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0Cell Reports
Article
Circulating NOD1 Activators and Hematopoietic NOD1 Contribute to Metabolic Inflammation and Insulin Resistance
Graphical Abstract
Authors
Kenny L. Chan, Theresa H. Tam,
Parastoo Boroumand.....
Stephen E. Girardin, Dana J. Philpott, Amira Klip
Correspondence
amira@sickkids.ca
In Brief
Chan et al. identify that activators of NOD1, a receptor for bacterial cell wall peptidoglycan, increase in the bloodstream during high fat feeding. Moreover, depleting NOD1 from the immune system prevents proinflammatory macrophage activation and neutrophil infiltration in adipose tissue during a high fat diet, to improve whole-body insulin sensitivity.
Highlights
• High fat diet (HFD) elevates circulating NOD1 activators
• Immune cell-specific NOD1 knockout protects against HFD-induced insulin resistance
• NOD1 mediates macrophage pro-inflammatory polarization but not infiltration in adipose
• Macrophages attract neutrophils into adipose via NOD1-dependent CXCL1 production
Chan et al., 2017, Cell Reports 18, 2415-2426 ciossMark March 7, 2017 © 2017 The Authors.
http://dx.d0i.0rg/l 0.1016/j.celrep.2017.02.027
CelPress
Cell Reports
Article
Circulating NOD1 Activators and Hematopoietic NOD1 Contribute to Metabolic Inflammation and Insulin Resistance
Kenny L. Chan,12Theresa H. Tam,1 Parastoo Boroumand,13 David Prescott,45 Sheila R. Costford,5 Nichole K. Escalante,4
Noah Fine,6 YuShan Tu,7 Susan J. Robertson,4 Dilshaayee Prabaharan,1 Zhi Liu,1 Philip J. Bilan,1 Michael W. Salter,27
Michael Glogauer,6 Stephen E. Girardin,4 5 Dana J. Philpott,4 and Amira Klip12 3,8,*
1Cell Biology Program, The Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada
2Department of Physiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada
3Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada
4Department of Immunology, University of Toronto, Toronto, Ontario M5S 1A8, Canada
5Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada 6Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, Toronto, Ontario M5S 3E2, Canada 7Neurosciences and Mental Health Program, The Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada 8Lead Contact
'Correspondence: amira@sickkids.ca http://dx.doi.org/10.1016/j.celrep.2017.02.027
SUMMARY
Insulin resistance is a chronic inflammatory condition accompanying obesity or high fat diets that leads to type 2 diabetes. It is hypothesized that lipids and gut bacterial compounds in particular contribute to metabolic inflammation by activating the immune system; however, the receptors detecting these "instigators" of inflammation remain largely undefined. Here, we show that circulating activators of NOD1, a receptor for bacterial peptidoglycan, increase with high fat feeding in mice, suggesting that NOD1 could be a critical sensor leading to metabolic inflammation. Hematopoietic depletion of NOD1 did not prevent weight gain but protected chimeric mice against diet-induced glucose and insulin intolerance. Mechanistically, while macrophage infiltration of adipose tissue persisted, notably these cells were less pro-inflammatory, had lower CXCL1 production, and consequently, lower neutrophil chemoat-traction into the tissue. These findings reveal macrophage NOD1 as a cell-specific target to combat diet-induced inflammation past the step of macrophage infiltration, leading to insulin resistance.
INTRODUCTION
Type 2 diabetes (T2D) is a systemic disease affecting more than 300 million people worldwide (Danaei et al., 2011). Preceding T2D is a prediabetic state characterized by obesity, insulin resistance, and as demonstrated over the last decade, chronic low-grade inflammation (Hotamisligil, 2006; Xu et al., 2003). With obesity or high fat diets (HFD), metabolic tissues such as adipose, liver, and skeletal muscle become infiltrated by immune
cells such as macrophages and neutrophils (Fink et al., 2014; Morinaga et al., 2015; Weisberg et al., 2003). A body of literature proposes that these immune cells provoke tissue inflammation upon pro-inflammatory polarization, contributing to insulin intolerance in peripheral tissues (Chawla et al., 2011; Olefsky and Glass, 2010). Remarkably, it remains unclear how these immune cells adopt a pro-inflammatory phenotype during HFD.
Recent studies show that the intestinal barrier becomes compromised during HFD, resulting in translocation of gut-derived bacterial DNA and lipopolysaccharide (LPS) into blood and tissues (Cani et al., 2007; Luck et al., 2015). Along with elevated levels of saturated fat, it has been postulated that these bacterial products activate pro-inflammatory pathways in immune cells during HFD (Chan etal., 2015; McPheeandSchertzer, 2015; Olefsky and Glass, 2010). This "metabolic endotoxemia'' is a potentially critical contributor to the chronic inflammation associated with obesity. Importantly, germ-free mice are protected against HFD-induced obesity and insulin resistance (Rabot et al., 2010), but become insulin resistant when colonized with gut microbiota from conventionally housed mice (Backhed et al., 2004). Consistent with the concept that bacterial products can cause insulin resistance, protection against metabolic inflammation and insulin resistance is observed in mice lacking Toll-like receptors TLR2 or TLR4, or the NLRP3 inflammasome, which detect specific bacterial components to initiate inflammation (Kuo et al., 2011; Shi et al., 2006; Vandanmagsar et al., 2011).
Another family of pattern recognition receptors consists of the nucleotide-binding oligomerization domain-containing proteins, NOD1 and NOD2. NOD1 is a widely expressed intracellular receptor for peptidoglycan from Gram-negative and some Grampositive bacteria (Girardin et al., 2003, 2005). Recently, we and others associated NOD1 with metabolic disease. Specifically, NOD1 expression is elevated in adipose tissue from patients exhibiting metabolic syndrome or gestational diabetes (Lappas, 2014; Zhou et al., 2015) and in circulating monocytes from patients with T2D (Shiny et al., 2013). Moreover, certain NOD1
Cell Reports 18, 2415-2426, March 7, 2017 © 2017 The Authors. 2415 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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Figure 1. Circulating NOD1 Activators Increase with Obesity and High Fat Diet
(A) Serum from mice fed low (LFD) or high fat diet (HFD) for 14 weeks was assessed for NOD1 stimulatory activity.
(B) Serum NOD1 stimulatory activity plotted against body weight with linear regression shown.
(C) Serum NOD1 stimulatory activity plotted against fasting blood glucose with linear regression shown. Results are means ± SEM, n = 8-9 mice per group. Unpaired Student's t test. *p < 0.05.
gene polymorphisms augment the correlation between dietary saturated fat intake and insulin sensitivity in humans (Cuda et al., 2012). Compellingly, whole body depletion of NOD1/2 (Schertzer et al., 2011) or NOD1 alone (Amar et al., 2011) reduces HFD-linked glucose and insulin intolerance in mice. Further, injection of synthetic NOD1 ligands provokes acute inflammation and insulin resistance in the absence of HFD (Schertzer et al., 2011), establishing a direct link between NOD1 activation and insulin resistance. However, and importantly, it is unknown if NOD1 activators circulate in serum during obesity. Moreover, neither the cell types in which NOD1 acts nor the mechanisms involved in eliciting insulin resistance acutely or in response to HFD are known.
Here, we investigate the hypothesis that NOD1 activators in blood are elevated in response to HFD, and NOD1 specifically in immune cells is required for the accompanying metabolic inflammation and insulin resistance. We show that NOD1 activators rise in serum of mice fed HFD, and hematopoietic NOD1 depletion achieved through bone marrow transplantation conferred protection against HFD-induced glucose and insulin intolerance. Although adipose tissue macrophage (ATM) numbers still increase in these chimeric mice, they are less pro-inflammatory compared to NOD1-expressing counterparts. Moreover, this phenotypic switch reduced neutrophil chemoattraction to the tissue through diminished production of local chemokines (CXCL1, CXCL2). We propose that NOD1 in ATMs senses dietary and/or gut-derived bacterial products that trigger pro-inflammatory polarization, leading to adipose tissue-specific inflammation and ensuing whole-body insulin resistance in obesity.
RESULTS
High Fat Feeding Increases Circulating NOD1 Activators
Previous reports have shown that HFD disrupts intestinal integrity, promoting movement of bacterial LPS into the circulation (Cani et al., 2007; Lucket al., 2015); however, the profile of other obesity-linked circulating factors remains poorly characterized. We hypothesized that circulating NOD1 ligands may be elevated in the blood with high fat feeding. Using a HEK293T cell-based
reporter assay that responds to NOD1 activation, we observed that serum from HFD-fed mice had a higher NOD1 activating capacity compared to low fat diet (LFD)-fed mice (Figure 1A), revealing that HFD elevates circulating NOD1 activators. Interestingly, serum NOD1 activating capacity correlated with individual body weight (Figure 1B) and fasting blood glucose concentrations (Figure 1C). We therefore addressed the possibility that circulating NOD1 ligands activate immune cells, contributing to diet-induced inflammation, which begets glucose and insulin intolerance.
Generation of a Mouse Model Lacking NOD1 in Immune Cells
To elucidate the contribution of NOD1 in immune cells to the pathogenesis of diet-induced insulin resistance, we generated mice lacking NOD1 specifically in hematopoietic cells (denoted knockout [KO]/wild-type [WT]) or wild-type controls (WT/ WT) by bone marrow transplantation. Nod1 expression in whole blood and bone marrow-derived macrophages was lower in the KO/WT compared to WT/WT chimeric mice, confirming efficient hematopoietic knockout (Figures S1A and S1B), without changes in Nod2 or Tlr4 expression, or Nod1 expression in non-hematopoietic tissues (Figures S1C-S1F). Importantly, as phenotypic differences between WT/WT and KO/WT mice may depend on the gut microbiota, which could release pepti-doglycan into circulation during HFD, we examined fecal bacteria composition. As reported for both mice and humans (Ley et al., 2006; Murphy et al., 2010), the Bacteroidetes to Firmi-cutes ratio decreased with HFD in our WT/WT mice relative to LFD-fed controls, and this change was not affected by the absence of NOD1 in immune cells (Figure S2A). WT/WT and KO/WT mice also had similar relative abundance of all genera analyzed, including Bacteroides, Clostridium, Lactobacillus, and Bifidobacterium (data not shown). Additionally, the relative abundance of bacterial DNA in adipose tissue was unchanged by diet or hematopoietic NOD1 depletion (Figures S2B-S2E). Thus, any metabolic or inflammatory changes observed in KO/WT mice would unlikely have arisen from stark differences in the microbiota or bacterial translocation.
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Figure 2. Hematopoietic NOD1 Deletion Prevents HFD-Induced Glucose and Insulin Intolerance
Mice with wild-type (WT/WT) or Nodi(KO/WT) immune systems were fed LFD or HFD for 18 weeks.
(A) Body weight measured weekly.
(B) Food intake measured weekly.
(C) Epididymal white adipose tissue (EWAT) and inguinal white adipose tissue (IWAT) weight.
(D) EWAT adipocyte cross-sectional area.
(E) Blood glucose levels following a 4 hr fast measured bi-weekly.
(F) Glucose tolerance test 16 weeks into diets.
(G) Insulin tolerance test 17 weeks into diets.
(H) Akt phosphorylation in EWAT following i.p. saline or insulin injections.
Results are means ± SEM, n = 8-11 (A-C, E-G), n = 3-5 (D), n = 2 (saline), or n = 3 (insulin) (H) mice per group. Two-way ANOVA, Tukey post-test. *p < 0.05, **p < 0.01, ***p < 0.001 (WT/WT- LFD versus WT/WT- HFD), #p < 0.05, ##p < 0.01, ###p < 0.001 (KO/WT- LFD versus KO/WT- HFD), fp < 0.05 (WT/ WT- HFD versus KO/WT- HFD). n.s., not significant. See also Figure S3.
NOD1 Deletion from Immune Cells Improves Glucose and Insulin Tolerance
To ascertain the role of hematopoietic NOD1 in whole-body glucose metabolism, WT/WT and KO/WT mice were fed HFD or LFD for 18 weeks. NOD1 deletion from immune cells did not affect food intake or weight gain in either the LFD or
HFD groups (Figures 2A, 2B, S3A, and S3B). Similarly, both visceral epididymal white adipose tissue (EWAT) and subcutaneous inguinal white adipose tissue (IWAT) expanded with HFD regardless of bone marrow genotype (Figure 2C; Table 1), with no differences in adipocyte size between WT/WT and KO/ WT mice (Figure 2D). Therefore, any alterations arising in glucose
Table 1. NOD1 Chimera Study Endpoint Tissue Weights
WT/WT KO/WT
Tissue LFD HFD LFD HFD
EWAT 937.7 ± 111.7 2,437.6 ± 187.5a 785.2 ± 112.4 2,534.2 ± 227.1b
IWAT 588.6 ± 113.5 1,981.1 ± 257.1a 475.9 ± 76.6 2,176 ± 35.8b
BAT 176.9 ± 27.3 193.2 ± 29.7 162.2 ± 10.6 185.8 ± 5.7
Soleus 16.4 ± 1.4 17.2 ± 0.7 16.3 ± 17.9 17.9 ± 0.9
Gastrocnemius 281.6 ± 12.5 290.4 ± 11.8 278.5 ± 7.3 290.6 ± 25.3
Quadriceps 347.5 ± 17.7 322.9 ± 14.8 342.8 ± 20.4 356.6 ± 39.9
Heart 128.5 ± 9.0 135.0 ± 3.5 132.3 ± 7.6 139.9 ± 5.4
Liver 1,124.1 ± 98.6 1,098.3 ± 76.8 1,161.4 ± 31.8 1,232.2 ± 66.0
Pancreas 114.6 ± 23.0 160.3 ± 13.5 136.7 ± 20.4 181.2 ± 31.5
Kidneys Spleen 356.1 ± 13.7 61.0 ± 2.0 347.0 ± 9.3 69.1 ± 2.3 340.3 ± 2.3 66.3 ± 7.9 349.1 ± 15.0 65.2 ± 3.5
Tissue weights in mgfrom WT/WT or KO/WT mice following 18 weeks of LFD or HFD. Results are means ± SEM, n = 8-9 mice per group. Two-way ANOVA, Tukey post-test. BAT, brown adipose tissue; EWAT, epididymal white adipose tissue; HFD, high fat diet; IWAT, inguinal white adipose tissue; KO, knockout; LFD, low fat diet; WT, wild-type. ap < 0.001 (WT/WT - LFD versus WT/WT - HFD). bp < 0.001 (KO/WT - LFD versus KO/WT - HFD).
or insulin tolerance in the absence of hematopoietic NOD1 cannot be attributed to changes in body weight or adiposity.
As expected, HFD-fed WT/WT mice displayed elevated fasting blood glucose levels compared to LFD-fed controls, but interestingly, these levels were consistently lower in KO/WT mice (Figure 2E). When challenged with a glucose tolerance test, WT/WT mice fed HFD again showed impaired glucose tolerance, which was partially but significantly alleviated in the absence of hematopoietic NOD1 (Figure 2F). Notably, these observations were not due to differences in insulin secretion (Figure S3C), suggesting that KO/WT mice have improved insulin sensitivity. Indeed, insulin tolerance tests revealed that HFD-induced insulin resistance in WT/WT mice was ameliorated in KO/WT mice (Figures 2G and S3D). Further, while HFD suppressed insulin-stimulated Akt phosphorylation in EWAT of WT/WT mice, insulin action was intact in the KO/WT mice (Figure 2H). Collectively, these results demonstrate that NOD1 depletion specifically from immune cells improves glucose and insulin tolerance at the adipose tissue and whole-body level, independent of changes in body weight or adiposity.
Hematopoietic NOD1 Regulates Adipose Tissue Proinflammatory Macrophage Polarization and Neutrophil Infiltration
A hallmark of metabolic inflammation is a rise in adipose tissue immune cells during HFD or obesity. Macrophages are the most widely characterized infiltrating cell type in EWAT during obesity, and their appearance and pro-inflammatory polarization correlate with impaired insulin sensitivity (Lumeng et al., 2007; Wentworth et al., 2010). We therefore assessed macrophage populations in the EWAT stromal vascular fraction (SVF) by flow cytometry in chimeric mice fed LFD or HFD. Total CD11b+ F4/80+ macrophage numbers were higher in HFD-fed WT/ WT mice as previously shown for non-chimeric WT mice (Weisberg et al., 2003), and importantly, a similar elevation in EWAT macrophages was observed in KO/WT mice (Figure 3A).
Thus, EWAT macrophage infiltration was not dependent on hematopoietic NOD1. HFD induced a marked increase in proinflammatory CD11c+ macrophages in the WT/WT mice as expected, but notably, this elevation of CD11c+ macrophages was lower in KO/WT mice (Figure 3B). The differential proportion of CD11c+ macrophages correlated with the Ly6C+ monocyte population in adipose tissue without changes in the CD11c+ dendritic cell count (Figures 3C and 3D). These results were confirmed by analyzing expression of the macrophage markers Itgam/CD11b and Emr1/F4/80 and of the pro-inflammatory marker Itgax/CD11c by qPCR in whole EWAT. Gene expression changes supported flow cytometry data (Figure 3F), reinforcing that the proportion of pro-inflammatory macrophages was reduced in mice lacking NOD1 in immune cells.
Interestingly, it was reported that Nod1 expression is required for neutrophil recruitment to sites of infection and injury (Dhar-ancy et al., 2010; Frutuoso et al., 2010). In line with reports on WT mice (Elgazar-Carmon et al., 2008; Hadad et al., 2013), EWAT neutrophil counts were higher in HFD-fed WT/WT mice, but strikingly, this infiltration was abolished in the absence of hematopoietic NOD1 (Figure 3E). Corroborating these observations, expression of elastase (Elane), a neutrophil-specific enzyme reported to trigger insulin resistance (Talukdar et al., 2012), was higher in EWAT of HFD-fed compared to LFD-fed WT/WT but not KO/WT mice (Figure 3F). Notably, in the liver, neutrophil infiltration was also dependent on hematopoietic NOD1 (Figure S4). Collectively, these data indicate that NOD1 in immune cells drives pro-inflammatory macrophage polarization and neutrophil infiltration in EWAT during HFD, promoting an inflammatory tissue environment associated with impaired insulin responsiveness.
NOD1-Deficient Neutrophils Do Not Have Impaired Migratory Capacity
The reduced neutrophil infiltration in EWAT during HFD evoked by hematopoietic NOD1 deletion may have arisen from lower
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Figure 3. Hematopoietic NOD1 Regulates Adipose Tissue Neutrophil and Pro-inflammatory Macrophage Count
EWAT was isolated following 18 weeks on LFD or HFD. Representative flow cytometry plots and quantification as a percentage of SVF cells are shown.
(A) CD11b+ F4/80+ cells (total macrophages).
(B) F4/80+ CD11c+ cells (pro-inflammatory macrophages).
(C) CD11b+ Ly6c+ cells (monocytes).
(D) CD11c+ MHC-II+ cells (dendritic cells).
(E) CD11b+ Gr-1+ cells (neutrophils).
(F) Itgam/CD11b, Emr1 /F4/80, Itgax/CD11c, E/ane/neutrophil elastase expression in whole EWAT by qPCR.
Results are means ± SEM, n = 4-9 (A-E) or n = 8-9 (F) mice per group. Two-way ANOVA, Tukey post-test. *p < 0.05, **p < 0.01, ***p < 0.001 (WT/WT - LFD versus WT/WT-HFD),#p< 0.05, ##p<0.01 (KO/WT-LFD versus KO/WT-HFD), tp<0.05, ftfp<0.001 (WT/WT-HFD versus KO/WT-HFD). n.s., not significant.
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Figure 4. NOD1-Deficient Neutrophils Do Not Have Impaired Migratory Capacity
(A) Following 18 weeks of feeding, CD11b+ Ly6G+ neutrophil percentages In blood were counted by flow cytometry. Representative plot shown.
(B) Transwell migration assay using WT or Nod1~'~ bone marrow-derived neutrophils (BMDN) migrating toward CXCL1.
(C) Expression of Cxcr2 and Cxcr4 in WT or NodiBMDN by qPCR.
Results are means ± SEM, n = 4-6 (A) or n = 3-4 (Band C) mice per group. Two-way ANOVA, Tukey post-test. *p < 0.05 (versus 0 mM, WT), #p < 0.05 (versus 0 mM, Nodi~'~). n.s., not significant.
blood neutrophil count, reduced neutrophil chemotactic ability, or from diminished levels of chemoattractants produced by tissue-embedded immune cells. Discriminating between these possibilities, we found no significant differences in blood neutrophil percentages due to diet or bone marrow genotype (Figure 4A).
To evaluate if neutrophil-autonomous NOD1 mediates chemotaxis, WT or NOD1-deficient bone marrow-derived neutrophils (BMDN) were allowed to migrate across a transwell membrane toward the neutrophil chemoattractant CXCL1. Both WT and NOD1-knockout BMDN transited toward CXCL1 to a similar extent and in a dose-dependent manner (Figure 4B). Additionally, the expression of neutrophil chemoattractant receptors Cxcr2 and Cxcr4 in BMDN was unchanged in the absence of NOD1 (Figure 4C). Together, these results suggest that NOD1-deficient neutrophils do not have impaired migratory capabilities, and thus, neutrophil infiltration into EWAT during HFD is unlikely to depend on NOD1 expression in neutrophils.
NOD1 Determines Production of Neutrophil Chemoattractants by Adipose Tissue Macrophages
To address the possibility that NOD1-deficient resident adipose tissue immune cells produce less neutrophil chemoattractants, we measured nuclear factor kB (NF-kB) activation and analyzed a panel of pro-inflammatory (Ccl2, Ccl5, Cxcll, Cxcl2, Cxcl12, Illb, Il6, Il12a, Nos2, Tnf) and anti-inflammatory (Argi, Il4, Il10) genes in whole EWAT. The neutrophil chemoattractants CXCL1, CXCL2, and CXCL12 were upregulated with HFD in EWAT from WT/WT mice, but remarkably did not change in KO/WT mice, and a lesser phosphorylation of the NF-kB p65 subunit in EWAT from KO/WT mice correlated with lower production of these chemokines (Figures 5A and 5B). Upon separation of adipocyte-rich and stromal vascular fractions (SVF), the increase in EWAT Cxcli expression tracked with the SVF
(Figure 5C). Cxcl2 expression was higher in both the adipocytes and SVF of HFD-fed WT/WT mice, but again, no diet-induced increase was observed in the SVF of KO/WT mice (Figure 5C). These data imply that stromal vascular cells, likely immune cells, are the source of neutrophil chemoattractants produced during HFD-induced NOD1 activation. We next aimed to identify more directly the non-adipocyte cell type responsible for che-mokine production, focusing on CXCL1, as its expression is elevated in the SVF and not adipocytes of HFD-fed WT/ WT mice. We hypothesized that macrophages were the main source of NOD1-dependent CXCL1 during HFD, as they comprise a substantial percentage of the EWAT SVF and were less inflammatory in KO/WT mice. By immunohistochemical detection in EWAT sections, CXCL1 protein colocalized with the macrophage marker F4/80 within adipose tissue, but not with the adipocyte cell body (Figure 5D). Notably, CXCL1 was enriched in crown-like structures (macrophages surrounding dying adipocytes), and these structures were less abundant in the HFD-fed KO/WT mice than WT/WT mice (Figures S5A-S5C). These observations suggest that HFD provokes a NOD1-dependent production of neutrophil chemoattractants by ATMs.
Finally, we generated NOD1-deficient macrophages from bone marrow (BMDM) of LFD-fed chimeric mice and challenged them ex vivo with the NOD1 activator peptidoglycan (PGN). PGN elevated CXCL1 expression and secretion in BMDM from WT/WT mice; however, these responses were significantly reduced in BMDM derived from KO/WT mice (Figures 6A and 6B), underscoring a role for NOD1 in CXCL1 production by macrophages. Further supporting this concept, neutrophils migrated toward cell culture supernatant containing secreted factors from PGN-treated BMDM, but migration was dampened when the BMDM originated from KO/WT mice (Figure 6C). Of note, neutrophils were not attracted to PGN alone (Figure 6D).
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Figure 5. Hematopoietic NOD1 Knockout Diminishes Expression of Neutrophil Chemoattractants by Adipose Tissue Macrophages
(A) NF-kB p65 phosphorylation in EWAT.
(B) Expression of pro-inflammatory and anti-inflammatory genes in whole EWAT.
(C) Expression of Cxcll and Cxcl2 in EWAT separated into adipocyte-rich and stromal vascular fractions (SVF).
(D) EWAT sections were stained for colocalization between Caveolin-1 (adipocyte marker), F4/80 (macrophage marker), and CXCL1. Images have been adjusted to have equal color balance to show colocalization, not intensity. Scale bar, 50 mm.
Results are means ± SEM, n = 4-6 (A and C), n = 4-9(B), orn = 3-5(D) mice per group. Two-way ANOVA,Tukey post-test, *p < 0.05 (WT/WT- LFD versus WT/ WT - HFD), tp < 0.05 (WT/WT-HFD versus KO/WT-HFD). n.s., not significant. See also Figure S5.
Interestingly, macrophages favored a non-inflammatory phenotype in the absence of NOD1, as M1 markers such as Nos2, Il1b, and Il6 were significantly lower in BMDM from KO/ WT mice (Figure 6E). Moreover, while Tlr4 and Tlr2 expression were unaffected by NOD1 depletion (Figure 6F), CXCL1 expression and secretion were reduced in BMDM from KO/WT mice in response to the TLR4 agonist LPS, or the saturated fatty acid palmitate, which has been proposed to signal through TLR2 and TLR4 (Huang et al., 2012) (Figures 6G and 6H). Altogether, these results suggest that NOD1 activation, potentially triggered by circulating saturated fats, or gut-derived PGN and LPS, is crit-
ical for CXCL1 production and neutrophil chemoattraction by ATMs in the context of HFD.
DISCUSSION
The results presented in this study reveal that circulating NOD1 activators increase during HFD in mice and that silencing NOD1 specifically in the immune system prevents pro-inflammatory polarization of EWAT macrophages and neutrophil recruitment and consequently alleviates diet-induced glucose and insulin intolerance.
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Figure 6. NOD1 Mediates CXCL1 Production and Neutrophil Attraction by Macrophages
Bone marrow-derived macrophages (BMDM) were isolated from LFD-fed WT/WTor KO/WT mice and treated with 10 mg/mL peptidoglycan (PGN) for 18 hr.
(A) Cxcll mRNA expression.
(B) Secreted CXCL1 protein.
(C) Neutrophil migration toward supernatant from vehicle (SN-Veh) or PGN-treated (SN-PGN) BMDMs.
(D) Neutrophil migration toward PGN.
(E) M1 and M2 macrophage marker expression.
(F) Tlr2 and Tlr4 mRNA expression.
(G and H) BMDM were treated with 0.5 mM palmitate (PA) or 100 ng/ml lipopolysaccharide (LPS) for 18 hr. (G) Cxcll mRNA expression. (H) Secreted CXCL1 protein.
Results are means ± SEM, n = 6-9 (A-C, E-H) or n = 2 (D) mice per group. Two-way ANOVA, Tukey post-test, *p < 0.05, **p < 0.01 ***p < 0.001.
High Fat Diet Increases Circulating NOD1 Activators
A key finding of this study was that HFD increases serum levels of NOD1 activators, potentially peptidoglycan fragments, which positively correlate with the increasing body weight and fasting blood glucose. Together, these findings suggest that weight gain increases levels of circulating NOD1 activators, which in turn could predict glycemia. The "infection hypothesis" posits that gut microbes or their constituents activate the immune system during chronic inflammatory diseases, including T2D (Musso et al., 2010; Stassen et al., 2008). As acute infections result in whole-body insulin resistance (Yki-Jarvinen et al., 1989), it is
conceivable that persistent, low-level release of gut-derived bacterial cell wall components into the bloodstream during HFD could initiate the chronic low-grade inflammation associated with insulin resistance. Thus, it is possible that increased translocation of gut-derived peptidoglycan leads to hematopoi-etic NOD1 activation and metabolic dysfunction.
Recent studies show that HFD can disrupt intestinal epithelial cell tight junctions, mediated by local pro-inflammatory cytokines produced by intestinal immune cells (macrophages, T cells, and eosinophils), promoting paracellular transport of LPS (Cani et al., 2008; Johnson et al., 2015; Luck et al., 2015). Here, we
report that serum NOD1 stimulatory activity increased with HFD. Importantly, the increase was not statistically different in KO/ WT mice (1.09 ± 0.26 to 1.59 ± 0.32 a.u.) compared to WT/ WT mice (1.00 ± 0.10 to 1.50 ± 0.14 a.u.), suggesting that the appearance of NOD1 activators in the circulation is not dependent on hematopoietic NOD1, but instead is diet-linked.
Our findings are consistent with the possibility that blood NOD1 activators emanate from the gut, rather than arising from changes in the composition of the gut microbiome. The gut mi-crobiota is sensitive to dietary changes, with HFD decreasing Gram-negative Bacteroidetes and increasing Gram-positive Firmicutes as seen in previous studies (Ley et al., 2006; Murphy et al., 2010) as well as the present study. Yet, the NOD1-activating motif (g-D-Glu-mDAP/iE-DAP), is present in peptido-glycan of all Gram-negative bacteria and only some Grampositive bacteria (Girardin et al., 2003, 2005); therefore, the dysbiotic microbiome associated with HFD is unlikely to induce greater NOD1 activity without alterations in bacterial product translocation. More in-depth analyses of the gut microbiota are required to identify if specific NOD1-activating genera or species increase with HFD.
Alternatively, the circulating NOD1 activators that increase with HFD may not be of gut bacterial origin. As NOD1 is an intracellular pattern recognition receptor, it is well-positioned to detect cellular danger signals or metabolites, whether endoge-nously produced orfrom the incoming diet. In this context, dietary lipids, but not those released through lipolysis, contribute to metabolic inflammation (Caspar-Bauguil et al., 2015), and saturated fatty acids, which are the predominant lipid components of the HFD, activate NOD1 in colonic epithelial cells in vitro (Zhao et al., 2007). Similarly, a mixture of palmitate and oleate activates pro-inflammatory gene expression and interferes with insulin signaling in cultured adipocytes, and these effects are blunted by NOD1 knockdown (Zhou et al., 2013). In the present study, we also show NOD1-deficient macrophages are protected from the pro-inflammatory effects of palmitate. Furthermore, acylation facilitates iE-DAP entry into cells and heightens NOD1 stimulation 100- to 1,000-fold (Lee et al., 2009). We surmise that dietary saturated fatty acids and bacterial peptidoglycan may synergistically activate NOD1 during HFD by a mechanism requiring further investigation.
Immune Cell-Specific NOD1 Knockout Alleviates Adipose Tissue Inflammation and Insulin Resistance
Another major finding of this study was that hematopoietic lineage-specific NOD1 deletion improved insulin sensitivity and reduced adipose tissue inflammation independent of body weight gain or EWAT macrophage infiltration during HFD. Hence, adipose tissue changes may dictate the whole body insulin response. While in some studies, muscle and liver also gain pro-inflammatory macrophages with HFD (Fink et al., 2014; Morinaga et al., 2015), the net gain is less compared to that in adipose tissue. One may speculate that NOD1 activation during HFD occurs preferentially in adipose tissue over other metabolic tissues as a result of the combined local and systemic environments. Of note, HFD has been associated with increased bacterial count within EWAT (Amar et al., 2011); however, there were no differences in bacterial DNA in EWAT with HFD in the WT/WT or KO/WT
mice, suggesting that NOD1 activation in our model does not result from bacteria translocation. As in other mouse models, alterations manifested in adipose tissue may be communicated to tissues directly responsible for controlling blood glucose levels (muscle and liver) through circulating factors (Bastard et al., 2006; Jung and Choi, 2014).
Hematopoietic NOD1 ablation significantly but incompletely relieved diet-induced insulin resistance, highlighting possible involvement of other pattern recognition receptors. TLR2, TLR4, and NLRP3 (Davis et al., 2011; Saberi et al., 2009; Shi et al., 2006; Vandanmagsar et al., 2011) have all been implicated in metabolic inflammation. Deleting each of these receptors reduces HFD-induced insulin resistance; however, the tissue inflammatory responses differ among genotypes. In contrast to ourfindings in KO/WT mice, hematopoietic TLR4 depletion reduces ATM infiltration and production of tumor necrosis factor alpha (TNF-a), interleukin-6 (IL-6), and interleukin-12 (IL-12), but exceptionally not CXCL1 (Saberi et al., 2009). While similar transcription factors (NF-kB, AP-1) are activated upon TLR4 and NOD1 stimulation (Kawai and Akira, 2007; Strober et al., 2006), it is not understood how each receptor evokes a unique gene expression profile. Our results reveal that hematopoietic NOD1 is required for HFD-induced CXCL1 expression within adipose tissue SVF cells, which begets neutrophil infiltration. In spite of the different consequences of TLR4 or NOD1 depletion in the hematopoietic lineage, previous studies have demonstrated synergistic effects of TLR4 and NOD1 agonists in vitro and in vivo (Farzi et al., 2015; Tada et al., 2005). Consistent with this notion, we find that responses to TLR agonism are partly reduced in macrophages from KO/WT mice.
Many previous reports demonstrate the critical role of pro-inflammatory CD11c+ macrophages in diet-induced insulin resistance (Lumeng et al., 2007; Patsouris et al., 2008). We find that compared to WT/WT controls, HFD-fed KO/WT mice have fewer crown-like structures, which we and others find to be regions most enriched with CD11c+ macrophages (Wentworth et al., 2010), and CXCL1 is expressed predominantly in these crown-like structures. We hypothesize that HFD drives a NOD1-dependent macrophage polarization to a CD11c+ phenotype, which consequently promotes crown-like structure formation and neutrophil chemoattractant production.
Importantly, we and others have reported that neutrophils can infiltrate adipose tissue as early as 3 to 7 days of HFD (Elgazar-Carmon et al., 2008; Fink et al., 2014; Talukdar et al., 2012). While total ATM numbers increase later, pro-inflammatory polarization of existing resident macrophages also occurs within this time frame (Fink et al., 2014; Lee et al., 2011). This early appearance of neutrophils and polarization of macrophages coincides with the earliest observation of diet-induced insulin resistance in mice (Lee et al., 2011), indicating that adipose tissue neutrophils and pro-inflammatory macrophages can be present at the onset of insulin resistance.
In our model, neutrophils were also depleted of NOD1, but we found that this receptor was not required for their migration toward CXCL1; however, it is possible that NOD1 regulates their inflammatory phenotype. While it was previously assumed that neutrophils are a largely homogeneous population of inflammatory cells, growing evidence proposes that neutrophils
can present a spectrum of phenotypes ranging from pro-inflammatory N1 to anti-inflammatory N2 cells (Fridlender et al., 2009; Ma et al., 2016). These studies describe neutrophil polarization during infection, cancer, and myocardial infarction, but equivalent data from HFD-fed mice are virtually lacking. Recently, it was reported that HFD promotes N1 polarization of lung neutrophils (Manicone et al., 2016). It is possible that NOD1 activation during HFD drives N1 polarization that initiates the release of inflammatory factors from neutrophils that contribute to insulin resistance. Notably, however, information on the role of neutrophils in the pathogenesis of metabolic inflammation is limited. Although neutrophils comprise a relatively small percentage of adipose tissue and skeletal muscle immune cells during HFD (Fink et al., 2014), one study showed that inhibiting adipose tissue neutrophil infiltration prevents HFD-induced hepatic insulin resistance (Hadad et al., 2013). This may be due to the secretion of neutrophil-specific elas-tase, which is causally associated with adipose tissue inflammation acting through TLR4 signaling (Talukdar et al., 2012). Very recently, Revelo et al. (2016) reported that HFD enhances release of neutrophil DNA extracellular traps, and DNA detection by TLR7 and TLR9 in macrophages contributes to adipose tissue inflammation and whole-body insulin resistance. These reports support a contribution by neutrophils to insulin resistance during HFD. We build on this hypothesis by showing that, with hematopoietic NOD1 depletion, a combination of diminished macrophage pro-inflammatory polarization and neutrophil infiltration in adipose tissue likely contributes to ameliorating metabolic inflammation and insulin resistance.
In conclusion, we have identified that HFD increases systemic NOD1 activators, which are sensed by immune cells to initiate a gene expression program, producing a distinct adipose tissue inflammatory phenotype associated with insulin resistance. NOD1 may be required particularly for the pro-inflammatory activity of infiltrating macrophages and consequent neutrophil chemoat-traction. Strategies to neutralize NOD1 ligands or selectively inhibit NOD1 in macrophages should be tested in the future to relieve this diet-induced insulin intolerance.
EXPERIMENTAL PROCEDURES
Bone Marrow Transplantation and Animal Procedures
Mouse protocols were approved by the Animal Care Committee (Protocol #20010547 to S.E.G. and D.J.P., University of Toronto and #1000036308 to A.K., The Hospital for Sick Children). Six- week-old male C57BL/6 mice were lethally irradiated (11 Gy) then reconstituted with 106 bone marrow cells from 9-week-old male littermate WT or Nod1~'~ (Millennium Pharmaceuticals) donors. Three weeks following bone marrow transplantation, mice were fed low fat (10% kcal from fat; D12450Ji, Research Diets) or high fat diet (60% kcal from fat; D12492i, Research Diets) for 18 weeks. Glucose and tolerance tests were performed 16 and 17 weeks into diet, respectively. Mice were euthanized by cervical dislocation 18 weeks into diet.
NOD1 Activity Reporter Cells
HEK-Blue mNOD1 cells (InvivoGen), which express NOD1 and a secreted embryonic alkaline phosphatase (SEAP) reporter gene were seeded overnight in a 96-well plate then incubated with HEK-Blue Detection media (InvivoGen) in the presence of 10% heat-inactivated serum samples. NOD1 stimulatory activity was measured using a microplate reader set to 630 nm.
Bone Marrow-Derived Macrophage Isolation and Culture
Bone marrow was extracted and differentiated into bone marrow-derived macrophages (BMDM) as described previously (Chan et al., 2015). Seven days following isolation, BMDM were treated as indicated for 18 hr.
Transwell Migration Assay
Bone marrow-derived neutrophils (BMDN) from WT or Nod1—/— mice were isolated as described previously (Vong et al., 2013). Transwell migration assays were performed as described (Mukovozov et al., 2015; Tole et al., 2009). Cells were incubated at 37°C for 1 hr, and migrated cells in the bottom chamber were lifted with EDTA and counted on a Z2 Coulter Counter (Beckman Coulter).
Flow Cytometry
EWAT stromal vascular fraction (SVF) was isolated as described (Orr et al., 2013), the supernatant fraction containing adipocytes decanted and saved for additional analyses. Blood samples were collected by cardiac puncture into EDTA and PBS, then fixed in PFA. Red blood cells were lysed, and cells were resuspended in fluorescence-activated cell sorting (FACS) buffer. Suspended SVF pellets or blood leukocytes were blocked and stained with indicated antibodies. Cells were acquired using Fortessa 4-laser flow cytometer (BD Biosciences) and data analyzed using FlowJo version 10 (FlowJo, LLC).
RNA was extracted from cells or tissues using TRIzol (Life Technologies), then cDNA synthesized using the SuperScript VILO cDNA kit (Life Technologies) according to manufacturer's instructions. qPCR reactions were performed with predesigned TaqMan probes (Life Technologies) on a StepOne Plus RealTime PCR System (Life Technologies). Gene expression was normalized to that of housekeeping genes, Abt1, Hprt, and/or Eef2.
Immunohistochemistry
EWAT samples were fixed in 10% formalin (Sigma-Aldrich) then paraffin-embedded and sections cut onto microscope slides for immunohistochemical staining. Images were acquired on a Leica TCS SP8 STED super-resolution microscope equipped with Leica Application Suite X (Leica Microsystems).
Immunoblotting
Prior to sacrifice, mice were intraperitoneally (i.p.) injected with saline or insulin, then EWAT was frozen in liquid nitrogen and stored at —80°C until lysis. EWAT was homogenized in lysis buffer, run on an SDS-PAGE gel, then probed with indicated antibodies. Blots were visualized on an Odyssey Fc Imager (LI-COR) and quantified using Odyssey Fc Image Studio version 4.0 (LI-COR).
CXCL1 in cell culture supernatant was analyzed using Mouse CXCL1/KC DuoSet ELISA (DY453, R&D Systems) according to manufacturer's instructions and quantified on a microplate reader set to 450 nm.
Statistical Analyses
Data are means ± SEM. An unpaired Student's t test or two-way ANOVA with Tukey post-test were used to detect differences in datasets containing one or two variables, respectively. Statistical significance was set at p < 0.05. Graphs were prepared and statistics analyzed using GraphPad Prism version 6 (GraphPad Software).
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures and five figures and can be found with this article online at http://dx.doi.org/ 10.1016/j.celrep.2017.02.027.
AUTHOR CONTRIBUTIONS
Conceptualization, K.L.C., D.J.P., and A.K.; Methodology, K.L.C., D.P., S.R.C., N.F., and S.J.R.; Investigation, K.L.C., T.H.T., P.B., D.P., S.R.C., N.K.E., N.F., Y.T., S.J.R., D.P., and Z.L.; Resources, M.W.S., M.G., and
S.E.G.; Writing - Original Draft, K.L.C., D.J.P., and A.K.; Writing - Review & Editing, K.L.C., T.H.T., P.B., S.R.C., N.K.E., Y.T., S.J.R., P.J.B., M.G., S.E.G., D.J.P., and A.K.; Funding Acquisition, D.J.P. and A.K.; Supervision, P.J.B., M.W.S., M.G., S.E.G., D.J.P., and A.K.
ACKNOWLEDGMENTS
This work was supported by a Foundation grant (FND-143203) to A.K. from the Canadian Institutes of Health Research (CIHR). A.K. is a Canada Research Chair in The Cell Biology of Insulin Action. K.L.C. was supported by a studentship from the Banting and Best Diabetes Centre (BBDC) of the University of Toronto and an Ontario Graduate Scholarship (OGS). T.H.T. was supported by a summer studentship from the BBDC. N.K.E. was supported by a CIHR Vanier Canada Graduate Studentship. M.W.S. holds the Northbridge Chair in Paediat-ric Research. We also acknowledge support from an operating CIHR grant (MT-11219) to M.W.S., a CIHR Bone Team grant (TBO-122068) to M.G., and a CIHR Team grant: Nod-like receptors: linking innate immunity to inflammation in chronic diseases (THC-135238) to D.J.P. and S.E.G. We sincerely thank Dr. Assaf Rudich and Dr. Yulia Haim for valuable discussion.
Received: August 11, 2016 Revised: December 9, 2016 Accepted: February 7, 2017 Published: March 7, 2017
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