Osteocytes reside as three-dimensionally (3D) networked cells in the lacunocanalicular structure of bones and regulate bone and mineral homeostasis. Despite of their important regulatory roles, in vitro studies of osteocytes have been challenging because: (1) current cell lines do not sufficiently represent the phenotypic features of mature osteocytes and (2) primary cells rapidly differentiate to osteoblasts upon isolation. In this study, we used a 3D perfusion culture approach to: (1) construct the 3D cellular network of primary murine osteocytes by biomimetic assembly with microbeads and (2) reproduce ex vivo the phenotype of primary murine osteocytes, for the first time to our best knowledge. In order to enable 3D construction with a sufficient number of viable cells, we used a proliferated osteoblastic population of healthy cells outgrown from digested bone chips. The diameter of microbeads was controlled to: (1) distribute and entrap cells within the interstitial spaces between the microbeads and (2) maintain average cell-to-cell distance to be about 19 µm. The entrapped cells formed a 3D cellular network by extending and connecting their processes through openings between the microbeads. Also, with increasing culture time, the entrapped cells exhibited the characteristic gene expressions (SOST and FGF23) and nonproliferative behavior of mature osteocytes. In contrast, 2D-cultured cells continued their osteoblastic differentiation and proliferation. This 3D biomimetic approach is expected to provide a new means of: (1) studying flow-induced shear stress on the mechanotransduction function of primary osteocytes, (2) studying physiological functions of 3D-networked osteocytes with in vitro convenience, and (3) developing clinically relevant human bone disease models.
In the current study we evaluated the effects of immunoproteasome inhibition using ONX 0914 (formerly PR-957) to ameliorate graft-versus-host disease (GVHD). ONX 0914, an LMP7-selective epoxyketone inhibitor of the immunoproteasome, has been shown to reduce cytokine production in activated monocytes and T cells and attenuate disease progression in mouse models of rheumatoid arthritis, colitis, systemic lupus erythematosus, and, more recently, encephalomyelitis. Inhibition of LMP7 with ONX 0914 in the B10.BR→CBA MHC-matched/minor histocompatibility antigen (miHA)-disparate murine blood and marrow transplant (BMT) model caused a modest but significant improvement in the survival of mice experiencing GVHD. Concomitant with these results, in vitro mixed lymphocyte cultures revealed that stimulator splenocytes, but not responder T cells, treated with ONX 0914 resulted in decreased IFN-γ production by allogeneic T cells in both MHC-disparate (B10.BR anti-B6) and miHA-mismatched (B10.BR anti-CBA) settings. In addition, a reduction in the expression of the MHC class I-restricted SIINFEKL peptide was observed in splenocytes from transgenic C57BL/6-Tg(CAG-OVA)916Jen/J mice exposed to ONX 0914. Taken together, these data support that LMP7 inhibition in the context of BMT modulates allogeneic responses by decreasing endogenous miHA presentation and that the consequential reduction in allogeneic stimulation and cytokine production reduces GVHD development.
Acute graft-versus-host disease (GVHD) is a major cause of nonrelapse morbidity and mortality following allogeneic blood and marrow transplantation (BMT). The majority of studies delineating the mechanisms involved in GVHD have focused on the alloreactive T cell response and their downstream cellular and inflammatory effector phases. While these efforts have provided important mechanistic insights that have been translated into therapies that target the T cell compartment, they ignore the role that the target organ plays in both initiating and propagating this disease. Acute gut GVHD affects more than 60% of patients and is a leading cause of death. The mechanisms for the early, conditioning-related, intestinal injuries that are the precipitating event leading to GVHD or how subsequent gut-specific GVHD actually disrupts intestinal homeostasis have not been fully explored. The transcription factor, hypoxia inducible factor-1 (HIF-1) has emerged as a common denominator for hypoxia and inflammation. Given that inflammatory bowel disease (IBD) and GVHD share many pathogenic mechanisms and intestinal epithelial HIF-1α afforded protection in IBD models, we hypothesize that the persistent activation of HIF-1 will protect the intestinal epithelium from radiation/chemotherapy and alloreactive T cell-induced damage. Since crypt damage is a hallmark of gut GVHD, we first determined whether loss of intestinal epithelial HIF-1α impaired intestinal regeneration after GVHD damage using conditional HIF-1α (HIF-1αIE) knockout mice that lack HIF-1α in the intestinal epithelium. In a major MHC mismatched B10.BR (H2k) into B6 (H2b) GVHD model, 8 days post-BMT, histopathologic assessment of H&E stained ileums of HIF-1αIE mice showed more severe crypt damage, loss of Paneth cells and fewer regenerating crypts as compared to wild-type (WT) mice. A two-fold increase of aberrant mitoses (p<0.02 vs WT) was observed in crypts of HIF-1αIE mice. Consistent with these findings, real-time PCR analysis demonstrated that loss of epithelial HIF-1α did not induce the expression of Lgr5 mRNA levels (p<0.05) and reduced Reg3γ mRNA levels in the ileum by 2.4-fold (p<0.05) relative to WT mice, post-BMT. Since intestinal crypts have been shown to undergo extensive proliferation to repair damage following injury, we examined the crypt proliferative response 21 days post-BMT using immunohistochemistry for the proliferative marker Ki67. Hyperplastic elongated crypts that are characteristic of regenerating crypts after radiation-induced damage were observed in Ki67-stained ileal sections of WT mice post-BMT whereas smaller "aberrant" appearing crypts with asymmetrically distributed Ki67-labeled cells and fewer regenerating crypts were evident in HIF-1αIE mice post-BMT. In control WT and HIF-1αIE (receiving T cell depleted BM), Ki67+-proliferating cells resided at the crypt base. The extent of irregular distribution of Ki67 within 50 crypts/ileum was graded independently with a grading score ranging 0 to 3 (0 = limited to the base of crypt, 1= symmetric distribution at crypt base and along each side of the elongated crypt, 2 = asymmetric distribution in bottom 2/3 of crypt and 3 = asymmetric distribution of Ki67-labeled cells in entire crypt without lumen). A modest but significant increase in the aberrant proliferative score was 1.72 in the HIF-1αIE post-BMT group versus 1.46 in the WT post-BMT group (p<0.05). Taken together, our findings suggest that intestinal epithelial HIF-1 may protect the intestinal stem cell niche and preserve intestinal regeneration in response to gut GVHD. Disclosures No relevant conflicts of interest to declare.
The failure to control both innate and adaptive immune responses in the gut has recently been implicated as a major pathogenic mechanism in the development of graft-versus-host disease (GVHD). Reduced oxygen availability in the intestine has been causally linked to gastrointestinal disease. During intestinal inflammation, increased metabolic activity of resident and infiltrating immune cells, bacteria and reduced blood flow may lead to a sharp decrease of oxygen, resulting in "inflammatory" hypoxia. The transcription factor family, hypoxia-inducible factor (HIF) originally discovered as a master regulator of the adaptive response to hypoxia, has recently emerged as a key regulator of the innate and adaptive immune responses. The HIF heterodimer consists of an oxygen-labile α subunit (HIFα) and a constitutively expressed HIF-1β subunit. Both HIF-1α and HIF-2α expression are markedly elevated in intestinal epithelial cells of patients with inflammatory bowel disease (IBD) and intestinal epithelial HIF-1 attenuates colitis in preclinical mouse models. Although HIF-2 has not been studied extensively in intestinal inflammation, it has emerged as a key regulator in intestinal iron homeostasis. Given that IBD and GVHD share many pathogenic mechanisms, we hypothesized that a sustained HIF response will protect the host intestinal epithelium from conditioning- and alloreactive T cell-induced gut damage. To determine the functional significance of intestinal epithelial HIF-1 and HIF-2 in gut GVHD, we generated conditional intestinal epithelial HIF-1α (HIF-1αΔIE) and HIF-2αΔIE vil-cre knockout (KO) mice on a C57BL/6 (B6) background lacking HIF-1α or HIF-2α in the host intestinal epithelium. Using a fully MHC mismatched B10.BR (H2k)→B6 (H2b) bone marrow transplant (BMT) model, loss of intestinal epithelial HIF-2 reduced the median survival time (43d) compared to wild-type (WT) recipients (58d, log-rank test, P < 0.005). Although intestinal epithelial HIF-1 deficiency shortened the median survival time (48.5d), it did not reach statistical difference. Loss of intestinal epithelial HIF-1 or HIF-2 worsened GVHD-induced histopathologic crypt damage compared to WT mice transplanted with T cell depleted bone marrow (BM) and enriched T cells (BM+T), 8d post- BMT. Pronounced subepithelial lifting, mucosal edema and sloughing were more evident in the villus tips of HIF-2αΔIE mice than HIF-1αΔIE mice. Hyperplastic crypts that are characteristic of regenerating crypts after radiation-induced damage were observed in Ki67-stained ileal/jejunal sections of WT mice post-BMT whereas fewer regenerating Ki67-labeled crypts were found in both HIF-1αΔIE and HIF-2αΔIE mice. In control T cell depleted BM groups (WT, HIF-1αΔIE and HIF-2αΔIE), Ki67+ -proliferating cells resided at the crypt base. Using quantitative real-time PCR analysis, we determined whether intestinal epithelial HIF-1 and HIF-2 differentially regulated the expression of Paneth cells and intestinal stem cell markers in the jejunum, 8d post-BMT. A 5-fold and 2-fold decrease in lysozyme (Lyz) mRNA levels occurred in WT (p<0.001) and HIF-1αΔIE BM+T mice (p<0.001) compared to their respective BM groups, 8d post-BMT. However, due to a 2-fold decrease in endogenous Lyz expression in HIF-1αΔIE (p<0.01) and HIF-2αΔIE (p<0.001) BM mice compared to WT BM mice, Lyz levels were not differentially changed among BM+T groups. Loss of either intestinal epithelial HIF-1 or HIF-2 reduced Reg3γ (p<0.05) and Sox9 (p<0.01) levels whereas only epithelial HIF-1 deficiency reduced Hes-1 by 2-fold (p<0.001) and prevented the recovery of the Lgr5 levels (p<0.01) compared to WT BM+T mice. In summary, we found that both intestinal epithelial HIF-1 and HIF-2 may protect the intestinal stem cell niche from GVHD-induced injury. Importantly, our results suggest that intestinal epithelial HIF-2 may be necessary in preventing GVHD-induced mortality. Disclosures No relevant conflicts of interest to declare.
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