Methods. The project will be organized in three working packages (WP). WP1. Determine if
the propensity of patients with PHPT to develop bone loss can be predicted by the
composition of stool microbiome (month 0-11). We will screen men and women age 30-80
years diagnosed with PHPT and evaluated by 3-site DXA (lumbar spine, hip, and distal
radius- both 1/3 and ultra-distal), and bone turnover markers. Two groups with 45
patients each will be enrolled: 1) Subjects with normal BMD or mild osteopenia (distal
radius T-score > -1.5), no fragility fractures and CTX levels that are associated with
normal bone turnover (100-250 pg/ml) (not bone looser). 2) Subjects with osteoporosis
(distal radius T-score < -2.5) with or without one or more fragility fractures) and CTX
levels that are associated with active disease (>500 pg/ml) (bone looser). Blood samples
and a faecal sample will be collected by each participant. Stool samples will be stored
immediately in a cooler with -20°C freezer packs and stored at -80°C until shipped to
COSMOSID for Metagenomic Shotgun sequencing and analysis of the microbiome community
structure.
Metagenomics Shotgun sequencing and analysis. The microbiome diversity and composition of
each stool sample will be determined by CosmosID Inc., Rockville, MD. Briefly, DNA from
human faecal samples will be isolated using the QIAGEN DNeasy PowerSoil Pro Kit. DNA
libraries will be prepared using the Illumina Nextera XT library preparation kit and
Library quantity assessed with Qubit (ThermoFisher). Libraries will be then sequenced on
an Illumina HiSeq platform 2x150bp. Bioinformatic analysis of sequencing reads will be
done by CosmosID bioinformatics platform that uses high performance data mining
algorithms and the highly curated dynamic comparator database GenBook®.
End points and interpretation: We will determine if PHPT patients loosing bone have a
higher frequency of one or more Th17 cell-inducing-SFB equivalent bacteria as compared to
control PHPT patients. This analysis will therefore yield evidence pertaining to species
and strains that may exacerbate bone loss in PHPT. The DXA endpoints will be BMD,
T-scores and Z-scores of the distal and 1/3 radius, lumbar spine and hip.
Power Calculation: Our analysis of the preliminary data set revealed that BL was abundant
at mean relative abundance of ~5.8% (SD 0.055). From these data we calculated that
enrolling 45 per group will provide 80% statistical power of detecting a difference for
BL if the mean relative abundance of BL is > 1.5-fold (8.7%) higher in PHPT patients with
low bone density as compared to control PHPT patients.
WP2. Evaluate how GM composition modulates immune system in humans (month 3-10). In order
to evaluate Th subsets, T cells will be analysed by real time RT-PCR measuring FOXP3,
IL-17A, TNF-alpha and IL-4 gene expression. Briefly, red cells will be lysed in
peripheral blood samples and total nucleated cells collected and frozen at -80°C until
RNA extraction. RNA will be isolated using TRIzol reagent, chloroform extraction, and
subsequent isopropanol precipitation according to standard procedure. Relative cytokine
expression will be determined using the 2-ΔΔCT method with normalization to β-Actin. In
order to evaluate inflammation we will measure IL-17A (the main species of IL-17 produced
by Th17 cells), free RANKL, OPG, TNF by ELISA technique. To assess bone turnover P1NP (a
marker of bone formation), CTX (a marker of bone resorption), 25OHvitamin D (25OHD) will
be measured by ELISA technique.
End points and interpretation. The data will reveal the existence of correlations between
rate of bone resorption, Th cell subset and serum levels of IL-17 and other cytokines.
Should we find that the composition of the GM is reflective of the activity of the
disease; the data will serve as the foundation for future intervention studies to
determine the efficacy of antibiotics or other interventions as, for example, with
probiotics in mitigating the skeletal complications of PHPT. We also expect that PHPT
patients with skeletal disease will have lower 25OHD levels than those without skeletal
disease. Should we find a correlation between 25OHD levels and bacterial species and
strains that favours Th17 cell formation; the data will provide novel mechanistic
information on how 25OHD levels affect the propensity to bone disease in PHPT. Should
this be the case, a very attractive subject for a future intervention study will be the
proposition that vitamin D replacement will restore a microbiota composition that is not
associated with skeletal disease.
Risk analyses and contingency plan. Oestrogen deficiency expands Th17 cells and their
production of IL-17 may contribute to postmenopausal bone loss in humans. This effect
might represent an important confounder, which may limit the assessment of the role of
IL-17 in the bone loss induced by PHPT in early postmenopausal women. If necessary, we
will manage this limitation by statistically adjusting for the effects of sex, age and
menopause.
WP3. Evaluate the causal relationship between GM composition and T cells activation
(month 6-10). In order to evaluate if Th cells are directly stimulated by proteins from
SFB equivalent bacteria we will test the peripheral blood mononuclear cells (PBMCs)
directly toward BL ATCC 15707 and evaluate the type of responding T cells by ELISPOT
technique. Briefly, PBMCs from 10 patients with increased Th17 cells in peripheral blood
will be tested against BL ATCC 15707 and the production of IL-17, TNF alpha and RANKL
will be tested by ELISPOT analyses for. These cytokines, within the PBMCs, are produced
specifically by T cells following their activation. ELISPOT technique allows testing
multiple proteins in a single experiment. TNF alpha and RANKL other than IL-17 have been
selected as candidate cytokines as the applicant and others have previously shown an
increased level of these cytokines in T cells from patients affected by post-menopausal
osteoporosis. Should we find a direct activation of T cells to BL ATCC 15707, the type of
T cell is responsible for activation will be further characterized thanks to the use of
single-cell RNA sequencing technique (scRNA-seq) with the the Fluidigm C1 system as it
allows a more in-depth sequencing per cell (about 200.000 readings). Briefly, PBMC cells
from 5 responders patients will be challenged with ATCC 15707 or with a control microbe
(B. fragilis) for 6 hours. After incubation T cells will be separated by total blood
nucleated cells by negative immunomagnetic cell isolation system, and evaluated with
scRNA-seq. The use scRNA-seq will enable us to characterize activated T- cells and their
gene expression End points and interpretation. We will determine if ATC C 15707 is able
to directly activate T cells and further we will characterise the type of responding T
cell. This will allow us to confirm the association data obtained in the first part of
the study and provide a mechanistic insight into the relationship between GM, immune
activation and bone loss.
Risk analyses and contingency plan. If T cells from patients with increased number of
Th17 in the peripheral blood will not positively answer to immune challenge with ATCC
15707, we will test T cells reactivity towards others microbes increased in the GM of
PHPT bone losers. GM strain able to stimulate Th17 cells will be chosen according to the
results obtained in WP1. In order to define a positive T cells activation we set a
threshold of 70% of positive results to immune challenge according with our preliminary
data and with previous results published by the PI.
