The Impact of Age and Body Fat on Graft-Versus-Host Disease (GVHD) in Mice

Allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially curative procedure performed for a variety of hematological diseases. Patient populations undergoing HSCT are generally skewed towards either young or old age due to the pathogenesis of hematological cancers. Several retrospective studies have identified age as one of the risk factors that correlate with treatment related mortality (TRM), graft versus host disease (GVHD) and tumor relapse. Aging is characterized by a gradual decline in immune cell function, known as immune senescence, yet is also hallmarked by a chronic, low-grade proinflammatory phenotype termed “inflammaging”. We hypothesized that aged animals develop more severe GVHD as a result of this inflammaging phenomenon. To investigate this, we first transplanted donor cells from B10.D2 mice (H-2^d) into either young (< 3 months old) or aged (>15 months old) BALB/c (H-2^d) recipients. While young recipients developed typical sclerodermatous chronic GVHD and died by 56 days post transplantation, aged mice developed severe acute GVHD and died at Day 7. Upon pathological examination, aged mice displayed massive lymphocytic infiltration associated with tissue necrosis in the gastrointestinal (GI) tract. Among several cytokines examined, elevated levels of serum TNF-α (97.89±9.83 versus 68.29±1.07 pg/ml, respectively) were observed in aged animals compared to young counterparts. Similarly, TNF-α and IL-6 gene expression levels were also increased in GI tract tissues. Additionally, we found greater frequencies of splenocyte derived TNF-α+ macrophages (CD45+CD19-F4/80+/CD11b+) in aged animals compared to young animals (26.2±1.00% versus 17.233±1.25%, respectively; P< 0.001) following allogeneic HSCT. Macrophage depletion using liposomal clodronate reduced serum TNF-α levels (97.89 ±9.83 versus 57.17±2.86 pg/ml) in aged mice following HSCT.

We observed that aged mice had markedly higher levels of visceral body fat compared to young mice. Based on the similarities in the inflammatory status between aged and obese animals, we next sought to verify whether the severity of GVHD can also be attributed to obesity. Eight week old recipient BALB/c mice were maintained on either a low fat diet (10% calories from fat) or a high fat diet (60% calories from fat) for three months. Each cohort of mice then underwent HSCT, following a conditional regimen of total body irradiation (Cs; 800 cGy) and adoptive transfer from donor B10.D2 mice. In line with the results observed from aged recipients, diet induced obese (DIO) mice died at Day 7 and demonstrated a severe acute GVHD response in the GI tract compared to lean mice based on histo-pathological scores correlating with significantly increased TNF-α and IL-6 gene expression in the GI tract. Flow analysis revealed an increase in total numbers of CD8+ T cells infiltrating in the GI tract in obese mice compared to lean mice (0.229± 0.025 x 10⁶ versus 0.071± 0.010 x 10⁶; P<0.01). In addition, obese mice demonstrated an increase in total numbers of TNF-a+ macrophages (CD45+F4/80+CD11b+TNF-α +; 5.18± 1.09 x 10⁴ versus 1.31± 0.75 x 10⁴ ; P<0.05) in the visceral fat tissues. Overall, these data implicate that age and body fat can predispose to severe acute GVHD, which is associated with increased production of proinflammatory cytokines mediated by dysregulated macrophages.