Journal of Diabetes and Its Complications 29 (2015) 617–620
Contents lists available at ScienceDirect
Journal of Diabetes and Its Complications journal homepage: WWW.JDCJOURNAL.COM
The evolving role of toll-like receptors in diabetic vascular complications
Diabetes is a pro-inflammatory state and confers an increased risk for both microvascular and macrovascular complications. Numerous studies have reported increased levels of both circulating and cellular biomarkers of inflammation, including the pattern recognition receptors, toll-like receptors (TLRs), especially TLR2 and TLR4, in diabetes (Jialal & Kaur, 2012). In this perspective, we discuss the evolving role of TLR2 and 4 in diabetes-related vascular complications. There is a paucity of data on the direct role of TLR-2 and TLR-4 in macrovascular complications of diabetes. Lu, Zhang, Li, Jin, and Huang (2013) showed that administration of Rs-LPS (Rhodobacter sphaeroides Lipopolysaccharide), a TLR4 antagonist, for 10 weeks to 14-week-old diabetic ApoE-knockout (KO) mice reduced atherosclerosis significantly as evidenced by en-face analysis following Sudan IV staining and more importantly by histological analysis of the aortic root intima. Rs-LPS administration also reduced vascular inflammation in lesions as evidenced by reduced IL-6 and MMP9 in the atherosclerotic plaques and a decrease in monocyte/macrophage content in atherosclerotic plaques in Rs-LPS-treated diabetic mice (Lu et al., 2013). Also using NOD mice a role for TLR4 in ameliorating diabetic cardiomyopathy was demonstrated. Using a TLR4 mutant mice model the authors showed that compared to WT-NOD the TLR4-deficent NOD mice had less cardiac lipid accumulation and decreased attenuation of cardiac systolic dysfunction compared to WT-NOD, as evidenced by changes in ejection fraction, fractional shortening etc. in the first 3 months following diabetes (Dong et al., 2012). Hence there is an urgent need for studies using the TLR2 and TLR4 knockout mice crossed with the LDL receptor KO or ApoE KO mice to clearly establish a role for both TLR2 and TLR4 in diabetic atherosclerosis as demonstrated for non-diabetic atherosclerosis mice. In addition for a role of inflammation in diabetic CVD, data also support an important role for inflammation in the genesis of microvascular complications especially nephropathy and retinopathy (Cheung, Mitchell, & Wong, 2010; Lin & Tang, 2014; Mudaliar, Pollock, & Panchapakesan, 2014; Tang & Kern, 2011; Wada & Makino, 2013). Studies from various groups have documented the presence of TLRs especially TLR 2 and 4 in numerous critical cell types in the kidney including podocytes, tubular epithelial cells, mesangial cells, glomerular endothelium and other classical cells such as macrophages and dendritic cells; in many of these cells these TLRs were up regulated with hyperglycemia (Kaur, Chien, & Jialal, 2012; Lin et al., 2012; Mudaliar et al., 2013; Sawa, Takata, Hatakeyama, Ishikawa, & Tsuruga, 2014). Our group has previously shown increased expression and activity of both TLR-2 and TLR-4 in type 1 diabetic patients with microvascular
Conflicts of interest: None. http://dx.doi.org/10.1016/j.jdiacomp.2015.04.002 1056-8727/Published by Elsevier Inc.
complications predominantly diabetic nephropathy, thus lending support to the notion that TLRs contribute to diabetic nephropathy (Devaraj, Jialal, Yun, & Bremer, 2011). In Table 1, we summarize the studies implicating a role of TLRs in diabetic nephropathy using genetic-deficient and mutant mice or inhibitors to TLR4 especially. Li et al. (2010) showed that diabetic rat kidneys had increased protein and mRNA expression of TLR-2, MyD88 and NF-κB. In a seminal study, Devaraj et al. (2011) demonstrated a decrease in the proinflammatory state of diabetes and incipient diabetic nephropathy after a diabetic duration of 14 weeks in TLR2KO mice. TLR2 expression and MyD88-dependent signalling were significantly decreased in diabetic STZ-TLR2KO macrophages compared to wild-type diabetic mice (WT-STZ) with no change in the non-MyD88 signalling cascade such as Trif and IRF3 in STZ-TLR2KO. Also, the NF-κB activity in peritoneal macrophages was significantly reduced in STZ-TLR2KO compared to diabetic WT mice. Further, the release of various inflammatory cytokines/chemokines such as IL1β, IL-6, IL-8, IP-10, and MCP-1 were significantly decreased in the diabetic TLR2KO mice compared to WT-STZ mice. In addition, STZ-TLR2KO mice showed a significant decrease in renal hypertrophy, and albuminuria compared to WT + STZ, increase in podocyte number, decrease in podocyte effacement, and a decrease in M1 macrophages as well as decreased inflammatory cytokines in the kidney lysates. Also, increase in nephrin and podocin, and decrease in transforming growth factor-β and laminin levels were reported in the STZ-TLR2KO mice versus the WT + STZ mice (Devaraj, Tobias, et al., 2011). This study clearly implicated the TLR2 pathway in the genesis of diabetic nephropathy by attenuating both renal inflammation and fibrosis and supports the data showing greater TLR2 activity in T1DM with microvascular complications and upregulation of TLR2 in TEC of diabetic mice (Mudaliar et al., 2013). Lin et al. (2012) reported increased expression of TLR4 but no change in TLR2 expression in the renal tubules of human kidneys with diabetic nephropathy. Only with the use of a complicated model of streptozotocin-induced diabetic mice with uninephrectomy, compared to WT-STZ mice, STZ- TLR4-KO mice, demonstrated significantly less albuminuria, macrophages, NF-κB activation, and MCP-1 expression. In addition they showed in human renal biopsies increased cytosolic HMGB1. Thus, in their study using TLR-4 knockout mice with uninephrectomy, they showed that deletion of TLR-4 conferred renal protection against diabetic nephropathy (Lin et al., 2012). It is important to point out that in this study no significant effect was observed with STZ-WT mice without the added insult of uninephrectomy. Kuwabara et al. (2012) induced diabetes in WT and TLR4KO mice using STZ for 2 weeks and then changed to a high-fat diet for an
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Table 1 Role of TLRs in diabetic nephropathy. Study
Type
(Devaraj, Tobias, et al., 2011) In vivo TLR2KO mice
(Lin et al., 2012)
(Kuwabara et al., 2012)
(Cha et al., 2013)
(Lin et al., 2013)
(Ma, Chadban, et al., 2014)
(Jialal et al., 2014)
(Ma, Wu, et al., 2014)
Background
Findings
STZ and STZ TLR2-/- mice for 14 weeks
Decreased albuminuria, TLR2 expression, MyD88 and cytokines and decreased kidney macrophage phenotype M1 and inflammation and restoration of nephrin, podocin, and podocyte number in STZ TLR2-/- mice compared to STZ-WT mice and decrease in Laminin and TGF-beta. 9 controls and 5 DN or uninephrectomized Renal cortical TLR4, not TLR2 elevated in DN and correlated Human kidney biopsies TLR4 def or wild-type STZ-induced diabetic with CD68 staining. Uninephrectomized TLR4-deficient and uninephrectomized mice for 12 weeks mice had significantly less albuminuria, renal dysfunction, TLR4mutant mice renal cortical NF-κB activation, tubular CCL-2 we use MCP-1 OR ccl2 not both expression, MCP-1 is fine and interstitial macrophage infiltration than wild-type animals Albuminuria was increased, and podocin was decreased in STZ-HFD In vivo TLR4KO mice WT and TLR4KO mice with STZ for compared to STZ–HFD-TLR4KO, along with decreased macrophages, 2 weeks ND followed by HFD for decreased mesangial expansion decreased MCP-1, PAI-1, CTGF and 6 weeks (8 weeks diabetes) S100a8 and S100a9 in STZ-HFD-TLR4KO. In vivo db/db mice and In Db/db mice with a DN without TLR4 GIT27 treatment decreased urinary albumin excretion, decreased vitro in cultured podocytes inhibition via GIT27 for 12 weeks proinflammatory cytokine synthesis, improved tissue lipid metabolism, decreased oxidative stress, and improved glomerulosclerosis compared with the control db/db group. In vitro, in podocytes, GIT27 decreased TLR4 and cytokines CRX-526 significantly reduced albuminuria, glomerular In vivo-eNOS-KO mice and in CRX-526 treatment for 8 weeks (TLR4 hypertrophy, glomerulosclerosis, and tubulointerstitial injury as vitro in proximal tubule cells inhibition) of eNOS KO mice with well as decreased CCL-2, CCL-5, osteopontin, decreased 12 weeks of STZ-induced diabetes macrophage infiltration, NFkB, TGF-beta and collagen deposition. In vitro, CRX-526 inhibited high glucose-induced osteopontin upregulation and NF-κB nuclear translocation in cultured human proximal tubular epithelial cells. WT-STZ mice vs. TLR4-/- STZ mice In STZ mice, TLR4-/- exhibited less albuminuria, inflammation, In vivo-TLR4KO mice and in followed for 12–24 weeks decreased glomerular hypertrophy and podocyte and tubular vitro in podocytes and injury as compared to diabetic wild-type controls. Marked tubular epithelial cells reductions in collagen, α-SMA, TGF-β and fibronectin in TLR-/STZ mice. In vitro, high glucose resulted in TLR4 activation in podocytes and tubular epithelial cells, NF-κB activation and consequent inflammatory and fibrogenic responses. In vivo-TLR4-KO mice STZ and TLR 4 STZ-TLR4 KO mice had significantly decreased macrophage and 17 weeks TLR4 immunostaining in kidney, MyD88, Interferon Regulatory Factor-3, NFKappaB activity, TNF-Alpha, IL-6, and MCP-1; significant decreases in fibrosis markers (collagen 4, and transforming growth factor-beta), and increased Podocyte numbers and podocin in STZ- TLR4KO mice vs. STZ-WT. In-vivo TLR2-KO mice and in STZ and kidney injury assessed at 6, Increased TLR2 gene expression, and its endogenous ligands and vitro in podocytes and TECs 12 and 24 weeks downstream cytokines in WT diabetic kidneys. TLR2KO mice protected against development of DN, with less albuminuria, inflammation, glomerular hypertrophy and hypercellularity, podocyte and tubular injury. Reduced interstitial collagen deposition, myofibroblast activation (α-SMA) and expression of fibrogenic genes (TGF-β and fibronectin) in TLR2KO. In vitro high glucose promoted TLR2 activation in podocytes and tubular epithelial cells (TECs) with resulting NF-κB activation, inflammation and TGF-β production.
additional 6 weeks. STZ and HFD treatment markedly increased albuminuria, increased podocyte injury (decreased podocin), mesangial expansion and macrophage infiltration and upregulated inflammation and fibrosis (MCP-1, fibronectin, connective tissue growth factor) in the glomeruli, even for this short duration of 8 weeks of diabetes, and all these effects were significantly reduced in the STZ-HFD -TLR4KO mice. Also they showed upregulation in the kidney of S100A8, a potent agonist for TLR4. This paper is unique in that it used HFD + STZ but only went for 8 weeks and hence did not see many differences when comparing diabetic TLR4KO vs. WT without HFD (Kuwabara et al., 2012). Studies using inhibitors against TLR4 have also shown a beneficial effect of TLR4 inhibition on diabetic nephropathy. Cha et al. (2013) demonstrated that GIT27, a compound thought to interfere with TLR4, TLR2 and TLR6 signalling pathways, attenuated renal inflammation and decreased glomerulosclerosis in db/db diabetic mice, which in turn attenuated the progression of diabetic nephropathy following
12 weeks of diabetes. In addition to a reduction in albuminuria and a decrease in renal macrophages, collagen 4, NFkB, MCP-1 and IL-6 they showed decreases in urinary excretion of nephrin, TNF and TGF-beta with GIT27 therapy. They also demonstrated that GIT27 treatment directly inactivated TLR4 expression in cultured podocytes, thereby resulting in a suppression of inflammatory cytokine secretion. Interestingly, GIT27 treatment was also associated with decreased oxidative stress in the kidney that paralleled the decrease in TLR4. Their study again points strongly towards TLR4 in contributing to the progression on diabetic nephropathy but does not convincingly rule out a role of TLR2, since this inhibitor blocks both TLR2 and 4 (Cha et al., 2013). Lin et al. (2013) using TLR4 antagonist CRX-526, a lipid A mimetic, that blocks TLR4/MD2 assembly showed that the compound could attenuate the progression of advanced diabetic nephropathy using the model of the eNOS (endothelial nitric oxide synthase) KO mice following 12 weeks of diabetes. CRX-526 reduced albuminuria and
I. Jialal, R. Pahwa / Journal of Diabetes and Its Complications 29 (2015) 617–620
blood urea nitrogen without altering blood glucose and systolic blood pressure in diabetic mice. TLR-4 inhibition also attenuated glomerular hypertrophy, glomerulosclerosis and tubulo-interstitial injury alongside inhibition of TGF-β overexpression and NF-κB activation, CCL2, Osteopontin, macrophage infiltration and collagen deposition (Lin et al., 2013). In a recent comprehensive study, Ma et al. (2014) report elevated expression of TLR4, its endogenous agonists (HMGB1, HSP70, biglycan) and downstream cytokines, chemokines and fibrogenic genes in diabetic nephropathy in WT mice with streptozotocin (STZ) diabetes followed up for up to 24 weeks. In STZ-TLR4KO mice there was an amelioration of DN, manifesting as less albuminuria, inflammation (decreased IL-6, TNF, MCP-1, CXCL10), glomerular hypertrophy and hypercellularity, podocyte and tubular injury as compared to diabetic wild-type controls. There were significant decreases in measures of fibrosis: decreased interstitial collagen deposition, myofibroblast activation (α-SMA) and expression of fibrogenic genes (TGF-β and fibronectin) in STZ-TLR4KO mice. In vitro, high glucose directly promoted TLR4 activation in podocytes and tubular epithelial cells, resulting in NF-κB activation and consequent inflammatory and fibrogenic responses. It also resulted in an increase in TLR4 endogenous agonists including, HMGB1, HSP70 and biglycan (Ma, Chadban, et al., 2014). Jialal, Major, & Devaraj (2014) showed that diabetic nephropathy is also mediated by TLR-4 using TLR-4 KO mice. They showed that STZ-TLR4KO mice had decreased fibrosis as measured by decreased TGF-β, alpha smooth muscle actin, laminin and collagen as well as decreased renal inflammation as measured by decreased NF-κB expression alongside decreases in downstream biomediators such as TNF-α, IL-6 and MCP-1 compared to wild-type STZ mice. Also diabetes-mediated decrease in podocytes in wild-type mice was restored in TLR4KO. A deficiency of this study was the failure to collect urine and report on albuminuria (Jialal et al., 2014). Recently the first study implicating TLR2 in the pathogenesis of DN (Devaraj, Tobias, et al., 2011) was confirmed by (Ma et al., 2014) using the similar STZ-TLR2KO mice model. They showed that the STZ-TLR2KO mice exhibited lesser proteinuria, inflammation, glomerular hypertrophy, podocyte injury, collagen deposition, myofibroblast activation, TGF-beta and fibronectin. In addition they showed that hyperglycemia stimulated TLR2 activation in both podocytes and TECs. Once again they reported an increase in HMGB1, HSP70 and biglycan in the WT-diabetic mice (Ma, Wu, et al., 2014). Hence the studies reviewed above and summarized in Table 1 make a compelling argument that TLRs, 2 and 4 have a role in the pathogenesis of DN and translation to human studies with safe inhibitors can be a welcome therapeutic advance in forestalling this common complication which is the leading cause of ESRD. The role of TLRs in diabetic retinopathy has not been studied in great depth so far. Tang et al. (2013) showed a crucial role for the adaptor molecule, MyD88, in mediating diabetes-induced inflammation. Chimeric mice with MyD88 deletion in bone marrow-derived cells showed decreased diabetes-induced leukostasis, inhibition of diabetes-mediated ICAM expression and retinal superoxide generation. Additionally, this study also showed that chimeric mice with TLR-2/4 deletion showed inhibition of diabetes-induced leukostasis, ICAM expression and retinal superoxide generation. Interestingly none of the knockout mice showed an amelioration of diabetic-induced increased retinal permeability and paradoxically unlike the TLR2/TLR4KO mice leuckocytes, the MyD88-KO mice leuckocytes resulted in greater killing of endothelial cells. This study points towards the possibility of both TLR-2 and TLR-4 equally contributing to diabetic retinopathy. Furthermore they showed that an agonist of TLR2 (Pam3Cys) but not TLR4 (endotoxin) or other TLR agonists induced both IL-8 and IL-6 secretion in diabetic retinal supernatants. Whilst they focused on the effect of leukocyte perturbations on retinal pathology their data support a role
619
especially for TLR2 in DR (Tang et al., 2013). Recently, our group showed that hyperglycemia upregulated TLR2 and TLR4 expression and activity including numerous biomediators of inflammation in retinal microvascular endothelial cells including leukocyte adhesion which was inhibited by various strategies including siRNA and small molecular weight inhibitors further underscoring a role of TLR-mediated inflammation in diabetic retinopathy (Rajamani & Jialal, 2014). This needs to be confirmed in-vivo using the genetically deficient TLR2 and TLR4 mice. The role of TLR-2 and TLR-4 in mediating diabetes-induced inflammation thereby resulting in macro/microvascular complications has been studied to some extent as evidenced by the studies discussed above. However, further studies are required to confirm and elucidate the mechanisms by which they participate in the macro and microvascular complications of diabetes, a pro-inflammatory state as we discussed previously including hyperglycemia, increase oxidative stress, PAMPS, DAMPS etc. (Jialal & Kaur, 2012).
References Cha, J. J., Hyun, Y. Y., Lee, M. H., Kim, J. E., Nam, D. H., Song, H. K., et al. (2013). Renal protective effects of toll-like receptor 4 signaling blockade in type 2 diabetic mice. Endocrinology, 154, 2144–2155. Cheung, N., Mitchell, P., & Wong, T. Y. (2010). Diabetic retinopathy. Lancet, 376, 124–136. Devaraj, S., Jialal, I., Yun, J. M., & Bremer, A. (2011). Demonstration of increased toll-like receptor 2 and toll-like receptor 4 expression in monocytes of type 1 diabetes mellitus patients with microvascular complications. Metabolism, 60, 256–259. Devaraj, S., Tobias, P., Kasinath, B. S., Ramsamooj, R., Afify, A., & Jialal, I. (2011). Knockout of toll-like receptor-2 attenuates both the proinflammatory state of diabetes and incipient diabetic nephropathy. Arterioscler Thromb Vasc Biol, 31, 1796–1804. Dong, B., Qi, D., Yang, L., Huang, Y., Xiao, X., Tai, N., et al. (2012). TLR4 regulates cardiac lipid accumulation and diabetic heart disease in the nonobese diabetic mouse model of type 1 diabetes. Am J Physiol Heart Circ Physiol, 303, H732–H742. Jialal, I., & Kaur, H. (2012). The role of toll-like receptors in diabetes induced inflammation: Implications for vascular complications. Curr Diab Rep, 12, 172–179. Jialal, I., Major, A. M., & Devaraj, S. (2014). Global toll-like receptor 4 knockout results in decreased renal inflammation, fibrosis and podocytopathy. J Diabetes Complications, 28, 755–761. Kaur, H., Chien, A., & Jialal, I. (2012). Hyperglycemia induces toll like receptor 4 expression and activity in mouse mesangial cells: Relevance to diabetic nephropathy. Am J Physiol Renal Physiol, 303, F1145–F1150. Kuwabara, T., Mori, K., Mukoyama, M., Kasahara, M., Yokoi, H., Saito, Y., et al. (2012). Exacerbation of diabetic nephropathy by hyperlipidaemia is mediated by toll-like receptor 4 in mice. Diabetologia, 55, 2256–2266. Li, F., Yang, N., Zhang, L., Tan, H., Huang, B., Liang, Y., et al. (2010). Increased expression of toll-like receptor2 in rat diabetic nephropathy. Am J Nephrol, 32, 179–186. Lin, M., & Tang, S. C. W. (2014). Toll -like receptors sensing and reacting to diabetic injury in the kidney. Nephrol Dial Transplant, 29, 746–754. Lin, M., Yiu, W. H., Li, R. X., Wu, H. J., Wong, D. W., Chan, L. Y., et al. (2013). The TLR4 antagonist CRX-526 protects against advanced diabetic nephropathy. Kidney Int, 83, 887–900. Lin, M., Yiu, W. H., Wu, H. J., Chan, L. Y., Leung, J. C., Au, W. S., et al. (2012). Toll-like receptor 4 promotes tubular inflammation in diabetic nephropathy. J Am Soc Nephrol, 23, 86–102. Lu, Z., Zhang, X., Li, Y., Jin, J., & Huang, Y. (2013). TLR4 antagonist reduces early-stage atherosclerosis in diabetic apolipoprotein E-deficient mice. J Endocrinol, 216, 61–71. Ma, J., Chadban, S. J., Zhao, C. Y., Chen, X., Kwan, T., Panchapakesan, U., et al. (2014). TLR4 activation promotes podocyte injury and interstitial fibrosis in diabetic nephropathy. PLoS One, 9, e97985. Ma, J., Wu, H., Zhao, C. Y., Panchapakesan, U., Pollock, C., & Chadban, S. J. (2014). Requirement for TLR2 in the development of albuminuria, inflammation and fibrosis in experimental diabetic nephropathy. Int J Clin Exp Pathol, 7, 481–495. Mudaliar, H., Pollock, C., Komala, M. G., Chadban, S., Wu, H., & Panchapakesan, U. (2013). The role of toll-like receptor proteins (TLR) 2 and 4 in mediating inflammation in proximal tubules. Am J Physiol Renal Physiol, 305, F143–F154. Mudaliar, H., Pollock, C., & Panchapakesan, U. (2014). Role of toll-like receptors in diabetic nephropathy. Clin Sci, 126, 685–694. Rajamani, U., & Jialal, I. (2014). Hyperglycemia induces toll-like receptor-2 and -4 expression and activity in human microvascular retinal endothelial cells: Implications for diabetic retinopathy. J Diabetes Res, 2014, 790902http://dx.doi. org/10.1155/2014/790902. Sawa, Y., Takata, S., Hatakeyama, Y., Ishikawa, H., & Tsuruga, E. (2014). Expression of toll-like receptor 2 in glomerular endothelial cells and promotion of diabetic nephropathy by Porphyromonas gingivalis lipopolysaccharide. PLoS One, 9, e97165. Tang, J., Allen Lee, C., Du, Y., Sun, Y., Pearlman, E., Sheibani, N., et al. (2013). MyD88-dependent pathways in leukocytes affect the retina in diabetes. PLoS One, 8, e68871.
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Tang, J., & Kern, T. S. (2011). Inflammation in diabetic retinopathy. Prog Retin Eye Res, 30, 343–358. Wada, J., & Makino, H. (2013). Inflammation and the pathogenesis of diabetic nephropathy. Clin Sci, 124, 139–152.
Research, University of California Davis Medical Center, 4635 2nd Ave, Research I Bldg, Room 3000, Sacramento, CA 95817-2218 Tel.: + 1 916 734 6590; fax: + 1 916 734 6593 E-mail address: [email protected]
Ishwarlal Jialal Laboratory of Atherosclerosis and Metabolic Research Department of Pathology, University of California Davis Medical Center, Sacramento, CA Veterans Affairs Medical Center, Mather, CA Corresponding author. Laboratory of Atherosclerosis and Metabolic
Roma Pahwa Laboratory of Atherosclerosis and Metabolic Research Department of Pathology, University of California Davis Medical Center, Sacramento, CA Veterans Affairs Medical Center, Mather, CA
Contents lists available at ScienceDirect
Journal of Diabetes and Its Complications journal homepage: WWW.JDCJOURNAL.COM
The evolving role of toll-like receptors in diabetic vascular complications
Diabetes is a pro-inflammatory state and confers an increased risk for both microvascular and macrovascular complications. Numerous studies have reported increased levels of both circulating and cellular biomarkers of inflammation, including the pattern recognition receptors, toll-like receptors (TLRs), especially TLR2 and TLR4, in diabetes (Jialal & Kaur, 2012). In this perspective, we discuss the evolving role of TLR2 and 4 in diabetes-related vascular complications. There is a paucity of data on the direct role of TLR-2 and TLR-4 in macrovascular complications of diabetes. Lu, Zhang, Li, Jin, and Huang (2013) showed that administration of Rs-LPS (Rhodobacter sphaeroides Lipopolysaccharide), a TLR4 antagonist, for 10 weeks to 14-week-old diabetic ApoE-knockout (KO) mice reduced atherosclerosis significantly as evidenced by en-face analysis following Sudan IV staining and more importantly by histological analysis of the aortic root intima. Rs-LPS administration also reduced vascular inflammation in lesions as evidenced by reduced IL-6 and MMP9 in the atherosclerotic plaques and a decrease in monocyte/macrophage content in atherosclerotic plaques in Rs-LPS-treated diabetic mice (Lu et al., 2013). Also using NOD mice a role for TLR4 in ameliorating diabetic cardiomyopathy was demonstrated. Using a TLR4 mutant mice model the authors showed that compared to WT-NOD the TLR4-deficent NOD mice had less cardiac lipid accumulation and decreased attenuation of cardiac systolic dysfunction compared to WT-NOD, as evidenced by changes in ejection fraction, fractional shortening etc. in the first 3 months following diabetes (Dong et al., 2012). Hence there is an urgent need for studies using the TLR2 and TLR4 knockout mice crossed with the LDL receptor KO or ApoE KO mice to clearly establish a role for both TLR2 and TLR4 in diabetic atherosclerosis as demonstrated for non-diabetic atherosclerosis mice. In addition for a role of inflammation in diabetic CVD, data also support an important role for inflammation in the genesis of microvascular complications especially nephropathy and retinopathy (Cheung, Mitchell, & Wong, 2010; Lin & Tang, 2014; Mudaliar, Pollock, & Panchapakesan, 2014; Tang & Kern, 2011; Wada & Makino, 2013). Studies from various groups have documented the presence of TLRs especially TLR 2 and 4 in numerous critical cell types in the kidney including podocytes, tubular epithelial cells, mesangial cells, glomerular endothelium and other classical cells such as macrophages and dendritic cells; in many of these cells these TLRs were up regulated with hyperglycemia (Kaur, Chien, & Jialal, 2012; Lin et al., 2012; Mudaliar et al., 2013; Sawa, Takata, Hatakeyama, Ishikawa, & Tsuruga, 2014). Our group has previously shown increased expression and activity of both TLR-2 and TLR-4 in type 1 diabetic patients with microvascular
Conflicts of interest: None. http://dx.doi.org/10.1016/j.jdiacomp.2015.04.002 1056-8727/Published by Elsevier Inc.
complications predominantly diabetic nephropathy, thus lending support to the notion that TLRs contribute to diabetic nephropathy (Devaraj, Jialal, Yun, & Bremer, 2011). In Table 1, we summarize the studies implicating a role of TLRs in diabetic nephropathy using genetic-deficient and mutant mice or inhibitors to TLR4 especially. Li et al. (2010) showed that diabetic rat kidneys had increased protein and mRNA expression of TLR-2, MyD88 and NF-κB. In a seminal study, Devaraj et al. (2011) demonstrated a decrease in the proinflammatory state of diabetes and incipient diabetic nephropathy after a diabetic duration of 14 weeks in TLR2KO mice. TLR2 expression and MyD88-dependent signalling were significantly decreased in diabetic STZ-TLR2KO macrophages compared to wild-type diabetic mice (WT-STZ) with no change in the non-MyD88 signalling cascade such as Trif and IRF3 in STZ-TLR2KO. Also, the NF-κB activity in peritoneal macrophages was significantly reduced in STZ-TLR2KO compared to diabetic WT mice. Further, the release of various inflammatory cytokines/chemokines such as IL1β, IL-6, IL-8, IP-10, and MCP-1 were significantly decreased in the diabetic TLR2KO mice compared to WT-STZ mice. In addition, STZ-TLR2KO mice showed a significant decrease in renal hypertrophy, and albuminuria compared to WT + STZ, increase in podocyte number, decrease in podocyte effacement, and a decrease in M1 macrophages as well as decreased inflammatory cytokines in the kidney lysates. Also, increase in nephrin and podocin, and decrease in transforming growth factor-β and laminin levels were reported in the STZ-TLR2KO mice versus the WT + STZ mice (Devaraj, Tobias, et al., 2011). This study clearly implicated the TLR2 pathway in the genesis of diabetic nephropathy by attenuating both renal inflammation and fibrosis and supports the data showing greater TLR2 activity in T1DM with microvascular complications and upregulation of TLR2 in TEC of diabetic mice (Mudaliar et al., 2013). Lin et al. (2012) reported increased expression of TLR4 but no change in TLR2 expression in the renal tubules of human kidneys with diabetic nephropathy. Only with the use of a complicated model of streptozotocin-induced diabetic mice with uninephrectomy, compared to WT-STZ mice, STZ- TLR4-KO mice, demonstrated significantly less albuminuria, macrophages, NF-κB activation, and MCP-1 expression. In addition they showed in human renal biopsies increased cytosolic HMGB1. Thus, in their study using TLR-4 knockout mice with uninephrectomy, they showed that deletion of TLR-4 conferred renal protection against diabetic nephropathy (Lin et al., 2012). It is important to point out that in this study no significant effect was observed with STZ-WT mice without the added insult of uninephrectomy. Kuwabara et al. (2012) induced diabetes in WT and TLR4KO mice using STZ for 2 weeks and then changed to a high-fat diet for an
618
I. Jialal, R. Pahwa / Journal of Diabetes and Its Complications 29 (2015) 617–620
Table 1 Role of TLRs in diabetic nephropathy. Study
Type
(Devaraj, Tobias, et al., 2011) In vivo TLR2KO mice
(Lin et al., 2012)
(Kuwabara et al., 2012)
(Cha et al., 2013)
(Lin et al., 2013)
(Ma, Chadban, et al., 2014)
(Jialal et al., 2014)
(Ma, Wu, et al., 2014)
Background
Findings
STZ and STZ TLR2-/- mice for 14 weeks
Decreased albuminuria, TLR2 expression, MyD88 and cytokines and decreased kidney macrophage phenotype M1 and inflammation and restoration of nephrin, podocin, and podocyte number in STZ TLR2-/- mice compared to STZ-WT mice and decrease in Laminin and TGF-beta. 9 controls and 5 DN or uninephrectomized Renal cortical TLR4, not TLR2 elevated in DN and correlated Human kidney biopsies TLR4 def or wild-type STZ-induced diabetic with CD68 staining. Uninephrectomized TLR4-deficient and uninephrectomized mice for 12 weeks mice had significantly less albuminuria, renal dysfunction, TLR4mutant mice renal cortical NF-κB activation, tubular CCL-2 we use MCP-1 OR ccl2 not both expression, MCP-1 is fine and interstitial macrophage infiltration than wild-type animals Albuminuria was increased, and podocin was decreased in STZ-HFD In vivo TLR4KO mice WT and TLR4KO mice with STZ for compared to STZ–HFD-TLR4KO, along with decreased macrophages, 2 weeks ND followed by HFD for decreased mesangial expansion decreased MCP-1, PAI-1, CTGF and 6 weeks (8 weeks diabetes) S100a8 and S100a9 in STZ-HFD-TLR4KO. In vivo db/db mice and In Db/db mice with a DN without TLR4 GIT27 treatment decreased urinary albumin excretion, decreased vitro in cultured podocytes inhibition via GIT27 for 12 weeks proinflammatory cytokine synthesis, improved tissue lipid metabolism, decreased oxidative stress, and improved glomerulosclerosis compared with the control db/db group. In vitro, in podocytes, GIT27 decreased TLR4 and cytokines CRX-526 significantly reduced albuminuria, glomerular In vivo-eNOS-KO mice and in CRX-526 treatment for 8 weeks (TLR4 hypertrophy, glomerulosclerosis, and tubulointerstitial injury as vitro in proximal tubule cells inhibition) of eNOS KO mice with well as decreased CCL-2, CCL-5, osteopontin, decreased 12 weeks of STZ-induced diabetes macrophage infiltration, NFkB, TGF-beta and collagen deposition. In vitro, CRX-526 inhibited high glucose-induced osteopontin upregulation and NF-κB nuclear translocation in cultured human proximal tubular epithelial cells. WT-STZ mice vs. TLR4-/- STZ mice In STZ mice, TLR4-/- exhibited less albuminuria, inflammation, In vivo-TLR4KO mice and in followed for 12–24 weeks decreased glomerular hypertrophy and podocyte and tubular vitro in podocytes and injury as compared to diabetic wild-type controls. Marked tubular epithelial cells reductions in collagen, α-SMA, TGF-β and fibronectin in TLR-/STZ mice. In vitro, high glucose resulted in TLR4 activation in podocytes and tubular epithelial cells, NF-κB activation and consequent inflammatory and fibrogenic responses. In vivo-TLR4-KO mice STZ and TLR 4 STZ-TLR4 KO mice had significantly decreased macrophage and 17 weeks TLR4 immunostaining in kidney, MyD88, Interferon Regulatory Factor-3, NFKappaB activity, TNF-Alpha, IL-6, and MCP-1; significant decreases in fibrosis markers (collagen 4, and transforming growth factor-beta), and increased Podocyte numbers and podocin in STZ- TLR4KO mice vs. STZ-WT. In-vivo TLR2-KO mice and in STZ and kidney injury assessed at 6, Increased TLR2 gene expression, and its endogenous ligands and vitro in podocytes and TECs 12 and 24 weeks downstream cytokines in WT diabetic kidneys. TLR2KO mice protected against development of DN, with less albuminuria, inflammation, glomerular hypertrophy and hypercellularity, podocyte and tubular injury. Reduced interstitial collagen deposition, myofibroblast activation (α-SMA) and expression of fibrogenic genes (TGF-β and fibronectin) in TLR2KO. In vitro high glucose promoted TLR2 activation in podocytes and tubular epithelial cells (TECs) with resulting NF-κB activation, inflammation and TGF-β production.
additional 6 weeks. STZ and HFD treatment markedly increased albuminuria, increased podocyte injury (decreased podocin), mesangial expansion and macrophage infiltration and upregulated inflammation and fibrosis (MCP-1, fibronectin, connective tissue growth factor) in the glomeruli, even for this short duration of 8 weeks of diabetes, and all these effects were significantly reduced in the STZ-HFD -TLR4KO mice. Also they showed upregulation in the kidney of S100A8, a potent agonist for TLR4. This paper is unique in that it used HFD + STZ but only went for 8 weeks and hence did not see many differences when comparing diabetic TLR4KO vs. WT without HFD (Kuwabara et al., 2012). Studies using inhibitors against TLR4 have also shown a beneficial effect of TLR4 inhibition on diabetic nephropathy. Cha et al. (2013) demonstrated that GIT27, a compound thought to interfere with TLR4, TLR2 and TLR6 signalling pathways, attenuated renal inflammation and decreased glomerulosclerosis in db/db diabetic mice, which in turn attenuated the progression of diabetic nephropathy following
12 weeks of diabetes. In addition to a reduction in albuminuria and a decrease in renal macrophages, collagen 4, NFkB, MCP-1 and IL-6 they showed decreases in urinary excretion of nephrin, TNF and TGF-beta with GIT27 therapy. They also demonstrated that GIT27 treatment directly inactivated TLR4 expression in cultured podocytes, thereby resulting in a suppression of inflammatory cytokine secretion. Interestingly, GIT27 treatment was also associated with decreased oxidative stress in the kidney that paralleled the decrease in TLR4. Their study again points strongly towards TLR4 in contributing to the progression on diabetic nephropathy but does not convincingly rule out a role of TLR2, since this inhibitor blocks both TLR2 and 4 (Cha et al., 2013). Lin et al. (2013) using TLR4 antagonist CRX-526, a lipid A mimetic, that blocks TLR4/MD2 assembly showed that the compound could attenuate the progression of advanced diabetic nephropathy using the model of the eNOS (endothelial nitric oxide synthase) KO mice following 12 weeks of diabetes. CRX-526 reduced albuminuria and
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blood urea nitrogen without altering blood glucose and systolic blood pressure in diabetic mice. TLR-4 inhibition also attenuated glomerular hypertrophy, glomerulosclerosis and tubulo-interstitial injury alongside inhibition of TGF-β overexpression and NF-κB activation, CCL2, Osteopontin, macrophage infiltration and collagen deposition (Lin et al., 2013). In a recent comprehensive study, Ma et al. (2014) report elevated expression of TLR4, its endogenous agonists (HMGB1, HSP70, biglycan) and downstream cytokines, chemokines and fibrogenic genes in diabetic nephropathy in WT mice with streptozotocin (STZ) diabetes followed up for up to 24 weeks. In STZ-TLR4KO mice there was an amelioration of DN, manifesting as less albuminuria, inflammation (decreased IL-6, TNF, MCP-1, CXCL10), glomerular hypertrophy and hypercellularity, podocyte and tubular injury as compared to diabetic wild-type controls. There were significant decreases in measures of fibrosis: decreased interstitial collagen deposition, myofibroblast activation (α-SMA) and expression of fibrogenic genes (TGF-β and fibronectin) in STZ-TLR4KO mice. In vitro, high glucose directly promoted TLR4 activation in podocytes and tubular epithelial cells, resulting in NF-κB activation and consequent inflammatory and fibrogenic responses. It also resulted in an increase in TLR4 endogenous agonists including, HMGB1, HSP70 and biglycan (Ma, Chadban, et al., 2014). Jialal, Major, & Devaraj (2014) showed that diabetic nephropathy is also mediated by TLR-4 using TLR-4 KO mice. They showed that STZ-TLR4KO mice had decreased fibrosis as measured by decreased TGF-β, alpha smooth muscle actin, laminin and collagen as well as decreased renal inflammation as measured by decreased NF-κB expression alongside decreases in downstream biomediators such as TNF-α, IL-6 and MCP-1 compared to wild-type STZ mice. Also diabetes-mediated decrease in podocytes in wild-type mice was restored in TLR4KO. A deficiency of this study was the failure to collect urine and report on albuminuria (Jialal et al., 2014). Recently the first study implicating TLR2 in the pathogenesis of DN (Devaraj, Tobias, et al., 2011) was confirmed by (Ma et al., 2014) using the similar STZ-TLR2KO mice model. They showed that the STZ-TLR2KO mice exhibited lesser proteinuria, inflammation, glomerular hypertrophy, podocyte injury, collagen deposition, myofibroblast activation, TGF-beta and fibronectin. In addition they showed that hyperglycemia stimulated TLR2 activation in both podocytes and TECs. Once again they reported an increase in HMGB1, HSP70 and biglycan in the WT-diabetic mice (Ma, Wu, et al., 2014). Hence the studies reviewed above and summarized in Table 1 make a compelling argument that TLRs, 2 and 4 have a role in the pathogenesis of DN and translation to human studies with safe inhibitors can be a welcome therapeutic advance in forestalling this common complication which is the leading cause of ESRD. The role of TLRs in diabetic retinopathy has not been studied in great depth so far. Tang et al. (2013) showed a crucial role for the adaptor molecule, MyD88, in mediating diabetes-induced inflammation. Chimeric mice with MyD88 deletion in bone marrow-derived cells showed decreased diabetes-induced leukostasis, inhibition of diabetes-mediated ICAM expression and retinal superoxide generation. Additionally, this study also showed that chimeric mice with TLR-2/4 deletion showed inhibition of diabetes-induced leukostasis, ICAM expression and retinal superoxide generation. Interestingly none of the knockout mice showed an amelioration of diabetic-induced increased retinal permeability and paradoxically unlike the TLR2/TLR4KO mice leuckocytes, the MyD88-KO mice leuckocytes resulted in greater killing of endothelial cells. This study points towards the possibility of both TLR-2 and TLR-4 equally contributing to diabetic retinopathy. Furthermore they showed that an agonist of TLR2 (Pam3Cys) but not TLR4 (endotoxin) or other TLR agonists induced both IL-8 and IL-6 secretion in diabetic retinal supernatants. Whilst they focused on the effect of leukocyte perturbations on retinal pathology their data support a role
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especially for TLR2 in DR (Tang et al., 2013). Recently, our group showed that hyperglycemia upregulated TLR2 and TLR4 expression and activity including numerous biomediators of inflammation in retinal microvascular endothelial cells including leukocyte adhesion which was inhibited by various strategies including siRNA and small molecular weight inhibitors further underscoring a role of TLR-mediated inflammation in diabetic retinopathy (Rajamani & Jialal, 2014). This needs to be confirmed in-vivo using the genetically deficient TLR2 and TLR4 mice. The role of TLR-2 and TLR-4 in mediating diabetes-induced inflammation thereby resulting in macro/microvascular complications has been studied to some extent as evidenced by the studies discussed above. However, further studies are required to confirm and elucidate the mechanisms by which they participate in the macro and microvascular complications of diabetes, a pro-inflammatory state as we discussed previously including hyperglycemia, increase oxidative stress, PAMPS, DAMPS etc. (Jialal & Kaur, 2012).
References Cha, J. J., Hyun, Y. Y., Lee, M. H., Kim, J. E., Nam, D. H., Song, H. K., et al. (2013). Renal protective effects of toll-like receptor 4 signaling blockade in type 2 diabetic mice. Endocrinology, 154, 2144–2155. Cheung, N., Mitchell, P., & Wong, T. Y. (2010). Diabetic retinopathy. Lancet, 376, 124–136. Devaraj, S., Jialal, I., Yun, J. M., & Bremer, A. (2011). Demonstration of increased toll-like receptor 2 and toll-like receptor 4 expression in monocytes of type 1 diabetes mellitus patients with microvascular complications. Metabolism, 60, 256–259. Devaraj, S., Tobias, P., Kasinath, B. S., Ramsamooj, R., Afify, A., & Jialal, I. (2011). Knockout of toll-like receptor-2 attenuates both the proinflammatory state of diabetes and incipient diabetic nephropathy. Arterioscler Thromb Vasc Biol, 31, 1796–1804. Dong, B., Qi, D., Yang, L., Huang, Y., Xiao, X., Tai, N., et al. (2012). TLR4 regulates cardiac lipid accumulation and diabetic heart disease in the nonobese diabetic mouse model of type 1 diabetes. Am J Physiol Heart Circ Physiol, 303, H732–H742. Jialal, I., & Kaur, H. (2012). The role of toll-like receptors in diabetes induced inflammation: Implications for vascular complications. Curr Diab Rep, 12, 172–179. Jialal, I., Major, A. M., & Devaraj, S. (2014). Global toll-like receptor 4 knockout results in decreased renal inflammation, fibrosis and podocytopathy. J Diabetes Complications, 28, 755–761. Kaur, H., Chien, A., & Jialal, I. (2012). Hyperglycemia induces toll like receptor 4 expression and activity in mouse mesangial cells: Relevance to diabetic nephropathy. Am J Physiol Renal Physiol, 303, F1145–F1150. Kuwabara, T., Mori, K., Mukoyama, M., Kasahara, M., Yokoi, H., Saito, Y., et al. (2012). Exacerbation of diabetic nephropathy by hyperlipidaemia is mediated by toll-like receptor 4 in mice. Diabetologia, 55, 2256–2266. Li, F., Yang, N., Zhang, L., Tan, H., Huang, B., Liang, Y., et al. (2010). Increased expression of toll-like receptor2 in rat diabetic nephropathy. Am J Nephrol, 32, 179–186. Lin, M., & Tang, S. C. W. (2014). Toll -like receptors sensing and reacting to diabetic injury in the kidney. Nephrol Dial Transplant, 29, 746–754. Lin, M., Yiu, W. H., Li, R. X., Wu, H. J., Wong, D. W., Chan, L. Y., et al. (2013). The TLR4 antagonist CRX-526 protects against advanced diabetic nephropathy. Kidney Int, 83, 887–900. Lin, M., Yiu, W. H., Wu, H. J., Chan, L. Y., Leung, J. C., Au, W. S., et al. (2012). Toll-like receptor 4 promotes tubular inflammation in diabetic nephropathy. J Am Soc Nephrol, 23, 86–102. Lu, Z., Zhang, X., Li, Y., Jin, J., & Huang, Y. (2013). TLR4 antagonist reduces early-stage atherosclerosis in diabetic apolipoprotein E-deficient mice. J Endocrinol, 216, 61–71. Ma, J., Chadban, S. J., Zhao, C. Y., Chen, X., Kwan, T., Panchapakesan, U., et al. (2014). TLR4 activation promotes podocyte injury and interstitial fibrosis in diabetic nephropathy. PLoS One, 9, e97985. Ma, J., Wu, H., Zhao, C. Y., Panchapakesan, U., Pollock, C., & Chadban, S. J. (2014). Requirement for TLR2 in the development of albuminuria, inflammation and fibrosis in experimental diabetic nephropathy. Int J Clin Exp Pathol, 7, 481–495. Mudaliar, H., Pollock, C., Komala, M. G., Chadban, S., Wu, H., & Panchapakesan, U. (2013). The role of toll-like receptor proteins (TLR) 2 and 4 in mediating inflammation in proximal tubules. Am J Physiol Renal Physiol, 305, F143–F154. Mudaliar, H., Pollock, C., & Panchapakesan, U. (2014). Role of toll-like receptors in diabetic nephropathy. Clin Sci, 126, 685–694. Rajamani, U., & Jialal, I. (2014). Hyperglycemia induces toll-like receptor-2 and -4 expression and activity in human microvascular retinal endothelial cells: Implications for diabetic retinopathy. J Diabetes Res, 2014, 790902http://dx.doi. org/10.1155/2014/790902. Sawa, Y., Takata, S., Hatakeyama, Y., Ishikawa, H., & Tsuruga, E. (2014). Expression of toll-like receptor 2 in glomerular endothelial cells and promotion of diabetic nephropathy by Porphyromonas gingivalis lipopolysaccharide. PLoS One, 9, e97165. Tang, J., Allen Lee, C., Du, Y., Sun, Y., Pearlman, E., Sheibani, N., et al. (2013). MyD88-dependent pathways in leukocytes affect the retina in diabetes. PLoS One, 8, e68871.
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Tang, J., & Kern, T. S. (2011). Inflammation in diabetic retinopathy. Prog Retin Eye Res, 30, 343–358. Wada, J., & Makino, H. (2013). Inflammation and the pathogenesis of diabetic nephropathy. Clin Sci, 124, 139–152.
Research, University of California Davis Medical Center, 4635 2nd Ave, Research I Bldg, Room 3000, Sacramento, CA 95817-2218 Tel.: + 1 916 734 6590; fax: + 1 916 734 6593 E-mail address: [email protected]
Ishwarlal Jialal Laboratory of Atherosclerosis and Metabolic Research Department of Pathology, University of California Davis Medical Center, Sacramento, CA Veterans Affairs Medical Center, Mather, CA Corresponding author. Laboratory of Atherosclerosis and Metabolic
Roma Pahwa Laboratory of Atherosclerosis and Metabolic Research Department of Pathology, University of California Davis Medical Center, Sacramento, CA Veterans Affairs Medical Center, Mather, CA
