Association of brain amyloidosis with pro-inflammatory gut bacterial taxa and peripheral inflammation markers in cognitively impaired elderly

Abstract

The pathway leading from amyloid-β deposition to cognitive impairment is believed to be a cornerstone of the pathogenesis of Alzheimer's disease (AD). However, what drives amyloid buildup in sporadic nongenetic cases of AD is still unknown. AD brains feature an inflammatory reaction around amyloid plaques, and a specific subset of the gut microbiota (GMB) may promote brain inflammation. We investigated the possible role of the GMB in AD pathogenesis by studying the association of brain amyloidosis with (1) GMB taxa with pro- and anti-inflammatory activity; and (2) peripheral inflammation in cognitively impaired patients. We measured the stool abundance of selected bacterial GMB taxa (Escherichia/Shigella, Pseudomonas aeruginosa, Eubacterium rectale, Eubacterium hallii, Faecalibacterium prausnitzii, and Bacteroides fragilis) and the blood expression levels of cytokines (pro-inflammatory cytokines: CXCL2, CXCL10, interleukin [IL]-1β, IL-6, IL-18, IL-8, inflammasome complex (NLRP3), tumor necrosis factor-alpha [TNF-α]; anti-inflammatory cytokines: IL-4, IL-10, IL-13) in cognitively impaired patients with (n = 40, Amy+) and with no brain amyloidosis (n = 33, Amy−) and also in a group of controls (n = 10, no brain amyloidosis and no cognitive impairment). Amy+ patients showed higher levels of pro-inflammatory cytokines (IL-6, CXCL2, NLRP3, and IL-1β) compared with both controls and with Amy− patients. A reduction of the anti-inflammatory cytokine IL-10 was observed in Amy+ versus Amy−. Amy+ showed lower abundance of E. rectale and higher abundance of Escherichia/Shigella compared with both healthy controls (fold change, FC = −9.6, p < 0.001 and FC = +12.8, p < 0.001, respectively) and to Amy− (FC = −7.7, p < 0.001 and FC = +7.4, p = 0.003). A positive correlation was observed between pro-inflammatory cytokines IL-1β, NLRP3, and CXCL2 with abundance of the inflammatory bacteria taxon Escherichia/Shigella (rho = 0.60, p < 0.001; rho = 0.57, p < 0.001; and rho = 0.30, p = 0.007, respectively) and a negative correlation with the anti-inflammatory E. rectale (rho = −0.48, p < 0.001; rho = −0.25, p = 0.024; rho = −0.49, p < 0.001). Our data indicate that an increase in the abundance of a pro-inflammatory GMB taxon, Escherichia/Shigella, and a reduction in the abundance of an anti-inflammatory taxon, E. rectale, are possibly associated with a peripheral inflammatory state in patients with cognitive impairment and brain amyloidosis. A possible causal relation between GMB-related inflammation and amyloidosis deserves further investigation.

Introduction

Neurodegenerative disorders, including Alzheimer's disease (AD), are characterized by the accumulation of neurotoxic proteins in the brain. In AD, these are amyloid-β (Aβ) and hyperphosphorylated tau, representing the major components of extracellular senile plaques and intracellular neurofibrillary tangles, respectively. The common feature of these proteins is the loss of their physiologic activity and the gain of toxic properties, promoting neurodegeneration. Aβ is widely believed to be the key in AD pathophysiology (Jack et al., 2013). In nongenetic cases of AD, the pathophysiological mechanisms of Aβ deposition and the ensuing neurodegeneration and cognitive symptoms remain to be elucidated, but neuroinflammation seems to play a key role (Heppner et al., 2015). Indeed, in addition to plaques and tangles, AD patients feature central inflammation, mediated by activated microglia, reactive astrocytes, and complement activation, that have been especially observed in the vicinity of amyloid plaques and even in the early stages of AD (Clark and Vissel, 2015, Heneka et al., 2015, Heppner et al., 2015, Latta et al., 2015, Stoeck et al., 2014). Enhanced inflammation occurs also in body fluids of AD patients, such as cerebrospinal fluid and blood (Kauwe et al., 2014, Monson et al., 2014, Nascimento et al., 2014, Vom Berg et al., 2012).

The increased interest in the complex network of inflammatory mediators and the immune system has allowed to identify a growing number of pro-inflammatory molecules involved in central nervous system disorders, such as interleukin (IL)-6, tumor necrosis factor-alpha (TNF-α), and the inflammasome complex (NLRP3). These have been found associated with cognitive impairment and AD pathology (Chen et al., 2015, Doecke et al., 2012, Leung et al., 2013, Ray et al., 2007, Soares et al., 2012b, Tan et al., 2013). However, the pathophysiological cascade linking inflammation with Aβ deposition is still unknown (Heppner et al., 2015). Some recent observations indicate that a specific subset of the gut microbiota (GMB) can drive neuroinflammation in rodents (Palm et al., 2015, Petra et al., 2015 Erny et al., 2015) and affect brain function and behavior in rodents and humans (Bercik et al., 2011, Diaz Heijtz et al., 2011, Li et al., 2009).

Alterations of GMB composition have been observed in multiple sclerosis (MS) and Parkinson's disease (PD), conditions also featuring neuroinflammation and protein misfolding. Indeed, the removal of GMB in animal models of multiple sclerosis prevents the development of relapsing-remitting demyelination (Berer et al., 2011) and oral ingestion of probiotics attenuates neuroinflammation (Luo et al., 2014, Toumi et al., 2014). In PD, the evidence is even stronger. The deposition of alpha (α)-synuclein, the underlying molecular pathology, has been found both in the digestive tract and enteric nervous system, already in the early phases of the disease (Del Tredici et al., 2010, Goedert et al., 2013, Lebouvier et al., 2010). Moreover, the gut mucosa of Parkinsonian patients shows increased permeability, signs of inflammation and invasion of coliform bacteria (Forsyth et al., 2011), and hosts a peculiar GMB composition, characterized by decreased abundance of Prevotellaceae and an increase in Enterobacteriaceae, which are also related to the severity of illness (Scheperjans et al., 2015). Importantly, enhanced inflammation, as a consequence of alterations in GMB composition, has been implicated in the initiation of α-synuclein misfolding (Olanow et al., 2014).

To our knowledge, no evidence of GMB alterations has been reported in AD patients yet; however, it has been recently suggested that bacterial endotoxins may play a key role in the inflammatory and pathologic processes associated with amyloidosis and AD (Asti and Gioglio, 2014, Vom Berg et al., 2012), as bacterial components, such as endotoxins, have been found within the typical senile plaque lesions of the AD brain (Asti and Gioglio, 2014, Schwartz, 2013).

The aim of this study was to test, in elder patients with cognitive impairment, the association between brain amyloidosis and (1) candidate GMB taxa with known inflammatory activity (pro-inflammatory: Escherichia/Shigella and Pseudomonas aeruginosa; anti-inflammatory: Eubacterium rectale, Eubacterium hallii, Faecalibacterium prausnitzii, and Bacteroides fragilis) (Bruzzese et al., 2014, Cantarel et al., 2015, De la Fuente et al., 2014, Friedland, 2015); and (2) peripheral inflammation markers implicated in the pathogenesis of AD (pro-inflammatory cytokines: CXCL2, CXCL10, IL-1β, IL-6, IL-18, IL-8, NLRP3, TNF-α; anti-inflammatory cytokines: IL-4, IL-10, IL-13) (Chen et al., 2015, Doecke et al., 2012, Leung et al., 2013, Ray et al., 2007, Soares et al., 2012b, Tan et al., 2013).