Autophagy gene Atg16L1 prevents lethal T cell alloreactivity mediated by dendritic cells.

Atg16L1 mediates the cellular degradative process of autophagy and is considered a critical regulator of inflammation based on its genetic association with inflammatory bowel disease. Here we find that Atg16L1 deficiency leads to an exacerbated graft-versus-host disease (GVHD) in a mouse model of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Atg16L1-deficient allo-HSCT recipients with GVHD displayed increased T cell proliferation due to increased dendritic cell (DC) numbers and costimulatory molecule expression. Reduced autophagy within DCs was associated with lysosomal abnormalities and decreased amounts of A20, a negative regulator of DC activation. These results broaden the function of Atg16L1 and the autophagy pathway to include a role in limiting a DC-mediated response during inflammatory disease, such as GVHD.

Interleukin-22 protects intestinal stem cells from immune-mediated tissue damage and regulates sensitivity to graft versus host disease.

Little is known about the maintenance of intestinal stem cells (ISCs) and progenitors during immune-mediated tissue damage or about the susceptibility of transplant recipients to tissue damage mediated by the donor immune system during graft versus host disease (GVHD). We demonstrate here that deficiency of recipient-derived IL-22 increased acute GVHD tissue damage and mortality, that ISCs were eliminated during GVHD, and that ISCs as well as their downstream progenitors expressed the IL-22 receptor. Intestinal IL-22 was produced after bone marrow transplant by IL-23-responsive innate lymphoid cells (ILCs) from the transplant recipients, and intestinal IL-22 increased in response to pretransplant conditioning. However, ILC frequency and IL-22 amounts were decreased by GVHD. Recipient IL-22 deficiency led to increased crypt apoptosis, depletion of ISCs, and loss of epithelial integrity. Our findings reveal IL-22 as a critical regulator of tissue sensitivity to GVHD and a protective factor for ISCs during inflammatory intestinal damage.

The central nervous system is a target of acute graft versus host disease in mice.

Despite significant advances in prevention and management, graft versus host disease (GVHD) is still a leading complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Although skin, gut, liver, thymus, and lung are GVHD targets, neurological complications (NC) have also been reported following allo-HSCT. We demonstrate that the central nervous system (CNS) can be a direct target of alloreactive T cells following allo-HSCT in mice. We found significant infiltration of the CNS with donor T lymphocytes and cell death of neurons and neuroglia in allo-HSCT recipients with GVHD. We also found that allo-HSCT recipients with GVHD had deficits in spatial learning/memory and demonstrated increased anxious behavior. These findings highlight CNS sensitivity to damage caused by alloreactive donor T cells and represent the first characterization of target cell subsets and NC during GVHD. Therefore, these clinically relevant studies offer a novel and rational explanation for the well-described neurological symptoms observed after allo-HSCT.

Proximal Plantar Plate of Lesser Toe Metatarsophalangeal Joint Vascular Supply.

BACKGROUND: The plantar plate is a major stabilizing structure of the metatarsophalangeal (MTP) joint with instability frequently occurring after a tear or attenuation of this structure. Commonly, a McGlamry elevator is used to strip the plantar plate from the plantar surface of the metatarsal to improve exposure of the MTP joint. The anatomy of the proximal plantar plate and vascular consequence of stripping the plantar plate from the metatarsal is not yet well understood. The purpose of this study is to describe the proximal attachment of the plantar plate anatomically and quantify the relative contribution of blood supply to the proximal plantar plate from both the metatarsal and the plantar fascia. METHODS: For anatomic evaluation, 6 lower extremity cadaver specimens without any gross evidence of foot and ankle deformity were utilized. For imaging analysis, 16 fresh frozen human adult cadaveric lower extremity specimens were used for this study, resulting in 35 MTP joints without deformity and 11 lesser MTP joints with cockup and/or crossover deformities. The specimens were prepared as described previously by Finney et al.5. RESULTS: From gross anatomic dissection, the plantar plate origin consists of a stout fibrous pedicle distinct from the surrounding synovial-type tissue that firmly anchors the plantar plate to the metatarsal. Based on nano-computed tomographic imaging, an average of 63.5% of the vascular supply to the proximal portion of the plantar plate entered from the metatarsal pedicle. The remaining 36.5% of the vascular supply entered from the plantar fascia. CONCLUSION: The proximal attachment of the plantar plate includes a stout fibrous pedicle anchoring the proximal portion of the plantar plate to the notch between the medial and lateral plantar condyles of the metatarsal head. The vascular supply of the proximal plantar plate is supplied from both the metatarsal pedicle and plantar fascia. LEVEL OF EVIDENCE: Level III, retrospective comparative study.

Presence of Neovascularization in Torn Plantar Plates of the Lesser Metatarsophalangeal Joints.

BACKGROUND: Recent surgical techniques have focused on anatomic repair of lesser toe metatarsophalangeal (MTP) plantar plate tears, yet it remains unknown whether the plantar plate has the biological capacity to heal these repairs. Therefore, a better understanding of the plantar plate vasculature in response to injury may provide further insight into the potential for healing after anatomic plantar plate repair. Recently, a study demonstrated that the microvasculature of the normal plantar plate is densest at the proximal and distal attachments. The purpose of this study was to compare the intact plantar plate microvasculature network to the microvasculature network of plantar plates in the presence of toe deformity using similar perfusion and nano-computed tomographic (CT) imaging methods. METHODS: Seven fresh-frozen human cadaveric lower extremities with lesser toe deformities including hammertoe or crossover toe were perfused using a barium solution. The soft tissues of each foot were counterstained with phosphomolybdic acid (PMA). Then using nano-CT imaging, the second through fourth toe metatarsophalangeal joints of 7 feet were imaged. These images were then reconstructed, plantar plate tears were identified, and 11 toes remained. The plantar plate microvasculature for these 11 toes was analyzed, and calculation of vascular density along the plantar plate was performed. Using analysis of variance (ANOVA), this experimental group was compared to a control group of 35 toes from cadaveric feet without deformity and the vascular density compared between quartiles of plantar plate length proximal to distal. A power analysis was performed, determining that 11 experimental toes and 35 control toes would be adequate to provide 80% power with an alpha of 0.05. RESULTS: Significantly greater vascular density (vascular volume/tissue volume) was found along the entire length of the plantar plate for the torn plantar plates compared to intact plantar plates (ANOVA, P < .001). For the first quartile of length (proximal to distal), the vascular density for the torn plantar plates was 0.365 (SD 0.058) compared to 0.281 (SD 0.036) for intact plantar plates; in the second quartile it was 0.300 (SD 0.044) vs 0.175 (SD 0.025); third quartile it was 0.326 (SD 0.051) vs 0.117 (SD 0.015); and fourth (most distal) quartile was 0.600 (SD 0.183) vs 0.319 (SD 0.082). CONCLUSION: Torn plantar plates showed increased vascular density throughout the length of the plantar plate with an increase in density most notable in the region at or just proximal to the attachment to the proximal phalanx. Our analysis revealed that torn plantar plates exhibit neovascularization around the site of a plantar plate tear that does not exist in normal plantar plates. CLINICAL RELEVANCE: The clinical significance of the increased vascularity of torn plantar plates is unknown at this time. However, the increase in vasculature may suggest that the plantar plate is a structure that is attempting to heal.

Microvasculature of the Plantar Plate Using Nano-Computed Tomography.

BACKGROUND:: Lesser toe plantar plate attenuation or disruption is being increasingly implicated in a variety of common clinical conditions. A multitude of surgical techniques and devices have been recently developed to facilitate surgical repair of the plantar plate. However, the microvascular anatomy, and therefore the healing potential in large part, has not been defined. We investigated the microvasculature of the plantar plate by employing a novel technique involving microvascular perfusion and nano-computed tomography (nano-CT) imaging. METHODS:: Twelve human adult cadaveric lower extremities were amputated distal to the knee. The anterior and posterior tibial arteries were perfused with a barium solution. The soft tissues of each foot were then counterstained with phosphomolybdic acid (PMA). The second through fourth toe metatarsophalangeal (MTP) joints of 12 feet were imaged with nano-CT at 14-micron resolution. Images were then reconstructed for analysis of the plantar plate microvasculature and calculation of the vascular density along the length of the plantar plate. RESULTS:: A microvascular network extends from the surrounding soft tissues at the attachments of the plantar plate on both the metatarsal and proximal phalanx. The midsubstance of the plantar plate appears to be relatively hypovascular. Analysis of the vascular density along the length of the plantar plate demonstrated a consistent trend with increased vascular density at approximately the proximal 29% and distal 22% of the plantar plate. CONCLUSION:: There is a vascular network extending from the surrounding soft tissues into the proximal and distal attachments of the plantar plate. CLINICAL RELEVANCE:: The hypovascular midportion of the plantar plate may play an important role in the underlying pathoanatomy and pathophysiology of this area. These findings may have significant clinical implications for the reparative potential of this region and the surgical procedures currently described to accomplish anatomic plantar plate repair.

Sex steroid blockade enhances thymopoiesis by modulating Notch signaling.

Paradoxical to its importance for generating a diverse T cell repertoire, thymic function progressively declines throughout life. This process has been at least partially attributed to the effects of sex steroids, and their removal promotes enhanced thymopoiesis and recovery from immune injury. We show that one mechanism by which sex steroids influence thymopoiesis is through direct inhibition in cortical thymic epithelial cells (cTECs) of Delta-like 4 (Dll4), a Notch ligand crucial for the commitment and differentiation of T cell progenitors in a dose-dependent manner. Consistent with this, sex steroid ablation (SSA) led to increased expression of Dll4 and its downstream targets. Importantly, SSA induced by luteinizing hormone-releasing hormone (LHRH) receptor antagonism bypassed the surge in sex steroids caused by LHRH agonists, the gold standard for clinical ablation of sex steroids, thereby facilitating increased Dll4 expression and more rapid promotion of thymopoiesis. Collectively, these findings not only reveal a novel mechanism underlying improved thymic regeneration upon SSA but also offer an improved clinical strategy for successfully boosting immune function.

A small-molecule c-Rel inhibitor reduces alloactivation of T cells without compromising antitumor activity.

Preventing unfavorable GVHD without inducing broad suppression of the immune system presents a major challenge of allogeneic hematopoietic stem cell transplantation (allo-HSCT). We developed a novel strategy to ameliorate GVHD while preserving graft-versus-tumor (GVT) activity by small molecule-based inhibition of the NF-κB family member c-Rel. Underlying mechanisms included reduced alloactivation, defective gut homing, and impaired negative feedback on interleukin (IL)-2 production, resulting in optimal IL-2 levels, which, in the absence of competition by effector T cells, translated into expansion of regulatory T cells. c-Rel activity was dispensable for antigen-specific T-cell receptor (TCR) activation, allowing c-Rel-deficient T cells to display normal GVT activity. In addition, inhibition of c-Rel activity reduced alloactivation without compromising antigen-specific cytotoxicity of human T cells. Finally, we were able to demonstrate the feasibility and efficacy of systemic c-Rel inhibitor administration. Our findings validate c-Rel as a promising target for immunomodulatory therapy and demonstrate the feasibility and efficacy of pharmaceutical inhibition of c-Rel activity.

Nrf2 regulates haematopoietic stem cell function.

Coordinating the balance between haematopoietic stem cell (HSC) quiescence and self-renewal is crucial for maintaining haematopoiesis lifelong. Equally important for haematopoietic function is modulating HSC localization within the bone marrow niches, as maintenance of HSC function is tightly controlled by a complex network of intrinsic molecular mechanisms and extrinsic signalling interactions with their surrounding microenvironment. In this study we demonstrate that nuclear factor erythroid 2-related factor 2 (Nfe2l2, or Nrf2), well established as a global regulator of the oxidative stress response, plays a regulatory role in several aspects of HSC homeostasis. Nrf2 deficiency results in an expansion of the haematopoietic stem and progenitor cell compartment due to cell-intrinsic hyperproliferation, which was accomplished at the expense of HSC quiescence and self-renewal. We further show that Nrf2 modulates both migration and retention of HSCs in their niche. Moreover, we identify a previously unrecognized link between Nrf2 and CXCR4, contributing, at least partially, to the maintenance of HSC function.

PLZF confers effector functions to donor T cells that preserve graft-versus-tumor effects while attenuating GVHD.

Efforts to limit GVHD mediated by alloreactive donor T cells after allogeneic bone marrow transplantation are limited by a concomitant decrease in graft-versus-tumor (GVT) activity and increased possibilities of tumor relapse. Using a novel approach, we adoptively transferred conventional T cells expressing the transcription factor promyelocytic leukemia zinc finger (PLZF), which confers effector properties resembling invariant natural killer T cells, such as copious production of cytokines under suboptimal stimulation. PLZF expression in T-cell allografts attenuates expansion of alloreactive T cells, leading to lower GVHD. Intact alloreactivity-driven antitumor cytokine responses result in preserved GVT effects, leading to improved survival. Our findings suggest that therapy with PLZF-overexpressing T cells would result in overall improved outcomes due to less GVHD and intact GVT effects.

Extrathymic development of murine T cells after bone marrow transplantation.

Restoring T cell competence is a significant clinical challenge in patients whose thymic function is severely compromised due to age or cytoreductive conditioning. Here, we demonstrate in mice that mesenteric LNs (MLNs) support extrathymic T cell development in euthymic and athymic recipients of bone marrow transplantation (BMT). Furthermore, in aged murine BMT recipients, the contribution of the MLNs to the generation of T cells was maintained, while the contribution of the thymus was significantly impaired. Thymic impairment resulted in a proportional increase in extrathymic-derived T cell progenitors. Extrathymic development in athymic recipients generated conventional naive TCRαß T cells with a broad Vß repertoire and intact functional and proliferative potential. Moreover, in the absence of a functional thymus, immunity against known pathogens could be augmented using engineered precursor T cells with viral specificity. These findings demonstrate the potential of extrathymic T cell development for T cell reconstitution in patients with limited thymic function.

Interleukin-22 drives endogenous thymic regeneration in mice.

Endogenous thymic regeneration is a crucial function that allows for renewal of immune competence after stress, infection, or immunodepletion. However, the mechanisms governing this regeneration remain poorly understood. We detail such a mechanism, centered on interleukin-22 (IL-22) and triggered by the depletion of CD4(+)CD8(+) double-positive thymocytes. Intrathymic levels of IL-22 were increased after thymic insult, and thymic recovery was impaired in IL-22-deficient mice. IL-22, which signaled through thymic epithelial cells and promoted their proliferation and survival, was up-regulated by radio-resistant RORγ(t)(+)CCR6(+)NKp46(-) lymphoid tissue inducer cells after thymic injury in an IL-23-dependent manner. Administration of IL-22 enhanced thymic recovery after total body irradiation. These studies reveal mechanisms of endogenous thymic repair and offer innovative regenerative strategies for improving immune competence.

Regulation of intestinal inflammation by microbiota following allogeneic bone marrow transplantation.

Despite a growing understanding of the link between intestinal inflammation and resident gut microbes, longitudinal studies of human flora before initial onset of intestinal inflammation have not been reported. Here, we demonstrate in murine and human recipients of allogeneic bone marrow transplantation (BMT) that intestinal inflammation secondary to graft-versus-host disease (GVHD) is associated with major shifts in the composition of the intestinal microbiota. The microbiota, in turn, can modulate the severity of intestinal inflammation. In mouse models of GVHD, we observed loss of overall diversity and expansion of Lactobacillales and loss of Clostridiales. Eliminating Lactobacillales from the flora of mice before BMT aggravated GVHD, whereas reintroducing the predominant species of Lactobacillus mediated significant protection against GVHD. We then characterized gut flora of patients during onset of intestinal inflammation caused by GVHD and found patterns mirroring those in mice. We also identified increased microbial chaos early after allogeneic BMT as a potential risk factor for subsequent GVHD. Together, these data demonstrate regulation of flora by intestinal inflammation and suggest that flora manipulation may reduce intestinal inflammation and improve outcomes for allogeneic BMT recipients.