The effect of obesity on acute GvHD outcomes following allogeneic HSCT

Immune processes, mediated by donor T cells, are thought to be central to the pathogenesis of both acute (a) and chronic (c) graft-versus-host disease (GvHD). Obesity, defined as a body mass index (BMI) ≥ 30, affects more than one-third of the US population and is often viewed as a state of low-grade inflammation. However, how obesity might impact allogeneic hematopoietic stem cell transplant (allo-HSCT) outcomes, including the development of GvHD, has not been clarified, mainly due to changing regimens surrounding allo-HSCT, the increasing age of patients, and limited sample size. A greater understanding is therefore needed, particularly given the growing incidence of obesity among patients undergoing allo-HSCT. The high incidence of both aGvHD and cGvHD prevents allo-HSCT from being completely curative for patients with hematologic diseases.

Lam Khuat and colleagues investigated the effect of obesity on the pathogenesis of GvHD following allo-HSCT in several mouse models, and analyzed the gut microbiota and serum GvHD markers in a clinical cohort of patients undergoing allo-HSCT. Here, we summarize the key points from their article, which was published in Science Translational Medicine.¹

Methods

Mice studies

Male and female BALB/c (H-2d) and C57BL/6 (H-2b) mice, aged 7 to 8 weeks old, were used as recipients of transplanted cells, and were fed 60% fat from lard (high-fat) or 10% fat (low-fat) diets for 4 to 6 months to create diet-induced obese (DIO) and control mice.

As expected, DIO mice were heavier, displayed a different distribution of body fat, and had increased serum glucose compared with controls (p < 0.0001). The strain combinations created are detailed in Table 1.

Table 1. Mouse models of allo-HSCT used*

aGvHD, acute GvHD; BM, bone marrow; cGvHD, chronic GvHD; GvHD, graft-versus-host disease; MHC, major histocompatibility complex. *adapted from Khuat et al.1
Strain combination	Donor	Recipient	MHC incompatibility	GvHD model
BALB/c +T →C57BL/6	BALB/c BM and T cells	C57BL/6	Major	Lethal aGvHD affecting gut, liver, and skin
B10.D2 +SC → BALB/c	B10.D2 BM and splenocytes	BALB/c	Minor	Sclerodermatous skin cGvHD
B10.D2 +CD8 → BALB/c	B10.D2 BM and CD8+ T cells	BALB/c	Minor	—
B10.D2 +CD4 → BALB/c	B10.D2 BM and CD4+ T cells	BALB/c	Minor	Sclerodermatous skin cGvHD
C3H.SW +CD8 → C57BL/6	C3H.SW BM and CD8+ T cells	C57BL/6	Minor	aGvHD affecting skin and liver
C3H.SW +T → C57BL/6	C3H.SW BM and T cells	C57BL/6	Minor	—

Patient studies

Posttransplant survival was assessed in 37 adult patients from the University of Minnesota undergoing unrelated donor allo-HSCT who subsequently developed aGvHD. Fecal microbiota diversity was examined in a different cohort of patients at the University of Minnesota who were participating in a prospective stool sample collection study. Blood samples from a further cohort of patients, who were undergoing allo-HSCT at The Tisch Cancer Institute at Mount Sinai, were also collected. Patient characteristics, stratified by body mass index (BMI) pretransplant, are summarized in Table 2.

Table 2. Characteristics for one cohort of patients at the University of Minnesota and the cohort at The Tisch Cancer Institute cohort, stratified by BMI*

ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; BMI, body mass index; MDS, myelodysplastic syndromes; MM, multiple myeloma; NHL, non-Hodgkin lymphoma; SLL, small lymphocytic lymphoma. *adapted from Khuat et al.1
Characteristic	University of Minnesota cohort		The Tisch Cancer Institute cohort
	BMI < 30 (n = 22)	BMI > 30 (n = 15)	BMI < 30 (n = 5)	BMI > 30 (n = 5)
Age, range	18–70	31–71	23–71	40–63
Donor relationship, n
Unrelated	22	15	—	—
Unrelated mismatched	—	—	5	4
Related mismatched	—	—	—	1
Conditioning, n
Nonmyeloblative	13	8	—	—
Full preparation	9	7	—	—
Diagnosis, n
Acute leukemia	12	9	4	2
NHL/SLL	2	1	—	—
MDS	4	3	—	1
Others	4	2	1	2

Key findings

As detailed in Table 3, in mouse models and adult patients undergoing allo-HSCT, obesity induces:

• more rapid aGvHD development
• more lethal aGvHD and worse survival
• increased levels of the inflammation markers, IL-6 and TNF
• higher levels of the serum GvHD marker ST2
• expansion of CD4⁺ T cells

Table 3. Obesity-induced changes after allo-HSCT*

aGvHD, acute GvHD; cGvHD, chronic GvHD; BMI, body mass index; DIO, diet-induced obese; GI, gastrointestinal; GvHD, graft-versus-host disease; IL-6, interleukin-6; TNF, tumor necrosis factor; TRM, transplant-related mortality. *Data from Khuat et al.1 †Outcome in DIO vs control mice or patients with BMI > 30 vs BMI < 30.
Outcome vs control†	Mouse strain/patient cohort	p value	Details
More rapid and severe aGvHD development	BALB/c +T → C57BL/6	p < 0.001	—
	B10.D2 → BALB/c	p < 0.001	Rapid lethal gut aGvHD, control mice had only skin cGvHD
	Univ. of Minnesota cohort	p < 0.05	—
Worse survival	BALB/c +T → C57BL/6	p < 0.0001	—
	B10.D2 → BALB/c	p < 0.0001	—
	Univ. of Minnesota cohort	—	1-year TRM: 26% in patients BMI > 30 vs 5% in patients BMI < 30
Worse GI damage	BALB/c +T → C57BL/6	p < 0.01	—
	B10.D2 → BALB/c	—	Destruction of epithelial mucus layer and GI crypts
Increased IL-6 and TNF levels	BALB/c +T → C57BL/6	p < 0.05	—
	B10.D2 → BALB/c	p < 0.05	—
Higher levels of ST2	BALB/c +T → C57BL/6	p < 0.001	—
	B10.D2 → BALB/c	p < 0.001	—
	Univ. of Minnesota cohort	—	1-year survival: 36% in patients BMI > 30 + elevated ST2 vs 81% in all other patients (p = 0.01)
	Tisch Cancer Institute cohort	p < 0.05	—
Expansion of donor CD4+ T cells	BALB/c +T → C57BL/6	p < 0.05	Mostly in mesenteric lymph nodes
	B10.D2 → BALB/c	p < 0.05	—

Obesity predisposes to CD4⁺ T cell-mediated gut aGvHD

• By comparing bone marrow (BM) transfer with whole splenocytes, or purified CD8⁺ or CD4⁺ T cells only, in the B10.D2 → BALB/c strain, the authors found that only DIO mice who received CD4⁺ T cells or whole splenocytes developed lethal aGvHD after allo-HSCT.
• Transplantation of total T cells (CD4⁺ and CD8⁺) or CD8⁺ T cells into the C3H.SW → C57BL/6 mice (a strain combination that results in CD8⁺ T cell-mediated aGvHD affecting the skin and liver), showed no difference in skin, liver, or gut pathology, or cytokine levels, between control and DIO mice.

Obesity increases gut permeability and epithelial cell apoptosis

• Two days after administration of a total body irradiation conditioning regimen, DIO mice displayed increased intestinal permeability and translocation of lipopolysaccharide compared with control mice (p < 0.05).
• There were higher numbers of apoptotic cells in the intestines of DIO mice than controls (p < 0.05), following allo-HSCT, indicating increased dendritic cell activation.

Obesity reduced gut microbiota diversity

• Assessment of bacterial taxa in both BALB/c and C57BL/6 strains revealed that the gut microbiota profiles of DIO-recipient mice were less diverse compared with controls.
• Specifically, the Clostridiaceae family of bacteria were lower in number in both DIO BALB/c (p < 0.01) and DIO C57BL/6 (p < 0.05) mice compared with controls, and also in mice fed a short-term high-fat diet compared to a low-fat diet.
• The relative abundance of GvHD-associated Akkermansia muciniphila was increased in BALB/c mice fed a short-term high-fat diet compared with those fed a low-fat diet. Levels were also higher in DIO BALB/c mice compared with controls.
• Increased amounts of enterococci, bacteria associated with poor GvHD outcomes, were found in fecal samples from DIO mice post-HSCT compared with controls (p = 0.0096).
• Differences in gut microbiota profiles were observed in the University of Minnesota patient cohort between those with a BMI > 30 and those with a lean BMI < 25 prior to allo-HSCT (p < 0.05).
• Sequencing analysis also showed a decrease in microbiota diversity in obese patients, alongside a lower abundance of genus Clostridium.

Prophylactic antibiotics reduce the negative effects of obesity on aGvHD development

Two weeks prior to allo-HSCT, DIO and control B10.D2 + SC → BALB/c mice transplanted with BM and splenocytes received ampicillin, vancomycin, and neomycin antibiotics in their drinking water. The following observations were made in antibiotic-treated DOI mice when compared with untreated DOI mice:

• GvHD lethality was markedly reduced (p < 0.01), with over 50% surviving > 50 days after allo-HSCT
• Clinical aGvHD scores were significantly reduced (p < 0.05)
• The proportion of A. muciniphila before (p < 0.05) and after (p < 0.01) allo-HSCT was reduced and the overall bacterial load was lower
• Levels of IL-6 (p < 0.05), TNF (p < 0.05), ST2 (p < 0.01), and endotoxin (p < 0.05) were lower
• Dendritic cell activation was decreased with fewer dendritic cells in the mesenteric lymph nodes
• Antibiotic-treated DIO mice who survived allo-HSCT developed sclerodermatous skin cGvHD, similar to control mice

Conclusions

In mouse models, obesity augmented the pathogenesis of lethal gut aGvHD, accompanied by CD4⁺ T cell expansion and excess production of proinflammatory cytokines. Data from patient cohorts confirmed these findings, with obesity correlating with poorer GvHD outcomes after allo-HSCT. The authors also concluded the following:

• In a mouse model of sclerodermatous skin cGvHD, obesity shifted the pathogenesis to a lethal rapid gut aGvHD associated with CD4⁺ T cell expansion and increased cytokine production.
• Obesity may predispose to greater intestinal permeability and cellular apoptosis after conditioning treatment, resulting in increased activation of intestinal antigen-presenting dendritic cells and heightened activation of the innate immune system after allo-HSCT.
• Obesity-associated alterations in the gut microbiota are likely to contribute to the exacerbation of aGvHD and poorer outcomes following allo-HSCT; however, antibiotic prophylaxis may offer partial protection from these effects.

Thus, obesity appears to have a multifactorial negative impact on allo-HSCT, namely due to gut aGvHD, with related changes in gut microbiota offering potential targets for therapeutic intervention.