Gut inflammation and dysbiosis in human motor neuron disease.

Amyotrophic lateral sclerosis (ALS) is a systemic disorder that involves dysfunction of multiple organs. Growing evidence has shown that neurodegenerative disorders with gut dysbiosis affect the central nervous system via pro-inflammatory mediators thus impacting gut-brain communications. We have demonstrated dysbiosis and increased intestinal permeability in the SOD1G93A ALS mouse model. In this study, we comprehensively examined the human gut microbiome in stool samples and evaluated infection and markers of intestinal inflammation in five patients with ALS and motor neuron disorders. Five patients we studied all had alteration in their gut microbiome characterized by a low diversity of the microbiome, compared to healthy cohorts with relatively intact abundance. Firmicutes and Bacteroidetes are the two major members of bacteria at the phylum level. Low Ruminococcus spp occurred in three patients with low Firmicutes/Bacteroidetes (F/B) ratio. A majority of patients had signs of intestinal inflammation. This is the first comprehensive examination of inflammatory markers in the stool of ALS patients. Studies in gut health and microbiome related to the onset and progression of ALS may reveal novel therapeutic targets for disease modulation.

Introduction

The human gut microbiome has been referred to as a “mega organ” harboring 1014 microbes and 4 x 106 genes, outnumbering human genes by 150:1 (Bhattacharjee and Lukiw 2013; Collins 2014). Emerging evidence suggests an altered gut microbiome may contribute to the development of neurodegenerative diseases, such as Parkinson's and Alzheimer's disease. The complex interplay of gastrointestinal ‐ central nervous system (CNS) communication involves autonomic and enteric nervous systems, neuroendocrine, and immune systems (Forsyth et al. 2011; Hsiao et al. 2013; Sampson et al. 2016). However, little is known about the intestinal microbiome in patients with amyotrophic lateral sclerosis (ALS) and other motor neuron disorders (MND). ALS is a progressive neurodegenerative disease causing loss of motor neurons in the brain, brainstem, and spinal cord, which leads to loss of voluntary skeletal muscle. The ALS death is due to respiratory failure, typically occurring two to 5 years from diagnosis (Forshew and Bromberg 2003; Miller et al. 2009).

Dysbiosis is defined as an imbalance in the structural and/or functional properties of the gut microbiota. We have demonstrated gut dysbiosis and increased intestinal permeability in the SOD1G93A ALS mouse model (Wu et al. 2015a). SOD1G93A mice harbor the human Cu/Zn superoxide dismutase gene mutation SOD1G93A recapitulating the neuronal and muscle impairment of human ALS. We have shown that using the bacterial product butyrate, a short chain fatty acid (SCFA), improves gut integrity and microbial homeostasis and prolongs lifespan of SOD1G93A mice (Zhang et al. 2017). In this study, we comprehensively examined the gut microbiome and evaluated markers of intestinal inflammation in five patients with MND.

Methods

Human subjects

This study was approved by the relevant research ethics committee at the University of Illinois at Chicago. Informed consent was obtained from the research subjects. JR follows patients 1, 2, and 5. Patients 3 and 4 were assessed by release of their medical records and direct email communication (patient 4) or email communication via a first‐hand relative (patient 3) when ALS progression made direct communication difficult. Stool testing was performed on seven samples from five patients. Testing was performed at Genova Diagnostics in four patients (patient No. 2 and No. 5 had repeat confirmatory tests) and at Doctors Data in one patient (patient No. 4). Each laboratory utilizes reference ranges generated from healthy cohorts. For the patient samples tested at Genova Diagnostics, the healthy cohort had 96 individuals recruited with preset exclusion criteria. They did not have intestinal diseases and had no abdominal symptoms. The stool samples of the healthy cohort were collected following the same steps and processed by Genova Diagnostics.

Stool sample collection

Human stool samples for microbiome test were collected at home using the kit provided by the Genova Diagnostics. Samples were stored at refrigerator before shipping to the company.

Bacterial diversity and abundance

Commensal Bacteria (PCR) (polymerase chain reaction, Genova Diagnostics) was performed for bacterial imbalance, diversity, and abundance. The 16s rRNA gene sequence (full or partial) for each microorganism was obtained from the NCBI GenBank database. Sequences of related organisms were aligned using Clustal W2 (http://www.clustal.org) to identify conserved regions (for genus probes) and regions of uniqueness (for species probes). Forward (5′–3′) and reverse (3′–5′) primers were chosen based on the level of specificity required of individual assays. The designed primer sequences were verified using NCBI BLAST analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi), once with the microorganism specified and once with the microorganism excluded from the search to ensure specificity of both the forward and reverse primer sequences. For proprietary right reasons, primer sequences are not shown here.

The relative abundances of commensal bacteria at phylum level from both patients and healthy controls were visualized by abundance bar plots (Xia and Sun, 2017). At the phylum level, bacteria include Bacteroidetes, Firmicutes, Actinobacteria, Proteobacteria, Euryarchaeota, Fusobacteria, and Verrucomicrobia. For each phylum, representative bacteria at the class level were tested: Bacteroides‐Prevotella group, Bacteroides vulgatus, Barnesiella spp., Odoribacter spp., and Prevotella spp. for Bacteroidetes; Anaerotruncus colihominis, Butyrivibrio crossotus, Clostridium spp., Coprococcus eutactus, Faecalibacterium prausnitzii, Lactobacillus spp., Pseudoflavonifractor spp., Roseburia spp., Ruminococcus spp., and Veillonella spp. for Firmicutes. Firmicutes and Bacteroidetes are the two major members of bacteria at the phylum level. Firmicutes/Bacteroidetes (F/B) ratio was used as an indicator of dysbiosis (Tamboli et al. 2004; Collins 2014; Sampson et al. 2016).

Infection

Bacterial and mycological culture were performed and observed by microscopy. Bacterial cultures are for Lactobacillus spp., Escherichia coli, Bifidobacterium, alpha haemolytic Streptococcus, Bacillus species, and gamma haemolytic Streptococcus. Parasitology EIA Tests were done for Cryptosporidium, Giardia lamblia, and Entamoeba histolytica. No infection by culture was reported at Genova Diagnostics in 4 patients. Yeast culture was tested at Doctors Data. Patient 4 was reported with candida albicans 2 + . Yeast antifungal susceptibility testing was done for patient No. 4.

Short chain fatty acids in stool samples

MALDI‐TOF MS (Matrix Assisted Laser Desorption Ionization Time‐of Flight Mass Spectrometry) was performed for the concentration of short chain fatty acids (SCFA) in stool samples. Total SCFAs (acetate, n‐butyrate, propionate) reference range >=23.3micromol/g, 29–34.5 is considered borderline low, n‐Butyrate concentration reference range>=3.6 micromol/g.

Inflammation and immunology

Fecal secretory IgA, calprotectin, and eosinophilic protein X were tested by EIA (enzyme immunoassay) for inflammatory biomarkers.

Diagnosis and data analysis

For each measurement performed by Genova Diagnostics, the generated value was compared with a reference range (RR) and displayed within a quintile distribution. RRs are usually generated based on two major theories: medical decision points or healthy cohort studies. Some biomarkers, such as calprotectin, are used to differentiate diseases. For those biomarkers, RRs are usually derived from medical literature. If the biomarkers are used to differentiate healthy and sick individuals, RRs are usually generated from studies using healthy cohorts. Results falling outside 2 standard deviations (SDs) from the mean are considered abnormal (see Table   1). Results falling outside 1 SD are considered borderline abnormal. It may change depending on if it is one‐tail or two‐tail distribution.

Table 1

Patient characteristics and stool analysis results

N	Agea	Gender	Diagnosis	Stool inflammatory markers/infection	Intestinal microbiome analysis	Short chain fatty acids (SCFA)
1	73	Male	BAD/CLLb	Calprotectin 77 mcg/g f EPX c 4.3 mcg/g sIgA e 335 mcg/g infection‐none	F/B ratiod 5 Low Diversity Low Beta‐glucuronidase 3350 U/gi	Total 92.8g n‐Butyrate 11.5h
2	82	Female	Bulbar‐onset definite ALSk	(Did twice of these tests) Calprotectin 143 mcg/g, 121 mcg/g EPX 1.6 mcg/g sIgA 208 mcg/g infection‐none	F/B ratio 113 Diversity Low Beta‐glucuronidase 591 U/g	Total 69 n‐Butyrate 12.3
3	53	Female	Bulbar‐onset definite ALS	Calprotectin <16 EPX 0.6 sIgA 449 mcg/g infection‐none	F/B ratio 9 Low Diversity Low Beta‐glucuronidase 9,991 U/g	Total 42.4 n‐Butyrate 7.5
4	46	Female	Definite ALS	Calprotectin <10 sIgA 56.8culture: candida albicans 2+	Dysbiosis by culture	Total 3.8 (4–18 mg/ml) n‐butyrate 0.43 Low (0.8–4.8 mg/ml)
5	39	Female	Definite ALS, Celiac disease (confirmed by intestinal biopsy)	(Did twice of these tests ×2) Calprotectin <16 × 2 EPX <DLj, 0.5 sIgA 121 mcg/g, 135 mcg/g infection‐none	F/B ratio 18, 10 Low Diversity Low × 2 Beta‐glucuronidase 3,737, 3,386 U/g	Total 34.5, 29 Borderline Low n‐Butyrate 12.2, 8.1 (>=3.6micromol/g)

In years.

Brachial Amyotrophic Diplegia/Chronic Lymphocytic Leukemia.

Eosinophil Protein X (reference range <= 4.6, 4.3 is borderline high).

Firmicutes/Bacteroidetes ratio (reference range 12–620, 18 is borderline low).

Fecal secretory IgA (reference range <885 mcg/g, 335 and 449 mcg/g are borderline elevated).

Calprotectin reference range <50 mcg/g.

Total SCFAs (acetate, n‐butyrate, propionate) reference range >=23.3micromol/g, 29–34.5 is considered borderline low.

n‐Butyrate concentration reference range>=3.6 micromol/g.

Beta‐glucuronidase reference range 368–6266 U/g.

Defined by El Escorial Criteria.

The bold fonts are used to shown the changed values.