Peripheral administration of the soluble TNF inhibitor XPro1595 modifies brain immune cell profiles, decreases beta-amyloid plaque load, and rescues impaired long-term potentiation in 5xFAD mice

Abstract

Clinical and animal model studies have implicated inflammation and peripheral immune cell responses in the pathophysiology of Alzheimer's disease (AD). Peripheral immune cells including T cells circulate in the cerebrospinal fluid (CSF) of healthy adults and are found in the brains of AD patients and AD rodent models. Blocking entry of peripheral macrophages into the CNS was reported to increase amyloid burden in an AD mouse model. To assess inflammation in the 5xFAD (Tg) mouse model, we first quantified central and immune cell profiles in the deep cervical lymph nodes and spleen. In the brains of Tg mice, activated (MHCII+, CD45high, and Ly6Chigh) myeloid-derived CD11b+ immune cells are decreased while CD3+ T cells are increased as a function of age relative to non-Tg mice. These immunological changes along with evidence of increased mRNA levels for several cytokines suggest that immune regulation and trafficking patterns are altered in Tg mice. Levels of soluble Tumor Necrosis Factor (sTNF) modulate blood-brain barrier (BBB) permeability and are increased in CSF and brain parenchyma post-mortem in AD subjects and Tg mice. We report here that in vivo peripheral administration of XPro1595, a novel biologic that sequesters sTNF into inactive heterotrimers, reduced the age-dependent increase in activated immune cells in Tg mice, while decreasing the overall number of CD4+ T cells. In addition, XPro1595 treatment in vivo rescued impaired long-term potentiation (LTP) measured in brain slices in association with decreased Aβ plaques in the subiculum. Selective targeting of sTNF may modulate brain immune cell infiltration, and prevent or delay neuronal dysfunction in AD.

Significance statement: Immune cells and cytokines perform specialized functions inside and outside the brain to maintain optimal brain health; but the extent to which their activities change in response to neuronal dysfunction and degeneration is not well understood. Our findings indicate that neutralization of sTNF reduced the age-dependent increase in activated immune cells in Tg mice, while decreasing the overall number of CD4+ T cells. In addition, impaired long-term potentiation (LTP) was rescued by XPro1595 in association with decreased hippocampal Aβ plaques. Selective targeting of sTNF holds translational potential to modulate brain immune cell infiltration, dampen neuroinflammation, and prevent or delay neuronal dysfunction in AD.

Keywords: Alzheimer's disease; Immune cell trafficking; Long-term potentiation; MHCII; Macrophage; Neuroinflammation; Soluble TNF; T cells; XPro1595.

Conflict of interest statement

Conflict of Interest: Malú G. Tansey is a former Xencor Inc. employee and co-inventor on patents covering the dominant negative TNF variants (XPro1595). She does not hold significant financial stake in the company and is not a consultant.

Copyright © 2017 Elsevier Inc. All rights reserved.

Figures

Fig. 1

Altered peripheral immune cell populations in the pro-inflammatory AD-like brain environment. A) Graphical depiction of select results from Table 2. At 7 months of age CCL2 and TNF mRNA expression is significantly increased in the hippocampus of Tg mice as compared to non-Tg mice. By flow cytometry, populations of immune cells within the brain were measured at 3.5, 5, 7, and 12 months of age in non-Tg and Tg mice. B) While there are no effects of genotype on frequency or number of CD11b+ (microglia and macrophage) or CD11b+CD45high (peripheral macrophage and activated microglia) immune cells (data not shown), there is a significant decrease in the frequency of MHCII+CD45highCD11b+ cells in the brain of Tg mice compared to that in non-Tg mice at 7 months of age, ****p < 0.0001. At 5 months of age the ratio of Ly6Chigh:Ly6Clow is significantly increased as compared to non-Tg mice * p < 0.05. C) At 12 months of age the frequency of CD3+ T cells in significantly increased in Tg mice as compared to non-Tg mice, ** p < 0.01. Contour plot inset shows CD4 and CD8 gating on the brain CD3+ T cell population. Within this population, subsets of CD4+ and CD8+ T cells were measured. D) In both non-Tg and Tg mice, the number of CD4+ T cells increases within the CNS with age. No effects were seen within the CD4+ T cell population. In Tg mice there was a significant increase in the number of CD8+ T cells within the CNS at 12 months of age, ***p < 0.001. E) Within the CD8+ T cell population, there is a significant decrease in the frequency of CD8+ effector T cell (CD62L−CD44−) population in Tg mice as compared to non-Tg mice at 3.5 months of age. By 5 months of age, the frequency of CD8+ effector cells has increased in Tg mice to non-Tg levels. At this same time point, there is a significant increase in the number of CD8+ effector T cells within the brain that can account for this return to non-Tg frequency levels, * p < 0.05.3.5 months: non-Tg n = 8, Tg n = 9; 5 months: non-Tg n = 11, Tg n = 11;7 months: non-Tg n = 11, Tg n = 13;12 months: non-Tg n = 10, Tg n = 9. Within these groups, subsets with robust MHCII and Ly6C staining were used for analysis of these markers in the brain: 3.5 months: non-Tg n = 8 (MHCII)/5 (Ly6C), Tg n = 9/4; 5 months: non-Tg n = 4/7, Tg n = 7/8; 7 months: non-Tg n = 8/6, Tg n = 11/7; 12 months: non-Tg n = 9/10, Tg n = 8/4. From the overall group, a subset of samples had reliable CD44 and CD62L staining and were used for analyses of those markers in brain 3.5 months non-Tg n = 5, Tg n = 3; 5 months: non-Tg n = 4, Tg n = 4; 7 months: non-Tg n = 5, Tg n = 5; 12 months: non-Tg n = 6, Tg n = 4. Data was analyzed across genotypes and age with two-way analysis of variance (ANOVA). Sidak’s multiple comparisons test was used for post hoc comparisons within each age.

Fig. 2

Altered naïve and effector T cell populations in deep cervical lymph nodes of 5xFAD mice. While there are no significant differences in DCLN in the number of CD4+ or CD8+ T cells (data not shown), there are significant changes within the CD4+ and CD8+ T cell populations. A) Tg mice show increased frequency of CD4+ naïve T cells, and decreased frequency of CD4+ effector T cells, significant difference at 5 months. DCLN CD4+ T cell subsets shift, with age, towards memory phenotypes. Frequency of both central and effector memory CD4+ T cells subsets are significantly increased at 12 months from 3.5, 5 and 7 months, significant effect of age, no effect of genotype (data not shown). B) Tg mice show increased frequency of CD8+ naïve T cells, and decreased frequency of CD8+ Effector T cells, significant difference at 5 months. DCLN CD8+ T cell subsets shift, with age, towards memory phenotypes. Frequency of central memory CD8+ T cells are significantly increased at 12 months from 3.5, 5 and 7 months, significant effect of age, no effect of genotype (data not shown). Frequency of effector memory CD8+ T cells are significantly increased in non-Tg mice as compared to Tg mice at 12 months of age, significant effect of age and of genotype (data not shown). 3.5 months: non-Tg n = 8, Tg n = 9; 5 months: non-Tg n = 11,Tg n = 11;7 months: non-Tg n = 11,Tg n = 13;12 months: non-Tg n = 10, Tg n = 9. Within these groups a subset of samples had reliable CD44 and CD62L staining and were used for analyses of those markers in DCLNs: 3.5 months non-Tg n = 5, Tg n = 3; 5 months: non-Tg n = 4, Tg n = 4; 7 months: non-Tg n = 5, Tg n = 5; 12 months: non-Tg n = 6, Tg n = 4 Data was analyzed across genotypes and age with two-way analysis of variance (ANOVA). Sidak’s multiple comparisons test was used for post hoc comparisons within each age.

Fig. 3

Inhibition of soluble TNF with XPro1595 decreases populations of activated CD11b+ immune cells and CD4+ T cells in the brain of 5xFAD mice. Tg and non-Tg mice were treated with XPro1595 (10 mg/kg s.c.) twice weekly for two months either from 5 to 7 months of age (A–E) or 2–4 months of age (F–H). A) Inhibition of soluble TNF with XPro1595 decreased MHCII+ populations within both activated (CD11b+ CD45high; frequency but not number) and quiescent (CD11b+ CD45low; frequency and number) immune cell populations, **p < 0.01; * p < 0.05. B) Ly6C, a marker of peripheral monocytes, displayed alterations with inhibition of soluble TNF. The ratio of Ly6Chigh:Ly6ClowCD11b+ brain immune cells in Tg mice is decreased, * p < 0.05. C) Brain T cells are decreased in frequency, but not number, with inhibition of soluble TNF, * p < 0.05. D) Within the T cell population the number of CD4+ T cells is decreased with inhibition of soluble TNF with a trend towards decreased CD4+ effector T cells, * p < 0.05. E) There are no effects of soluble TNF inhibition on the CD8+ T cell population. F) Inhibition of soluble TNF from 2 to 4 months of age decreased the activated (CD45high) population and increased the quiescent (CD45low) population, * p < 0.05. G) There is a trend towards a decreased ratio of Ly6Chigh:Ly6ClowCD11b+ brain immune cells in Tg mice dosed from 2 to 4 months. H) No significant effects are seen in T cell populations at this age. Statistical analysis: significance was determined across treatment using non-parametric t-test.

Fig. 4

Inhibition of soluble TNF with XPro1595 increases the naïve T cell population in the deep cervical lymph nodes (DCLNs) of 5xFAD mice. There is no significant difference in the frequency or number of CD8+ (D) or CD4+ (A) T cells with in the DCLC following XPro1595 or saline treatment within genotype. B) Within the CD4+ T cell niche, the number but not frequency of naïve T cells is increased in Tg mice following inhibition of soluble TNF with XPro1595 as compared to saline treated Tg mice, * p < 0.05. C) The frequency of CD4+ effector T cells is unchanged in Tg mice following inhibition of soluble TNF with XPro1595 as compared to saline-treated Tg mice. No effects are seen on population cell counts or within the CD4+ central memory or effector memory populations. E) Within the CD8+ T cell niche, the frequency and count of naïve T cells is increased in Tg mice following inhibition of soluble TNF with XPro1595 as compared to saline treated Tg mice, * p < 0.05. F) The frequency but not count of effector T cells is decreased in Tg mice following inhibition of soluble TNF with XPro1595 as compared to saline treated Tg mice, * p < 0.05. No effects are seen on population cell counts or on the CD8+ central memory populations. Statistical analysis: significance was determined across treatment using non-parametric t-test

Fig. 5

Inhibition of soluble TNF with XPro1595 decreases amyloid beta in the subiculum and decreases pro-inflammatory mRNA in the hippocampus of 5xFAD mice. Amyloid-β (6E10) (A) and disintegration of multiple neuronal elements (measured by amino-cupric silver staining, NeuroScience Associates) (B) density was calculated in the hippocampus (yellow) and subiculum (dorsal: magenta; and ventral: green; posterior (when dorsal and ventral join): not shown) in saline- and XPro1595-treated Tg animals. A) No effect of soluble TNF inhibition was detected in the hippocampus; however, within the subiculum there was a significant reduction in amyloid burden specifically within the dorsal subiculum, * p t-test. qPCR mRNA data was normalized to non-tg saline and analyzed across genotypes and drug treatment conditions with two-way analysis of variance (ANOVA). Sidak’s multiple comparisons test was used for post hoc comparisons within genotype.

Fig. 6

Inhibition of soluble TNF with peripheral XPro1595 administration in vivo rescued LTP impairment in 5xFAD mice. To determine if peripheral administration of XPro1595 modulates synaptic function in Tg mice, brain slices were harvested for analysis of CA3-CA1 synaptic strength curves (A (non-Tg); B (Tg)) and LTP levels (F (non-Tg); G (Tg)). At four months of age, basal synaptic strength deficits were relatively mild in saline-treated Tg mice, characterized by a modest downward shift in the synaptic strength curve and a significant reduction (p

Fig. 7

Specific targeting of sTNF/TNFR1 signaling rescues AD-like synaptic deficits and modulates AD-like associated immune cell population alterations. As Tg mice age, the brain becomes an inflamed environment with altered regulation of immune cell activation and peripheral immune cell trafficking to the CNS. While no evidence of peripheral inflammation is evident in plasma, there is evidence of altered T cell retention in the deep cervical lymph nodes to Tg mice, suggesting that the pro-inflammatory environment or potentially increased antigen presence is modulating peripheral immune cell populations. In vivo administration of peripheral XPro1595 to neutralize sTNF rescues LTP deficits and decreases amyloid (6e10) reactivity in the brain. These effects are associated with alterations in the brain inflammatory environment and immune cell traffic to the brain. In addition, XPro1595-treated group show further decrease in populations of activated CD11b+ immune cells as well as a decrease in CD4+ T cells suggesting these may be adaptive immune responses to the ongoing AD-like pathology.