4 DISCUSSION
The initial understanding of IP was limited to eyes, testes and brain,
which are organs with blood tissue barriers. Later, research expanded
the concept of IP, which actually exists in many tissues, including hair
follicles, intestinal mucosa, lung and so on. IP actively guides and
controls the immune response through various mechanisms to maintain the
integrity of immune tolerance microenvironment.
Fujisaki3 first revealed bone marrow IP sites by using
high-resolution in vivo imaging in animal experiments. The main
component cells of IP are Tregs, which are characterized by the
expression of FoxP3 in the nucleus. Through the binding of membrane
CXCR4 receptors with a large amount of chemokine CXCL12 in the HSCs
niche, Tregs aggregate to bone marrow near the endosteum surface of
trabecular bone to surround HSCs12, 13, where the
transplanted allogeneic HSCs could survive for a long time (30 days)
without myeloablative conditioning regimen before transplantation, which
is the gold standard experiment for IP identification. Once the Tregs
were removed from bone marrow by deleting the CXCR4 receptor in mice,
TNF-α and IFN-γ were enhanced, while IL-10 was decreased in the bone
marrow3. Then, the transplanted allogeneic HSCs
described in the above experiment could not survive in the osteoblast
niche. HSCs located in IP immune tolerance microenvironment are
LT-HSCs3. Subsequent studies showed that LT-HSCs were
over mobilized with stress induction after Tregs were
depleted12, 14. Therefore, in bone marrow, IP not only
provides an immune tolerance microenvironment to protect LT-HSCs from
various immune factors but also reduces the overmobilization of LT-HSCs
during stress and maintains the static state of LT-HSCs, which are the
stem cells maintaining lifelong hematopoiesis of bone marrow.
It has been proven in AA patients that the ability of Tregs to migrate
to bone marrow was impaired, and the proportion of Tregs in the bone
marrow detected by flow cytometry was also
decreased15,16. However, there has been no research
linking the immune abnormalities of AA with local immune tolerance
status of IP. Interestingly, a series of immune disorders of AA reported
in the literature and in our previous studies are strikingly similar to
the excessive immune status due to the abnormal IP. The increased
expression of TNF-α and IFN-γ in CD4+ and
CD8+ T cells was similar to the changes of bone marrow
cytokines in the IP damage assay (Treg deletion)3,
17,18. The Th1/Th2 polarization detected in a previous study of an AA
animal model was highly consistent with the immune disorder caused by
impaired IP function19,20.
Drawing on other experimental methods studying the function of IP in
local tissues 4,5,21, we tested the distribution of
Tregs in the bone marrow osteoblast niche by immunohistochemistry. There
were significantly fewer Tregs on the endosteal surface of bone marrow
than in normal controls and MDS patients, accompanied by an increase in
Th1/Th2, TNF-α, IFN-γ and IL-17 in bone marrow, while in another bone
marrow failure disease, MDS, they were not. All these results suggested
that IP structure and function were impaired in bone marrow of children
with AA.
The LT-HSCs protected by IP are usually in a static state to maintain
the hematopoietic stem cell pool3. Generally, LT-HSCs
are appropriately transition into activated ST-HSCs which in turn
differentiate into various blood cells22. This
transition increases during stress. Recent research has shown that Tregs
regulate the level of reactive oxygen species in LT-HSCs to prevent
LT-HSCs from being over mobilized and exhausted during oxidative
stress11,13. Our experiment showed significantly
reduced LT-HSCs in AA, which can be attributed to the loss of the
protective effect of IP on LT-HSCs. Various clinical evidences also
support that the ability to maintain HSCs against stress in AA is
reduced. Patients usually develop AA after infection, exposure to
radioactive substances or certain drugs, and the severity of the disease
is often aggravated during infection1, 23. In clinical
practice, we found that 60%-70% of children with NSAA naturally
evolved into SAA during the course of continuous exposure to various
environmental stresses24, 25. After the depletion of
LT-HSCs, there are not enough reserve HSCs to be mobilized, resulting in
the corresponding reduction of ST-HSCs. Therefore, we also detected a
decrease of ST-HSCs in the bone marrow of children with AA.