Unlocking the Mysteries of Immune Tolerance: Insights from Unique Bone Marrow Niches

Investigation reveals how stem cells abundant in nitric oxide within specialized vascular niches transform regenerative medicine and immune treatments.

Study: Bone marrow niches orchestrate stem-cell hierarchy and immune tolerance. Image Credit: sciencepics / Shutterstock

In a recent investigation published in the journal Nature, a team of scientists disclosed hierarchical arrangements of stem cells within bone marrow (BM) niches that regulate regeneration, immune privilege, and therapeutic capabilities.

Background

BM stem cells inhabit niches, specialized microenvironments where they engage with various cellular and molecular constituents to modulate their functions. Distinct categories of BM niches have been characterized, including endosteal and sinusoidal areas, yet their exact contributions to stem cell hierarchy and immune tolerance remain debated.

Recent studies have shown that these niches can enforce a hierarchy among hematopoietic stem cells (HSCs), impacting their regenerative abilities and immune characteristics. Hematopoietic stem cells (HSCs) exhibit diverse regenerative abilities and immune tolerances, prompting inquiries into the elements that direct these variances. The phenomenon of immune privilege, observed in tissues such as the testis and placenta, points to possible mechanisms for stem cell safeguarding.

Research has also highlighted the role of niche-specific regulatory T (Treg) cells and immunosuppressive factors as crucial players in establishing this immune tolerance. More investigations are required to clarify these ambiguities and assess therapeutic ramifications.

About the Study

A variety of genetically altered mouse models were utilized to examine bone marrow HSCs and their niches. These included models such as C57BL/6J, BALB/cJ, and B10.A mice, in addition to specific transgenic strains like leptin receptor-cre recombinase (Lepr-cre), nerve/glial antigen 2-cre estrogen receptor (Ng2-creERTM), and phosphodiesterase 4D interacting protein-cre estrogen receptor (Pdzk1ip1-creER).

All mice were housed under specific pathogen-free conditions, with experiments carried out on subjects aged 6-12 weeks. Tamoxifen treatments facilitated conditional deletions in designated models, adhering to precise protocols for efficient gene alterations.

Flow cytometry was utilized to measure nitric oxide (NO) concentrations in HSCs. Bone marrow cells, harvested by grinding tibias and femurs, were treated with red blood cell (RBC) lysis buffer and stained with fluorescently labeled antibodies against markers such as CD200 receptor (CD200R), stem cell antigen 1 (SCA1), and signaling lymphocytic activation molecule family member 1 (CD150).

Specialized dyes such as 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM) were applied to detect NO levels, while additional dyes were used to assess mitochondrial functionality and autophagy.

For intracellular evaluations, fixed BM cells were permeabilized and stained with antibodies for proteins including endothelial nitric oxide synthase (eNOS) and microtubule-associated protein 1A/1B-light chain 3 beta (LC3B). Similarly, BM mesenchymal cells were examined using markers like CD200 and vascular endothelial growth factor receptor (VEGFR). The samples underwent processing through advanced flow cytometry systems and were analyzed with FlowJo software.

Transplantation assays were conducted to examine the regenerative potential of NO-high (NOhi) and NO-low (NOlow) HSCs. Sorted HSCs, categorized by NO levels, were injected into irradiated mice alongside competitor BM cells. These trials uncovered distinct “sleeping beauty-like” reconstitution patterns in NOhi HSCs, marked by early dormancy followed by strong regeneration in later phases.

Serial transplantation studies assessed long-term engraftment and hierarchical properties. Non-conditioned recipients also received 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiD)-labeled HSCs for evaluating homing and survival.

Confocal microscopy offered three-dimensional visuals of BM vasculature and cell placement. This imaging validated that NOhi HSCs preferentially engage with specialized CD200hi capillaries concentrated in the metaphysis, a characteristic that distinguishes them.

these categories from others like type-H vessels and sinusoids. Markers such as CD200 highlighted capillaries, while HSCs were observed using specific fluorescent indicators. Statistical evaluations confirmed consistency and relevance across experiments.

Study Outcomes

In this research, NO expression surfaced as a critical trait differentiating potent, late-rising HSCs from their less efficient, exhaustion-prone variants. NOhi HSCs, which made up around 10-15% of bone marrow HSCs, demonstrated enhanced regenerative capability and immune privilege. These NOhi HSCs displayed elevated autophagic activity, reduced mitochondrial superoxide levels, and improved antioxidant features, ensuring their prolonged viability and functionality. They were exceptionally quiescent, expressed immunomodulatory molecules like CD200R, and showed substantial reconstitution over time, particularly following serial transplantation.

NOhi HSCs were predominantly found in distinct bone marrow niches associated with ciliated, CD200hi capillaries. These vascular structures, abundant in the metaphysis, were marked by elevated levels of the immune-checkpoint molecule CD200 and other angiocrine regulators. Sophisticated imaging indicated that NOhi HSCs preferentially engaged with these capillaries, while less effective NOlow HSCs localized near type-H vessels and sinusoids.

Functional investigations confirmed that CD200hi capillaries influenced NOhi HSC abundance and functionality through a signaling pathway involving CD200, eNOS, and autophagy. Conditional deletion of CD200 or the cilia-associated protein IFT20 in endothelial cells disrupted NOhi HSC maintenance, decreased eNOS levels, and lowered autophagic activity. This resulted in notable reductions in bone marrow cellularity, regenerative potential, and immune tolerance of HSCs, emphasizing the crucial role of these niches.

Additional analysis revealed that NOhi HSCs retained elevated basal and induced autophagic activity, which supported their quiescence and regenerative capabilities. eNOS depletion in NOhi HSCs hindered these autophagic processes, diminished mitochondrial superoxide levels, and negated their late-rising reconstitution ability. This underscored the importance of the CD200R–eNOS–autophagy axis in modulating NOhi HSC functions.

Transplantation experiments demonstrated that NOhi HSCs were more robust in allogeneic scenarios, gaining benefits from their relation to immunoprotective niches. CD200hi capillaries sustained activated regulatory T-cell populations and displayed high expressions of immune-checkpoint molecules, which contributed to the immune privilege of NOhi HSCs. This observation accentuates the significance of vascular niches in both regeneration and immune modulation. Deleting CD200 or IFT20 in vessels compromised immune protection, resulting in decreased engraftment of NOhi HSCs in allogeneic recipients.

In summary, these insights categorize NOhi HSCs as highly immune-privileged, primitive stem cells governed by distinct vascular niches. The interaction of CD200hi capillaries, eNOS signaling, and autophagy establishes a hierarchical framework within the BM, revealing new therapeutic targets for improving stem cell-based regenerative medicine and transplantation results.

Conclusions

This research indicates that NOhi HSCs embody a notably potent, immune-privileged subpopulation regulated by specialized CD200hi capillaries in BM niches. These capillaries, prevalent in the metaphysis, orchestrate stem-cell hierarchy through CD200, eNOS, and autophagy signaling. NOhi HSCs showcase late-rising regenerative potential and resilience in allogeneic contexts, highlighting their clinical significance.

The results also spotlight a “sleeping beauty-like” regenerative pattern in NOhi HSCs, underlining their capacity to reactivate after periods of dormancy. The findings emphasize the essential role of vascular niches in sustaining stem-cell equilibrium and immune tolerance, proposing innovative therapeutic strategies to enhance transplantation outcomes and regenerative medicine by targeting these specialized microenvironments.