Lean Recipients Research Articles

Obesity-Induced Microbiome Alterations Result in Severe Gastrointestinal Graft-Versus-Host Disease Following Allogeneic Hematopoietic Stem Cell Transplantation

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains a viable treatment option for many cancers but its clinical utility is limited due to the occurrence of graft-versus-host disease (GVHD). Understanding the impact of obesity on immune function has become increasingly important in the setting of the current obesity pandemic. We report here that obesity has a negative and selective impact on acute gut GVHD. Diet-induced obese (DIO) mice exhibited increased gut permeability, endotoxin translocation and radiation-induced gastrointestinal damage. After allo-HSCT, DIO recipients across strains and sex had markedly increased pro-inflammatory cytokines (IL-6, TNF), GVHD biomarker ST2, MHC class II expression and exhibited rapid mortality associated with severe acute gut pathology. This obesity-associated lethal acute gut GVHD was dependent on donor CD4 T cells and occurred even in minor MHC mismatch strain combination in which only a delayed skin chronic GVHD resulted in lean recipients. Pro-inflammatory cytokine blockade targeting both IL-6 and TNF ameliorated obesity-associated acute gut GVHD while maintaining graft-versus-tumor (GVT) effects. Microbiome assessment of DIO mice revealed markedly reduced microbiome diversity and decreased Clostridiaceae abundance. Additionally, DIO mice had a significant increase of GVHD-associated Akkermansia muciniphila before and after allo-HSCT compared to the controls. Extended antibiotic treatment of DIO mice protected from the endotoxin translocation, cytokine storm as well as gut GVHD pathology but did not protect later development of chronic skin GVHD. These results demonstrate that obesity alters the microbiome and imparts differential effects on GVHD following allo-HSCT with decreased survival and this inferior outcome can be pre-empted by combined pro-inflammatory cytokine blockade or antibiotic pretreatment. Disclosures Pai: Roche-Genentech: Employment. Holtan:CSL Behring: Consultancy; Incyte: Consultancy; Bristol-Myers Squibb: Consultancy; Janssen: Consultancy. Ferrara:ViraCor: Consultancy; Incyte: Consultancy; Kamada: Consultancy; Mallinckrodt: Consultancy; Enlivex: Consultancy; Xenikos: Consultancy; CSL Behring: Consultancy; National Institutes of Health: Research Funding. Levine:Novartis: Honoraria; Kamada: Research Funding; bluebird bio: Consultancy; National Cancer Institute: Research Funding; Incyte: Consultancy, Research Funding; Ironwood: Honoraria; Biogen: Other: non-financial support; Viracor: Patents & Royalties: biomarker patent. Abedi:Abbie: Speakers Bureau; Gilead: Speakers Bureau; Celgene: Speakers Bureau; BMS: Speakers Bureau; Takeda: Speakers Bureau. Blazar:Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Alpine Immune Sciences, Inc.: Research Funding; Abbvie Inc: Research Funding; Leukemia and Lymphoma Society: Research Funding; Childrens' Cancer Research Fund: Research Funding; KidsFirst Fund: Research Funding; Tmunity: Other: Co-Founder; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Cell-intrinsic TLR4-dependent bone marrow lympho-myeloid distortions in obesity

Abstract Obesity compromises bone marrow (BM) progenitor function and the mechanistic underpinnings are just beginning to be established. Here we show that following 16 weeks of high fat diet, mice exhibit poor emergency hematopoiesis in a toll-like receptor 4 (TLR4)-dependent manner, and that adoptive transfer of obese BM to lean recipients restores rebound capability. Genes associated with a myeloid signature (c-fms, pu.1, ikaros) are enhanced and lymphoid signature (flt3, meis1, e47) reduced. The skewed lympho-myeloid outgrowth of discrete BM subsets is apparent by 6 weeks of high fat diet, earlier than is commonly recognized. Using mixed BM chimeras we demonstrate that obesity-associated subversion of lineage-c-kit+Sca-1- (LKSneg) precursors to the myeloid lineage and common lymphoid progenitors (CLP) to the B lineage depends on BM cell subset-autonomous TLR4. The endogenous TLR4 ligands responsible for BM dysfunction in obesity are unclear. Here, we show that exposure of lean mice to lipopolysaccharide (LPS) at levels similar to that observed in obesity recapitulates key aspects of BM lympho-myeloid disruption. Together, our results establish a mechanistic role for BM cell-intrinsic TLR4 early in obesity-driven BM dysfunction, and demonstrate the biological importance of one obesity-associated TLR4 ligand.

Attenuation of Ischemia-Reperfusion Injury and Improvement of Survival in Recipients of Steatotic Rat Livers Using CD47 Monoclonal Antibody.

Despite the efficacy of orthotopic liver transplantation in the treatment of end-stage liver diseases, its therapeutic utility is severely limited by the availability of donor organs. The ability to rehabilitate marginal organs, such as steatotic allografts, has the potential to address some of the supply limitations of available organs for transplantation. Steatotic livers are more susceptible to ischemia-reperfusion injury (IRI), which is exacerbated by the thrombospondin-1/CD47 pathway through inhibition of nitric oxide signaling. We postulated that CD47 blockade with a monoclonal antibody specific to CD47, clone 400 (CD47mAb400) may reduce the extent of IRI in steatotic liver allografts. Orthotopic liver transplantation was performed using steatotic liver grafts from Zucker rats transplanted into lean recipients. Control IgG or the CD47mAb400 was administered to the donor livers at procurement. Serum transaminases, histological changes, and animal survival were assessed. Hepatocellular damage, oxidative and nitrosative stress, and inflammation were also quantified. Administration of CD47mAb400 to donor livers increased recipient survival and resulted in significant reduction of serum transaminases, bilirubin, triphosphate nick-end labeling staining, caspase-3 activity, oxidative and nitrosative stresses, and proinflammatory cytokine expression of TNF-α, IL-6 and IL-1β. We conclude that administration of CD47mAb400 to donor grafts may reduce IRI through CD47 blockade to result in improved function of steatotic liver allografts and increased survival of recipients and represent a novel strategy to allow the use of livers with higher levels of steatosis.

High-Fat Diet-Induced Obesity Enhances Allograft Rejection.

Obesity promotes a state of low-grade inflammation that exacerbates chronic inflammatory diseases, such as asthma and inflammatory bowel disease. In transplantation, the survival of organs transplanted into obese patients is reduced compared with allografts in lean recipients. However, whether this is due to increased alloimmunity remains to be addressed conclusively. We used a mouse model of high-fat diet (HFD)-induced obesity and assessed immune responses to allogeneic stimulation in vitro, allogeneic splenocyte immunization in vivo, and allogeneic heart transplantation. Our results indicate that HFD altered the composition and phenotype of splenic antigen-presenting cells that led to their enhanced capacity to stimulate T cells. Immunization with allogeneic splenocytes in vivo resulted in increased alloreactivity, as determined by IFNγ production. Moreover, cardiac allograft rejection in HFD mice was modestly accelerated compared to aged-matched control animals fed a low-fat diet, correlating with enhanced alloreactive T cell function. Our results highlight the increased alloresponse triggered by HFD-induced obesity and its negative impact on transplant outcome.

Compromised emergency hematopoiesis in obesity requires TLR4

Abstract Following acute injury/infection, bone marrow (BM) precursors must responsively redirect from steady-state to emergency hematopoiesis. Prospective and retrospective studies establish obesity as risk factor for infection but the mechanisms are poorly understood. Here, we show in a murine model of obesity that moderate changes in steady-state BM hematopoiesis become exaggerated during hematopoietic duress. At steady-state, diet-induced obese mice have a 30% decrease in BM hematopoietic stem cells (HSCs) with a parallel increase in splenic HSCs. HSCs are functionally competent in vitro and following competitive transfer in vivo. BM lymphoid and myeloid progenitors in obese mice are numerically intact but the transcriptional profiles and differentiation potential are strikingly myeloid-biased, accompanied by myeloid skewing of peripheral blood. Responses to acute hematopoietic challenge are deficient. Emergency hematopoiesis following chemotherapeutic ablation is reduced 60% in obesity. Likewise, emergency myelopoiesis in response to endotoxin is characterized by accumulation of immature rather than mature myeloid cells in obese mice. The obesity-associated inflammatory environment plays a key role as transfer of obese BM to lean recipients restores emergency immune response to chemotherapeutic challenge. Moreover, mice deficient in toll-like receptor 4 (TLR4), a central regulator of inflammation, retain robust hematopoietic rebound responses despite diet-associated weight gain. Thus, in obesity, moderate changes in steady-state blood cell production mask major defects in hematopoietic stress responses, via a TLR4-dependent mechanism.

Obesity Accelerates Allograft Rejection Linked to a Th17 Bias in Systemic Immune Responses.

Background: Obesity has been conceptualized as a state of chronic low-grade inflammation. We hypothesized that obesity will accelerate allograft rejection due to obesity-related inflammation. Methods: Diet-induced obese (DIO) C57Bl/6 mice were maintained for 14 wks on high-fat diet (60% kcal% fat) while lean littermates received standard chow. To test the hypothesis of an accelerated alloimmune response, we grafted tail skins in a fully MHC mismatched donor/recipient combination of DBA®C57Bl/6 (n=5/group). Isografts (C57Bl/6®C57Bl/6) served as controls (n=4/group). In parallel, we tested systemic immune responses by ELISA at serial time intervals. Results: Allogenic skin grafts were rejected significantly more rapidly in obese recipients (median graft survival: 7 vs. 9 d. in lean controls; Log-rank test: p=0.015). Of note, accelerated rejection was not related to surgical/technical issues or a compromised perfusion of skin transplants as isogenic grafts survived indefinitely in obese and lean recipients (Fig.1). Next, we explored if obesity impacted Th1, Th2 or Th17 dominated immune responses. While IFN-γ and IL-10 production was comparable following transplantation, splenocytes of obese recipients produced significantly more IL-17A (n=4/group by d. 6; p<0.05; Fig.2). Conclusion: Obese mice reject skin transplants more rapidly associated with an increased production of IL-17A. Those findings are of critical significance in the context of a rapidly increasing proportion of obese transplantat candidates.Fig. 1:: Allogenic and isogenic graft survival (obese allogenic vs. control p=0.015)Fig. 2:: IL-17A production from splenocytes (* p<0.05)

Adipose Tissue Macrophages Promote Myelopoiesis and Monocytosis in Obesity

Obesity is associated with infiltration of macrophages into adipose tissue (AT), contributing to insulin resistance and diabetes. However, relatively little is known regarding the origin of AT macrophages (ATMs). We discovered that murine models of obesity have prominent monocytosis and neutrophilia, associated with proliferation and expansion of bone marrow (BM) myeloid progenitors. AT transplantation conferred myeloid progenitor proliferation in lean recipients, while weight loss in both mice and humans (via gastric bypass) was associated with a reversal of monocytosis and neutrophilia. Adipose S100A8/A9 induced ATM TLR4/MyD88 and NLRP3 inflammasome-dependent IL-1β production. IL-1β interacted with the IL-1 receptor on BM myeloid progenitors to stimulate the production of monocytes and neutrophils. These studies uncover a positive feedback loop between ATMs and BM myeloid progenitors and suggest that inhibition of TLR4 ligands or the NLRP3-IL-1β signaling axis could reduce AT inflammation and insulin resistance in obesity.

Endoplasmic reticulum stress is a mediator of posttransplant injury in severely steatotic liver allografts

Hepatic steatosis continues to present a major challenge in liver transplantation. These organs have been shown to have increased susceptibility to cold ischemia/reperfusion (CIR) injury in comparison with otherwise comparable lean livers; the mechanisms governing this increased susceptibility to CIR injury are not fully understood. Endoplasmic reticulum (ER) stress is an important link between hepatic steatosis, insulin resistance, and metabolic syndrome. In this study, we investigated ER stress signaling and blockade in the mediation of CIR injury in severely steatotic rodent allografts. Steatotic allografts from genetically leptin-resistant rodents had increased ER stress responses and increased markers of hepatocellular injury after liver transplantation into strain-matched lean recipients. ER stress response components were reduced by the chemical chaperone taurine-conjugated ursodeoxycholic acid (TUDCA), and this resulted in an improvement in the allograft injury. TUDCA treatment decreased nuclear factor kappa B activation and the proinflammatory cytokines interleukin-6 and interleukin-1β. However, the predominant response was decreased expression of the ER stress cell death mediator [CCAAT/enhancer-binding protein homologous protein (CHOP)]. Furthermore, activation of inflammation-associated caspase-11 was decreased, and this linked ER stress/CHOP to proinflammatory cytokine production after steatotic liver transplantation. These data confirm ER stress in steatotic allografts and implicate this as a mediating mechanism of inflammation and hepatocyte death in the steatotic liver allograft.

Cold Ischemia and Reperfusion Injury in the Steatotic Livery Is Mediated by Acute Endoplasmic Reticuluim Stress

Background: Hepatic steatosis is an increasingly prevalent problem in liver transplantation (LT) leading to increased cold ischemia and reperfusion (CIR) injury and severe allograft dysfunction. Hepatic steatosis is strongly associated with endoplasmic reticulum (ER) stress which can trigger cell death via multiple pathways. We hypothesized that acute ER stress gene expression following CIR injury may mediate injury in steatotic livers following liver transplantation. Methods: Steatotic livers from obese Zucker rats were transplanted into lean recipients (cold preservation-2hours, reperfusion-12hr) (n=5).

Studies of regional adipose transplantation reveal a unique and beneficial interaction between subcutaneous adipose tissue and the intra-abdominal compartment

To the Editor: Obesity is associated with insulin resistance and type 2 diabetes, with accumulation of intra-abdominal fat carrying a more severe disease risk than accumulation of subcutaneous fat. It remains unclear whether increased visceral fat has an adverse metabolic effect due to its location or to the unique properties of intra-abdominal adipocytes. Konrad et al. [1] reported that increasing intra-abdominal fat mass by transplantation of epididymal fat from normal mice into lean recipients improved fasting glucose tolerance and insulin sensitivity, achieving an effect opposite to the expectedmetabolicconsequenceofincreasedintra-abdominal fat. This suggests that obesity-induced alterations in adipose tissue function rather than mass are responsible for the adverse metabolic consequences of obesity. We hypothesised that the intrinsic properties of adipocytes are responsible for their metabolic effects, irrespective of their anatomical location. We have addressed this using regional adipose tissue cross-transplantation in which subcutaneous (inguinal) and intra-abdominal (epididymal) fat pads from donor mice were transplanted into the subcutaneous (group 1) or intra-abdominal (group 2) compartment of recipient mice on a high-fat diet. Our studies revealed that transplantation of intra-abdominal fat into either the intraabdominal or subcutaneous space had no effect on the metabolism of a recipient animal, whereas transplantation of subcutaneous fat into the intra-abdominal space had a significant protective effect on adiposity, insulin sensitivity and glucose tolerance.

Cyclic RGD Peptides With High Affinity for α5β1 Integrin Protect Genetically Fat Zucker Rat Livers From Cold Ischemia/Reperfusion Injury

We tested a hypothesis that interactions between fibronectin (FN), a key extracellular matrix component, and its integrin α5β1 receptor are important in the development of ischemia/reperfusion (I/R) injury of steatotic liver transplants. We examined the effect of a cyclic RGD peptide (cRGD), with high affinity for α5β1 integrin, in a well-established steatotic rat liver model of ex vivo cold ischemia followed by isotransplantation. In this model, cRGD peptides were administered through the portal vein of steatotic Zucker rat livers prior to and after cold ischemic storage. Lean Zucker recipients of fatty orthotopic liver transplants (OLTs) received an additional course of cRGD peptides 1 hour posttransplantation. cRGD peptide therapy significantly inhibited the recruitment of monocyte/macrophages, and repressed the expression of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α and interferon (IFN)-γ. Moreover, it resulted in selective inhibition of inducible nitric oxide synthase (iNOS), and MMP-9 expression. Importantly, cRGD peptide therapy improved the function and histologic preservation of steatotic liver grafts, extending their 14-day survival in lean recipients from 50% in untreated to 100% in cRGD-treated OLTs. Thus, cRGD peptide-mediated blockade of FN-α5β1 interaction protects against severe I/R injury otherwise experienced by steatotic OLTs.

Steatotic liver transplantation in the mouse: a model of primary nonfunction

Background The number of potential donor organs deemed suboptimal for transplantation because of hepatic steatosis is rising as the obesity rate increases. However, no mouse transplant model has been described within the framework of hepatic steatosis. We describe the development of and our initial experience with a steatotic mouse orthotopic liver transplant model using the ob/ob mouse. This model is technically achievable and functionally mimics primary nonfunction. Materials and methods Adapting techniques of a nonarterialized murine transplant model, C57BL6 ob/ob mice aged 5–7 weeks (26–35 g) and lean controls served as liver donors and recipients. Orthotopic liver transplantation (OLT) was performed using a two-cuff technique at the infrahepatic cava and portal vein. The suprahepatic cava was anastomosed end to end, and the bile duct was stented. The hepatic artery was not reconstructed. Results Lean-to-lean OLT was performed with 70% ( n = 10) long-term survival. ob/ob-to-age-matched lean recipients had 0% ( n = 10) survival because of size discrepancy. ob/ob livers were transplanted to size-matched lean recipients (>3 months old) with short-term survival of 30% ( n = 10). These mice survived the operation, awakened, but expired within 24 h. Serum transaminases revealed a significantly higher injury profile in the recipients of the steatotic livers, and histology showed massive centrilobular coagulative necrosis with hemorrhage, the overall picture being that of primary nonfunction. Conclusions This novel use of the ob/ob mouse for OLT provides us with a model for steatotic transplantation with primary nonfunction as the end point and may help to better understand the response of the steatotic liver to the insult of transplantation.

Fibronectin-α4β1 Integrin-Mediated Blockade Protects Genetically Fat Zucker Rat Livers from Ischemia/Reperfusion Injury

We tested a hypothesis that interactions between fibronectin (FN), the major extracellular matrix component, and its integrin alpha 4 beta 1 receptor is important in the development of ischemia/reperfusion injury of steatotic liver transplants. We examined the effect of connecting segment-1 (CS1) peptide-facilitated blockade of FN-alpha 4 beta 1 interaction in a well-established steatotic rat liver model of ex vivo cold ischemia followed by iso-transplantation. In this model, CS1 peptides were administered through the portal vein of steatotic Zucker rat livers before and after cold ischemic storage. Lean Zucker recipients of fatty liver transplants received an additional 3-day course of CS1 peptides after transplant. CS1 peptide therapy significantly inhibited the recruitment of T lymphocytes, neutrophil activation/infiltration, and repressed the expression of proinflammatory tumor necrosis factor-alpha and interferon-gamma. Moreover, it resulted in selective inhibition of inducible nitric oxide synthase expression, peroxynitrite formation, and hepatic necrosis. Importantly, CS1 peptide therapy improved function/histological preservation of steatotic liver grafts, and extended their 14-day survival in lean recipients from 40% in untreated to 100% in CS1-treated OLTs. Thus, CS1 peptide-mediated blockade of FN-alpha 4 beta 1 interaction protects against severe ischemia/reperfusion injury experienced otherwise by steatotic OLTs. These novel findings document the potential of targeting FN-alpha 4 beta 1 in vivo interaction to increase the transplant donor pool through modulation of marginal steatotic livers.