration may only occur following sustained, chronic infections. The impact of polymicrobial sepsis on the brain has also been investigated. For example, cecal ligation results in sustained neuroinflammation in wild type mice (Singer et al., 2016) and amyloid plaque deposition in APP/PS1-21 transgenic mice (Basak et al., 2021). Cognitive deficits were also observed, but these occurred in the absence of neuronal loss or changes in synaptic density in the hippocampus (Singer et al., 2016). In this study we show that a real live, low-grade Salmonella typhimurium infection enhances neuroinflammation and synaptic loss in the ME7 prion model and that pre-existing neuronal damage may be needed for bacteria to enhance deterioration. Our study used adult mice and did not investigate the effect of aging. Bento-Torres et al. (2017) showed that older animals show a more robust immune response to prion disease, but if older animals with ME7 prion disease show further enhanced neuropathology remains to be investigated.