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Xavier S. Revelo,1 Sue Tsai,1 Helena Lei,1 Helen Luck,1 Magar Ghazarian,1 Hubert Tsui,2 Sally Y. Shi,1
Stephanie Schroer,1 Cynthia T. Luk,1 Gloria H.Y. Lin,3 Tak W. Mak,3 Minna Woo,1,4 Shawn Winer,1,5
and Daniel A. Winer15
OBESITY STUDIES
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associated with HFD feeding (15). Despite evidence of increased numbers and functional activity of pathogenic
CD8+ T cells, the mechanisms by which T-cell homeostasis
is regulated in inamed VAT are largely unknown. Moreover, it is unclear whether adipocyte cell death in VAT,
manifested as a dying cell surrounded by macrophages
(the so-called crown-like structures [CLS]), is dependent
on CD8+ T cellmediated killing mechanisms.
Perforin is a cytotoxic effector molecule primarily
released by CD8+ T cells and natural killer (NK) cells to
eliminate infected or dangerous cells via the perforingranzyme cell death pathway (16). Perforin-mediated cytotoxicity is also involved in the homeostatic regulation of
CD4+ and CD8+ T cells in vivo, in a physiologic function
distinct from pathogen clearance (17). Interestingly, expression of perforin and associated granzymes is increased
in VAT of HFD-fed mice, likely related to increased CD8+
T-cell activation (15). In humans, impaired perforindependent cytotoxic function leads to excessive T-cell activation, severe hyperinammation, and the often fatal
disorder hemophagocytic lymphohistiocytosis (18,19). Indeed, perforin-decient mice are susceptible to increases
in CD8+ T-cell activation and IFN-g production in response to distinct stimuli (20). Notably, this heightened
immune activation seems to be mediated via signaling of
the T-cell receptor and results from increased antigenspecic stimulation (20). Perforin-mediated regulation
of CD8+ T-cell activation and expansion may also include
a regulatory feedback on dendritic cells, which prime or
initiate T-cell responses during an immune response
(21,22).
Since obesity-mediated insulin resistance is associated
with increased perforin expression, increased inammation, and antigen-specic immunity in VAT, we investigated the effects of perforin deciency in diet-induced
obesity. Here, we show that mice lacking perforin
(Prf1null) placed on HFD develop worsened obesity, glucose tolerance, and insulin resistance when compared
with wild-type (WT) controls. These changes are accompanied by dysregulation of T-cell homeostasis in VAT with
increases in T-cell number and cytokine production, local
proliferation, and reduced T-cell apoptosis. Interestingly,
the ability to form CLS in VAT, a hallmark of adipocyte
cell death, was not reduced in Prf1null mice, suggesting
that perforin is not necessary for the formation of CLS
in VAT. These ndings suggest that a dominant role of
perforin in obesity-associated insulin resistance is to restrict
T-cell expansion and activation in inamed VAT, providing
further evidence of a critical role for adaptive immune cells
in governing obesity and glucose homeostasis.
RESEARCH DESIGN AND METHODS
Mice
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To determine a potential role for perforin in the regulation of inammation and insulin resistance during
diet-induced obesity, we rst assessed perforin expression
by intracellular staining in immune cells in the VAT,
spleen, and BM from mice fed either NCD or HFD. One
of the dominant cells expressing perforin was CD8+ T cells
in the VAT (Fig. 1A) and spleen (Supplementary Fig. 1A).
Compared with NCD controls, HFD-fed mice showed an
increased frequency of perforin+ CD8+ T cells in the VAT
upon anti-CD3/CD28 stimulation (Fig. 1A) and in the
spleen when left unstimulated (Supplementary Fig. 1A).
Perforin expression was also detected in NK cells (NK1.1+
CD32) and in CD4+ Foxp3+ T cells at low levels in all tissues (Supplementary Fig. 1B). In contrast, perforin was
nondetectable in CD4+ Foxp32 T, gd T, CD19+, CD11b+,
and CD11c+ cells (Supplementary Fig. 1B). No differences were detected in the frequency of NK or CD4+
Foxp3+ T cells expressing perforin between NCD- and
HFD-fed mice in the VAT, spleen, and BM (Supplementary Fig. 1B). Next, to assess the function of perforin
in diet-induced obesity, we compared Prf1null with agematched C57BL/6 WT mice fed HFD starting at 6 weeks
of age. Relative to HFD-fed WT mice, Prf1null mice receiving
the same diet had slight but signicant increases in body
weight between 6 and 18 weeks of age; although by 20
weeks of age, this difference was not statistically signicant
(Fig. 1B). To determine the location of this increase in body
weight, organs and adipose tissues from WT and Prf1null
mice were harvested and weighed after 10 and 20 weeks of
HFD feeding. Prf1null mice had increased subcutaneous fat
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Figure 1Perforin deciency results in increased body weight and adiposity. A: Frequency of perforin+ (Prf-1+) cells within the VAT CD8+ Tcell subsets in WT mice fed either an NCD or HFD for 10 weeks (n = 3 experiments, 10 mice each). Perforin expression was determined in
nonstimulated (NS) or CD3/CD28-stimulated (Stim) VAT cells. B: Body weights of WT and Prf1null mice fed HFD between 6 and 26 weeks of
age (n = 20). Organ weights of WT and Prf1null mice fed HFD (n = 6) for 10 (C) or 20 (D) weeks. E: Abdominal adipose volume of WT and
Prf1null mice fed HFD for 10 weeks determined using MRI (n = 5). F: Relative diameter of adipocytes harvested from WT and Prf1null mice fed
HFD for 10 weeks (n = 75 cells from two mice). Representative adipose tissue histology with the scale bar is set at 100 mm (G), and counting
of CLS per 1003 low power eld (LPF) (H) from WT and Prf1null mice fed HFD for 10 weeks (n = 3 mice; 1012 LPF per mouse). Expression
of lipogenic genes FABP4, CEBP-a, PPAR-g, and SREBP, relative to the housekeeping gene 18s in VAT (n = 3) (I) and liver (n = 5) (J) of WT
and Prf1null mice fed HFD for 10 weeks. K: Concentrations of triglycerides in the liver of WT and Prf1null mice fed HFD for 10 weeks (n = 4).
Data (AK) are means 6 SEM. eVAT, epididymal adipose tissue; SAT, subcutaneous adipose tissue. *P < 0.05, signicance of difference
between WT and Prf1null mice.
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Figure 2Perforin regulates whole-body glucose and insulin metabolism in mice fed an HFD. A: Fasting glucose of WT and Prf1null mice
fed HFD for 10 (n = 20) or 20 weeks (n = 7). B: Blood glucose concentrations (left) and calculated areas under the curves (AUC, right) during
a GTT on WT and Prf1null mice fed HFD for 10 weeks (n = 20). C: Blood glucose concentrations (left) and calculated areas under the curves
(AUC, right) during a GTT on WT and Prf1null mice fed HFD for 20 weeks (n = 7). D: Fasting insulin of WT and Prf1null mice fed HFD for 10 (n =
20) or 20 weeks (n = 7). E: Blood glucose concentrations (left) and calculated areas under the curves (AUC, right) during an ITT with 1.5 units $ kg21
i.p. insulin in WT and Prf1null mice fed HFD for 13 weeks (n = 5). F: Western blot analysis of total and pAkt in VAT, muscle, and liver from
HFD-fed WT and Prf1null (n = 3) after injection of 1.5 units $ kg21 i.p. insulin. Data (AF) are means 6 SEM. *P < 0.05, signicance of
difference between WT and Prf1null mice.
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inammation and associated worsening of insulin resistance observed in the HFD-fed Prf1null mice.
To determine whether the increased VAT T-cell
numbers in Prf1null mice, particularly in pathogenic
CD8+ T cells, were associated with altered cell turnover,
we assessed apoptotic changes and the proliferative potential of WT and Prf1null T cells after 10 weeks of HFD
feeding. Compared with WT controls, Prf1null mice had
a marked decrease in the percentage of early apoptotic
VAT CD8+ T cells but not splenic CD8+ T cells (Fig. 6A),
suggesting that Prf1null mice have a decreased ability to
regulate CD8+ T-cell numbers in VAT through a reduced
induction of apoptosis. Next, we assessed the effect of
perforin deciency on the proliferative potential of T cells.
Although CD3/CD28-stimulated WT and Prf1null splenic
CD8+ T cells proliferated similarly in vitro (Fig. 6B),
Prf1null mice showed a dramatic increase in the in vivo
proliferation of CD8+ T cells in VAT as determined by EdU
incorporation (Fig. 6C). Unrestrained CD8+ T-cell proliferation in Prf1null mice was only observed in VAT as no
differences were detected between WT and Prf1null mice in
the proliferation of CD8+ T cells in the spleen, LNs,
and BM (Supplementary Fig. 4A). Enhanced in vitro
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Figure 4Perforin deciency drives the accumulation of proinammatory immune cells and release of cytokines in VAT. A: Total, CD4+, and
CD8+ T-cell and macrophage (mac) inltration in VAT of WT and Prf1null mice fed HFD for 10 and 20 weeks. B: Frequency of VAT macrophages
(CD11b+ F4/80+) with M1 phenotype (CD11c+) in WT and Prf1null mice after 10 or 20 weeks of HFD feeding. C: CD4+-to-CD8+ ratio in VAT of WT
and Prf1null mice after 10 or 20 weeks of HFD feeding. Prf1null mice tended to have a lower CD4+-to-CD8+ ratio at 10 weeks (P = 0.09) of HFD
feeding. D: Proportion of VAT CD3+ T cells from HFD-fed WT and Prf1null mice positive for CD4 and intracellular Foxp3 (regulatory T cells
[Tregs]). Intracellular staining of IFN-g in CD4+ and CD8+ T cells and NK cells (E) and IL-17 in CD4+ and gd+ T cells (F). Data (AF) show
means 6 SEM of two to three experiments (three to ve pooled mice per group). *P < 0.05, signicance of difference between WT and
Prf1null mice.
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Figure 5Perforin deciency promotes the production of proinammatory cytokines in VAT and systemically. A: Fold changes in cytokine
production by SVCs harvested from VAT from WT and Prf1null mice (n = 3 experiments, nine mice). B: Serum levels of inammatory
cytokines in WT and Prf1null mice fed HFD for 10 (left) or 20 (right) weeks (n = 4). Data (A and B) show means 6 SEM. *P < 0.05, signicance
of difference between WT and Prf1null mice.
DISCUSSION
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Figure 6Impaired apoptosis and heightened local CD8+ T-cell proliferation in the VAT of Prf1null mice. A: Percentage of early apoptotic
splenic and VAT CD8+ T cells and dexamethasone-treated thymocyte control in WT and Prf1null mice fed HFD for 10 weeks determined by
Annexin V staining with propidium iodide. B: Representative histogram (left) and frequency (right) of splenic CD8+ T cells proliferating after
CD3/CD28 activation as determined by a carboxyuorescein succinimidyl ester (CFSE) proliferation assay in WT and Prf1null mice fed HFD
for 10 weeks (n = 4). C: Representative dot plots of CD8+ EdU+ VAT T cells (left) and EdU incorporation of CD4+ and CD8+ T cells in VAT
(right) of WT and Prf1null mice fed HFD for 10 weeks (n = 4) 20 h after EdU injection. D: Frequency of CD8+ T cells expressing CD107a in the
VAT of HFD-fed WT and Prf1null (n = 36) mice. E: Mean uorescence intensity (MFI) of CD44 expression by CD4+ and CD8+ T cells in the
VAT of HFD-fed WT and Prf1null (n = 5) mice. F: Frequency of effector memory (CD44hi CD62L2) CD4+ and CD8+ T cells in the VAT of HFDfed WT and Prf1null (n = 3) mice. Data (AF) are means 6 SEM. *P < 0.05, signicance of difference.
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Figure 7CD8+ T cellderived perforin regulates glucose homeostasis. Body weights (A), GTT (B), and fasting insulin (C) in HFD-fed
CD8null recipients 2 weeks after adoptive transfer of splenic CD8+ T cells from either WT or Prf1null mice (n = 5). D: Western blot analysis of
total and pAkt in VAT, muscle, and liver from HFD-fed CD8null recipients (n = 3) after injection of 1.5 units $ kg21 i.p. insulin. E: Expression of
lipogenic genes FABP4, CEBP-a, PPAR-g, and SREBP relative to the housekeeping gene 18s in VAT of HFD-fed Ragnull recipients 5 days
after adoptive transfer of splenic CD8+ T cells from either WT or Prf1null mice (n = 3). Liver weight (F) and hepatic triglyceride levels (G) in
HFD-fed CD8null recipients 2 weeks after adoptive transfer of splenic CD8+ T cells from either WT or Prf1null mice (n = 5). Body weight (H),
intraperitoneal GTT (I), and fasting insulin (J) in age-matched WT (n = 15 for GTT and n = 10 for insulin) and NKnull (n = 5 for GTT and n = 2 for
insulin) mice fed HFD for 12 weeks. Data (AI) are means 6 SEM. *P < 0.05, signicance of difference.
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