The effects of human endotoxemia on the functional capacity of hematopoietic stem and progenitor cells

Sepsis is a major medical challenge characterized by a systemic inflammatory
response, and is associated with a high mortality rate and increasing
incidence. The immune response in sepsis is highly variable between patients
and can comprise both a hyperinflammatory and a immunosuppressive state. The
experimental human endotoxemia model is widely used to study on the effects of
systemic inflammation in vivo in humans, because it bears hallmarks of both the
hyperinflammatory and a immunosuppressive state of sepsis, the latter
exemplified by the development of endotoxin tolerance. To date, mechanistic
research into systemic inflammation in humans has been limited to the
peripheral blood compartment, as tissue-residents cells are not easily
available for harvesting. A discrepancy in duration of ex vivo and in vivo
endotoxin tolerance of circulating leukocytes, and the lack of correlation
between ex vivo and in vivo responses to endotoxin administration in humans,
suggest that immune cells in other compartments, rather than circulating blood
leukocytes, are important effectors of the in vivo inflammatory response. The
function of bone marrow cells in the in vivo inflammatory response is currently
unknown. Hematopoietic stem and progenitor cells in bone marrow express
Toll-like receptors and thereby are assumed to have immunomodulatory functions.
Macrophages derived from human hematopoietic stem and progenitor cells exhibit
reduced inflammatory cytokine production after being exposed to a TLR2 agonist
prior to macrophage development. These data suggest a pivotal role for the bone
marrow compartment in regulation of the inflammatory response. In this study,
we will investigate whether LPS induces changes in function of bone marrow
cells and their downstream effector cells. Furthermore, we will also
investigate other compartments such as the blood and the lungs. To
comprehensively investigate underlying mechanisms behind functional changes in
these cell types, we will use state-of-the-art systems biology techniques,
including transcriptomics, epigenetics, and metabolomics.

Primary objective:
- To characterize hematopoietic progenitor cells in human bone marrow before
and after endotoxemia (by evaluating hematopoietic lineage differentiation and
functional capacity).

Secondary Objectives:
- To determine the effects of endotoxemia on the functional capacity of
alveolar macrophages in the sputum.
- To determine whether endotoxemia induces changes in the transcriptome
(inflammatory transcriptional pathways) of human hematopoietic stem and
progenitor cells , blood leukocytes, and alveolar macrophages.
- To determine whether endotoxemia induces changes in the epigenome (complement
of chemical compounds altering gene expression) of human stem and hematopoietic
progenitor cells, blood leukocytes, and alveolar macrophages.
- To determine whether endotoxemia induces changes cellular metabolism of
hematopoietic stem and progenitor cells, blood leukocytes, and alveolar
macrophages.
- To determine the correlation of gene polymorphisms relating to innate immune
function (e.g. autophagy ATG2b and ATG5 SNPs) with immune function and
induction of endotoxin tolerance.
- To determine the life-span and transit times of different subsets of
leukocytes and their progenitors in the bone marrow of humans at homeostasis
and during endotoxemia.

An explorative randomized placebo-controlled pilot study in 12 healthy male
volunteers.

The study takes place at the research unit of the intensive care department of
the Radboud University Medical Center, Nijmegen. The duration of the study
period for each individual subject will be three weeks. Volunteers will be
recruited and are subjected to a medical examination (including interview,
medical history and physical examination). After medical approval and informed
consent subjects will be randomly allocated to the LPS group (n=8) or to the
placebo group (n=4).

On first challenge day, all subjects will be challenged with a bolus LPS of
2ng/kg (n=8) or a placebo solution (n=4). On second challenge day, only the LPS
group (n=8) will be subjected to a second LPS challenge to determine the extent
of in vivo endotoxin tolerance.

To characterize the hematopoietic stem and progenitor cells, subjects will
undergo a bone marrow examination three times; one week before (baseline), 4
hours after, and 7 days after the first challenge. Furthermore, blood will be
withdrawn and sputum (containing alveolar macrophages) will be collected. In
addition, deuterium glucose will be used to label mitotic cells to track
downstream inflammatory leukocytes.

This study investigates the effects of experimental endotoxemia on the systemic
inflammatory response and endotoxin tolerance in different compartments of the
human body (bone marrow, blood, and lungs). As such, LPS will be used as a
challenge agent to induce systemic inflammation. No investigational
(medication) treatment is given. The eight subjects in the LPS group will be
challenged with LPS twice, on day 8 and day 15. The four subjects in the
placebo group will receive a placebo challenge on day 8 and they won*t be
challenged on day 15. There is no need for a blinded procedure as the effects
of LPS injection will be profound.

Subjects will visit the research unit a total of 6 times; for screening, first
labeling day, first bone marrow examination, second labeling day, first
challenge day, and second challenge day. Volunteers will be recruited and are
subjected to a medical examination (including interview, medical history and
physical examination). The administration of a lipopolysaccharide induces
flu-like symptoms for approximately 3-4 hrs. This model of systemic
inflammation has been applied for more than 10 years in our department and
thousands of subjects worldwide have participated in endotoxemia trials. During
the LPS challenge, subjects will be under constant supervision of an
experienced physician with continuous monitoring of blood pressure and heart
rate. The endotoxemia protocol and associated risks are identical to earlier
endotoxemia studies performed in our institute. Bone marrow examinations will
be performed by a highly experienced physician assistant of the hematology
department. The bone marrow examinations in a previous study were very well
tolerated by the subjects. A maximum of 500 ml blood will be drawn during the
study, which is comparable to previous studies and never resulted in adverse
events. The labeling of leukocytes is safe, non-invasive, and the group of
prof. Koenderman is very experienced in using this technique. The subjects will
not benefit directly from participation to the study. This study will yield a
comprehensive insight into inflammatory signaling in the bone marrow and will
thereby improve our understanding of systemic inflammation, endotoxin
tolerance, and sepsis, possibly revealing new therapeutic targets to improve
sepsis outcome. The risks to the subjects in this study is classified as a
*negligible risk* (low risk on minor harms). A subject fee is provided.