Spleen-derived interleukin-10 downregulates the severity of high-fat diet-induced non-alcoholic fatty pancreas disease.

Obesity is associated with systemic low-grade inflammation and is a risk factor for non-alcoholic fatty pancreas disease (NAFPD), but the molecular mechanisms of these associations are not clear. Interleukin (IL)-10, a potent anti-inflammatory cytokine, is released during acute pancreatitis and is known to limit inflammatory responses by downregulating the release of proinflammatory mediators. The origin of IL-10 that suppresses pancreatitis has not been investigated. Since obesity is known to reduce expression of proinflammatory cytokines in the spleen, we examined whether spleen-derived IL-10 regulates NAFPD caused by high-fat (HF) diet-induced obesity. The following investigations were performed: 1) IL-10 induction from spleen was examined in male mice fed a HF diet; 2) triglyceride content, expression of pro- and anti-inflammatory cytokines and infiltration of M1 and M2 macrophages were determined to evaluate ectopic fat accumulation and inflammatory responses in the pancreas of splenectomy (SPX)-treated mice fed HF diet; 3) exogenous IL-10 was systemically administered to SPX-treated obese mice and the resulting pathogenesis caused by SPX was assessed; and 4) IL-10 knockout (IL-10KO) mice were treated with SPX and ectopic fat deposition and inflammatory conditions in the pancreas were investigated. Obesity impaired the ability of the spleen to synthesize cytokines, including IL-10. SPX aggravated fat accumulation and inflammatory responses in the pancreas of HF diet-induced obese mice and these effects were inhibited by systemic administration of IL-10. Moreover, SPX had little effect on fat deposition and inflammatory responses in the pancreas of IL-10KO mice. Our findings indicate that obesity reduces IL-10 production by the spleen and that spleen-derived IL-10 may protect against the development of NAFPD

SPX has little effect on pro-inflammatory cytokines in the pancreas of IL-10-deficient mice.

<p>Pancreatic content of pro-inflammatory cytokines in each group (<i>n</i> = 6). *<i>P</i><0.05 vs. the Sham group (wild-type or IL-10KO mice). Treatment groups: Sham, fed a HF diet, given serum albumin, and sham-operated; SPX, fed a HF diet, given serum albumin, and splenectomized; SPX+IL-10, fed a HF diet, given recombinant mouse IL-10, and splenectomized. Wild-type, wild-type mice; IL-10KO, IL-10-deficient mice.</p

Effects of splenectomy on daily food intake, body weight, epididymal WAT weight, serum adiponectin levels, glucose and lipid metabolism.

*<p><i>P</i><0.05 vs. the Standard-Sham group. Treatment groups: Standard-Sham, fed standard chow and sham-operated (<i>n</i> = 6); Standard-SPX, fed standard chow and splenectomized (<i>n</i> = 6).</p

Systemic administration of IL-10 diminishes SPX-induced fat accumulation, infiltration of macrophages, and pro-inflammatory responses in the pancreas.

<p>(<b>A</b>) Representative images of H&E staining, oil-red O staining, CD11c staining, and CD206 staining in intra-lobular areas in pancreas sections from each group. Scale bar = 100 µm. (<b>B−E</b>) TG content (<b>B</b>), M1/M2 ratios (<b>C</b>), content of pro- and anti-inflammatory cytokines (<b>D</b>), and IL-10/IL-1β ratios (<b>E</b>) in the pancreas of each group (<i>n</i> = 6). *<i>P</i><0.05 vs. the Standard-Sham group, ^#<i>P</i><0.05 vs. the HF-Sham group. Treatment groups: Standard-Sham, fed standard chow, given serum albumin, and sham-operated; HF-Sham, fed a HF diet, administered serum albumin, and sham-operated; HF-SPX, fed a HF diet, given serum albumin, and splenectomized; HF-SPX+IL-10, fed a HF diet, given IL-10, and splenectomized; Pair-fed, administered serum albumin, sham-operated, and fed the same amount of food as that consumed by the HF-SPX group.</p

Effects of SPX on islets and fat accumulation in the pancreas.

<p>(<b>A</b>) Representative images of insulin staining (left), Mallory-Azan staining (middle), and H&E staining (right) of pancreas sections from mice in each group. (<b>B−E</b>) Insulin staining area (<b>B</b>), intra-islet fibrosis area (<b>C</b>), intra-lobular fibrosis area (<b>D</b>), hydroxyproline content (<b>E</b>), α-SMA-positive cells (<b>F</b>), TG content (<b>G</b>) in the pancreas and serum leptin level (<b>H</b>) in each group (<i>n</i> = 6). *<i>P</i><0.05 vs. the Standard-Sham group. Scale bar = 100 µm. Treatment groups: Standard-Sham, fed standard chow and sham-operated; Standard-SPX, fed standard chow and splenectomized.</p

SPX has little effect on the infiltration of macrophages in intra-lobular area of IL-10-deficient mice.

<p>(<b>A and B</b>) Representative images of CD11c staining (<b>A</b>) and CD206 staining (<b>B</b>) in intra-lobular areas in pancreas sections from each group. Scale bar = 100 µm. (<b>C−E</b>) CD11c-positive areas (<b>C</b>), CD206-positive areas (<b>D</b>), and M1/M2 ratios (<b>E</b>) in intra-lobular areas in each group (<i>n</i> = 6). *<i>P</i><0.05 vs. the Sham group (wild-type or IL-10KO mice), ^#<i>P</i><0.05 vs. SPX mice (wild-type). Treatment groups: Sham, fed a HF diet, given serum albumin, and sham-operated; SPX, fed a HF diet, given serum albumin, and splenectomized; SPX+IL-10, fed a HF diet, given recombinant mouse IL-10, and splenectomized. Wild-type, wild-type mice; IL-10KO, IL-10-deficient mice.</p

Systemic administration of IL-10 diminishes SPX-induced alterations in pancreatic islets.

<p>(<b>A</b>) Representative images of insulin staining (left) and Mallory-Azan staining (right) in pancreas sections from each group. Scale bar = 100 µm. (<b>B−F</b>) Insulin-staining area (<b>B</b>), intra-islet (<b>C</b>) and intra-lobular (<b>D</b>) fibrosis area, hydroxyproline content (<b>E</b>) and α-SMA-positive cells (<b>F</b>) in the pancreas in each group (<i>n</i> = 6). *<i>P</i><0.05 vs. the Sham-Standard group, ^#<i>P</i><0.05 vs. the Sham-HF group. Treatment groups: Standard-Sham, fed standard chow, given serum albumin, and sham-operated; HF-Sham, fed a HF diet, administered serum albumin, and sham-operated; HF-SPX, fed a HF diet, given serum albumin, and splenectomized; HF-SPX+IL-10, fed a HF diet, given IL-10, and splenectomized; Pair-fed, administered serum albumin, sham-operated, and fed the same amount of food as that consumed by the HF-SPX group.</p

SPX has little effect on changes in the pancreatic islets in IL-10-deficient mice.

<p>(<b>A</b>) Representative images of insulin staining (left), Mallory-Azan staining (middle), and α-SMA staining (right) in pancreas sections from each group. Scale bar = 100 µm. (<b>B−F</b>) Insulin-staining area in the pancreas (<b>B</b>), intra-islet fibrosis area (<b>C</b>) and intra-lobular fibrosis area (<b>D</b>), hydroxyproline content (<b>E</b>) and α-SMA-positive cells (<b>F</b>) in each group (<i>n</i> = 6). *<i>P</i><0.05 vs. the Sham group (wild-type or IL-10KO mice), ^#<i>P</i><0.05 vs. SPX mice (wild-type). Treatment groups: Sham, fed a HF diet, given serum albumin and sham-operated; SPX, fed a HF diet, given serum albumin, and splenectomized; SPX+IL-10, fed a HF diet, given recombinant mouse IL-10 and splenectomized. Wild-type, wild-type mice; IL-10KO, IL-10-deficient mice.</p

Systemic administration of IL-10 diminishes SPX-induced fat accumulation, infiltration of macrophages, and pro-inflammatory responses in the islets.

<p>(<b>A</b>) Representative images of CD11c staining (upper sections) and CD206 staining (lower sections) in intra-islet areas in pancreas sections from each group. Scale bar = 100 µm. (<b>B−D</b>) CD11c-positive areas (<b>B</b>), CD206-positive areas (<b>C</b>), and M1/M2 ratios (<b>D</b>) in the islets in each group (<i>n</i> = 6). *<i>P</i><0.05 vs. the Standard-Sham group, ^#<i>P</i><0.05 vs. the HF-Sham group. (<b>E</b>) Serum levels of IL-1β, MCP-1 and IL-10 in sham and SPX mice fed standard chow or a HF diet. *<i>P</i><0.05 vs. the Standard-Sham group, ^#<i>P</i><0.05 vs. the Standard-SPX group. (<b>F</b>) Serum cytokine changes relative to the Standard-Sham group. *<i>P</i><0.05 vs. IL-1β and MCP-1. (<b>G</b>) Serum leptin levels in each group (n = 6). *<i>P</i><0.05 vs. the Standard-Sham group, ^#<i>P</i><0.05 vs. the HF-Sham group, ^$<i>P</i><0.05 vs. the HF-SPX group. Treatment groups: Standard-Sham, fed standard chow, given serum albumin, and sham-operated; HF-Sham, fed a HF diet, administered serum albumin, and sham-operated; HF-SPX, fed a HF diet, given serum albumin, and splenectomized; HF-SPX+IL-10, fed a HF diet, given IL-10, and splenectomized; Pair-fed, administered serum albumin, sham-operated, and fed the same amount of food as that consumed by the HF-SPX group.</p

SPX has little effect on the infiltration of macrophages in islets of IL-10-deficient mice.

<p>(<b>A and B</b>) Representative images of CD11c staining (<b>A</b>) and CD206 staining (<b>B</b>) in intra-islet areas of pancreas sections from each group. Scale bar = 100 µm. (<b>C−E</b>) CD11c-positive areas (<b>C</b>), CD206-positive areas (<b>D</b>), and M1/M2 ratios (<b>E</b>) in the islets in each group (<i>n</i> = 6). *<i>P</i><0.05 vs. the Sham group (wild-type or IL-10KO mice), ^#<i>P</i><0.05 vs. SPX mice (wild-type). Treatment groups: Sham, fed a HF diet, given serum albumin, and sham-operated; SPX, fed a HF diet, given serum albumin, and splenectomized; SPX+IL-10, fed a HF diet, given recombinant mouse IL-10, and splenectomized. Wild-type, wild-type mice; IL-10KO, IL-10-deficient mice.</p