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. 2020 Feb 15;204(4):879-891.
doi: 10.4049/jimmunol.1900582. Epub 2020 Jan 10.

Neuroimmune/Hematopoietic Axis With Distinct Regulation by the High-Mobility Group Box 1 in Association With Tachykinin Peptides

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Neuroimmune/Hematopoietic Axis With Distinct Regulation by the High-Mobility Group Box 1 in Association With Tachykinin Peptides

Marina Gergues et al. J Immunol. .
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Abstract

Hematopoiesis is tightly regulated by the bone marrow (BM) niche. The niche is robust, allowing for the return of hematopoietic homeostasis after insults such as infection. Hematopoiesis is partly regulated by soluble factors, such as neuropeptides, substance P (SP), and neurokinin A (NK-A), which mediate hematopoietic stimulation and inhibition, respectively. SP and NK-A are derived from the Tac1 gene that is alternately spliced into four variants. The hematopoietic effects of SP and NK-A are mostly mediated via BM stroma. Array analyses with 2400 genes indicated distinct changes in SP-stimulated BM stroma. Computational analyses indicated networks of genes with hematopoietic regulation. Included among these networks is the high-mobility group box 1 gene (HMGB1), a nonhistone chromatin-associated protein. Validation studies indicated that NK-A could reverse SP-mediated HMGB1 decrease. Long-term culture-initiating cell assay, with or without NK-A receptor antagonist (NK2), showed a suppressive effect of HMGB1 on hematopoietic progenitors and increase in long-term culture-initiating cell assay cells (primitive hematopoietic cells). These effects occurred partly through NK-A. NSG mice with human hematopoietic system injected with the HMGB1 antagonist glycyrrhizin verified the in vitro effects of HMGB1. Although the effects on myeloid lineage were suppressed, the results suggested a more complex effect on the lymphoid lineage. Clonogenic assay for CFU- granulocyte-monocyte suggested that HMGB1 may be required to prevent hematopoietic stem cell exhaustion to ensure immune homeostasis. In summary, this study showed how HMGB1 is linked to SP and NK-A to protect the most primitive hematopoietic cell and also to maintain immune/hematopoietic homeostasis.

Figures

FIGURE 1.
FIGURE 1.
Array validation and heatmap of the gene arrays. (A) Time-course studies were done with BM stromal cells stimulated with 10 nM SP. Total RNA was extracted and then studied by real-time PCR for APP and ATF-6. The results are presented as the mean ± SD (n = 5); each experiment was performed with stromal cells from a different donor. Each donor was tested in triplicate, and the mean of each was incorporated as one experimental point. The control (vehicle) values are shown for each time point. *p < 0.05 versus 24 and 48 h time points. (B) Heat map of microarray gene expression profile of all genes based on SP stimulation or vehicle treated in three biological replicates (each with a different BM donor). (C) Downregulated genes from the data in (B), and (D) upregulated genes based on the data in (B).
FIGURE 2.
FIGURE 2.
Functional network with the highest and lowest gene expression. (A) IPA established a network of upregulated and downregulated genes (Supplemental Table I). (B) String analysis established the identity of proteins linked to NID-1.
FIGURE 3.
FIGURE 3.
Genes selected by IPA with functional link between HMGB1 and hematopoiesis. The genes linked to hematopoiesis were established as a network using IPA. (B) The genes in (A) with a cut off score of 53 were used to establish a network of lymphocyte development. (C) Similar network for lymphocyte development using a score of 31. (D) Network established with the genes shown in (A) for leukocyte development.
FIGURE 4.
FIGURE 4.
Time-course and dose-response effects of SP on HMGB1 mRNA and effects of NK-A and/or SP on HMGB1 expression in BM stroma. (A) Real-time PCR using total RNA in time-course studies with stromal cells stimulated with SP (10 nM) in media with 10% FCS. The values of controls at each time point in which stroma was unstimulated and stimulated with vehicle (PBS) were similar and are there plotted together. The data were normalized with β-actin. Each experimental point is presented as the mean ± SD (n = 4), each with a different BM donor. Each donor was tested in triplicates, and the data point was entered as the mean value. (B) The studies in (A) were repeated, except with BM stroma stimulated for 16 h with different concentrations of SP in media containing 10% FCS. The results represent four independent experiments (mean ± SD), each with a different BM donor. (C) BM stromal cells were stimulated with 10 nM SP and/or 10 nM NK-A. Controls were stimulated with vehicle. At 16 h, total RNA was isolated and then analyzed by real-time PCR for HMGB1 mRNA. The values of unstimulated and vehicle were similar and were therefore plotted together (n = 8). The results are shown for four independent experiments (mean ± SD), each with stroma from a different donor. Each donor was tested in triplicates, and the mean was used for each experimental point. (D) Western blots for HMGB1 were performed with whole cell extracts from BM stroma stimulated for 16 h with 10 nM SP and/or 10 nM NK-A. Controls were unstimulated or treated with vehicle. The membranes were stripped and reprobed for β-actin. Densitometric analyses of normalized bands for three independent studies are shown below the graph. Each study was performed with a different BM donor. (E) The studies in (C) were repeated with 10 nM VIP replacing NK-A. The normalized values are presented as mean ± SD (n = 4); each experiment was performed with a different donor. *p < 0.05 versus other concentrations of SP, **p < 0.05 versus SP stimulation, ***p < 0.05 versus SP alone.
FIGURE 5.
FIGURE 5.
Effects of HMGB1 and NK-A on LTC-ICs. (A) Dose-response analyses for HMGB1 levels in pLVX–HMGB1/GS–transduced stroma, which were cultured with different concentrations of Dox. After 24 h of Dox treatment, Western blot for HMGB1 was performed with whole cell extracts. The normalized band densities for two independent experiments are shown below the blot. (B) BM stroma and MSCs were analyzed for baseline NK-A by ELISA. The results are presented as mean ± SD pg/ml (n = 9). Cells were obtained from three different BM donors, with each donor studied in triplicate. (C) BM stroma was cultured in with 100 nM SR48968 or vehicle. At different times, media were collected and then studied for HMGB1 mRNA by real-time PCR. The results are presented as the mean ± SD (n = 3). Each experiment was performed with stroma from a different donor. (D) The studies in (C) were repeated, and instead, the culture media were analyzed for SP level by ELISA. The results are presented as the mean ± SD picogram per milliliter (n = 3). (E) LTC-IC cultures were established with stromal cells and transduced with an inducible HMGB1 (pLVX–HMGB1/GS) or vector alone. The transduced stroma was studied with or without 10 nM NK-2 antagonist (SR48968). HMGB1 was induced with 10 ng and 100 ng of Dox at initiation of culture. At 6, 10, and 16 wk, mononuclear cells were analyzed for CFU-GM in methylcellulose cultures. The results for cultures with vehicle, media alone, and virus without the HMGB1 insert (virus) were similar, and the data were combined (controls). The data are the mean ± SD (n = 4), each experiment performed with a different donor. Each experiment was performed with stroma from a different donor and tested in sextuplets, and the mean was added as one experimental point. *p < 0.05 versus Dox. (E–H) The cultures in (C) were repeated, except for HMGB1 induction with 10 ng Dox, in the presence or absence of 100 nM SR48968 (NK-2 antagonist). The total number of experiments and the results were similarly presented for cultures without antagonists (E), and those cultures with antagonists at weeks 6 (F), 10 (G), and 16 (H).
FIGURE 6.
FIGURE 6.
Effects of HMGB1 on hematopoiesis in humanized NSG female BALB/c. (A) The outline of humanizing five NSG mice with CD34+ cells from BM aspirates and injection with HMGB1 inhibitor (Gly) is shown in the diagram. (B) Mean ± SD (n = 5) body weight of mice. (C) Total nucleated cells/femur (mean ± SD; n = 5). (D) Percentage of CD34+CD38 cells/femur is shown in addition to the absolute numbers of CD45+ cells on top of each bar (mean ± SD; n = 5). (E) Percentage of CD3+CD4+ T cells, CD19 B cells, CD33+ myeloid cells, and HLA-DR (mean ± SD; n = 5). (F) Percentage of CD3+CD8+ T cells (mean ± SD; n = 5); (G) Percentage of CD56+ NK cells (mean ± SD; n = 5). (H) CFU-GM/1 × 105 nucleated cells from femurs. The results are presented as the mean ± SD (n = 5). Each clonogenic assay was performed in duplicate, and the mean value was included as one experimental point. (I) Spleen weight, mean ± SD (n = 5). (J) H&E staining of spleen sections from mice treated with Gly or vehicle. The images (original magnification ×40) were taken with an Evos FL2 Auto Imager and represented five mice. (K) Summary of key findings. *p < 0.05 versus Gly treatment, **p > 0.05 versus Gly treatment

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