How and where does GLP-1 exert its actions that impact the immunes system and inflammation. The answer remains controversial, and existing published findings in part may reflect suboptimal reagents and challenging experimental techniques.

                                                                                      Lymphocytes

Hadjiyanni and colleagues detected widespread expression of mRNA transcripts encoding a full length GLP-1R in immune cells from bone marow, spleen, thymus and peripheral lymph nodes. Immune GLP-1R expression appeared to be relatively higher in cells from NOD mice compared to cells contained from C57BL/6 mice. GLP-1R activation weakly increased cyclic AMP formation in isolated immune populations but failed to affect lymphocyte survival, proliferation, or migration. Although no major changes in cell numbers or immune cell function were observed in Glp1r-/- lymphocytes, Glp1r-/- thymocytes exhibited a hypoproliferative response, whilst peripheral Glp1r-/- lymphocytes were hyperproliferative in response to mitogenic stimulation. As these studies were carried out in "normal mice", and in non-diabetic NOD mice, further analysis of the role of the GLP-1R in the context of immune dysfunction, such as T1DM, or other inflammatory or immunological disorders appears warranted. Glucagon-like peptide-1 receptor (GLP-1R) signaling selectively regulates murine lymphocyte proliferation and maintenance of peripheral regulatory T-cells Diabetologia 2010 Apr;53(4):730-40.

Bernardo Yusta and colleagues analyzed Glp1r expression within the gut, with a focus on the gut immune system. Surprisingly, Yusta observed that intestinal intraepithelial lymphocyte (IEL)-enriched fractions, and to a greater extent, IELs purified by FACS analysis, expressed abundant levels of the full length mouse Glp1r. IELs expressed a functional GLP-1R. In the basal non-stimulated state, and following activation by CD3/CD28, exendin-4 producing dose-dependent cAMP accumulation in IELs to robust levels mirroring those achieved with forskolin. Although intestinal IEL subsets were not different in mice treated with GLP-1R agonists, or in Glp1r-/- mice, basal intestinal gene expression was profoundly abnormal in the Glp1r-/- gut mucosa, reflecting a shift to a pro-inflammatory gene expression profile. Furthermore, Glp1r-/- mice exhibit enhanced intestinal colonic damage following exposure to low dose detran sulfate. GLP-1R agonists directly suppressed the production of infammatory cytokines from GLP-1R+ IELs, but had no effect on splenocytes or IELs from Glp1r-/- mice. The basal pro-inflammatory gene expression signature in Glp1r-/- mice could be almost completely reversed following bone marrow transplantation of GLP-1R+/+ door marrow into Glp1r-/- mice, reflecting selective repopulation of Glp1r-/- mice with GLP-1+/+ IELs. Furthermore, exendin-4 markedly suppressed the expression of pro-inflammatory cytokines (mRNA and protein) in IELs from Glp1r+/+ mice but not from splenocytes or IELs from Glp1r-/- mice. Intriguingly, although gut barrier function was normal in the small bowel, Glp1r-/- mice exhibited significant basal perturbations in the microbiome, notably increased levels of Bacteroides and Actinobacteria and decreased abundance of Proteobacteria and Firmicutes. Hence, the IEL GLP-1R controls mucosal inflammation in the small and large bowel. GLP-1 receptor (GLP-1R) agonists modulate enteric immune responses through the intestinal intraepithelial lymphocyte (IEL) GLP-1R Diabetes 2015 March 3, 2015,doi:10.2337/db14-1577

Marx and colleagues examined the actions of GLP-1(1-37), a non-classical weak agonist at the GLP-1 receptor, and exendin-4, a potent GLP-1 receptor agonist, on human CD4+ lymphocyte migration ex vivo. GLP-1(1-37) reduced SDF-1-stimulated lymphocyte migration in a dose-dependent manner, effects not seen with a scrambled GLP-1(1-37) peptide. The modest induction of PI3-kinase activity induced by SDF-1, and the phosphorylation of cofilin and myosin light chain(MLC) was also attenuate by the classical GLP-1 receptor agonist GLP-1(7-37). Similarly, GLP-1(1-37) attenuated the extent of SDF-1-induced F-actin formation in CD4+ lymphocytes. An immunoreactive GLP-1R protein was detected using GLP-1R antisera (Upstate) in human CD4+ lymphocytes and endothelial cells and a functional role for the GLP-1R was invoked by demonstrating that exendin-4 also modestly reduced SDF-1-enhanced lymphocyte migration. Knock down of GLP-1R expresion abrogated the effect of GLP-1(1-37) to reduce ICAM-3 expression, an indirect readout for lymphocyte cell migration. The effects ofexendin-4 were not reported in the same GLP-1R knockdown experiments. See Glucagon-like peptide-1(1-37) inhibits chemokine-induced migration of human CD4-positive lymphocytes Cell Mol Life Sci. 2010 Oct;67(20):3549-55

Macrophages

Several studies have demonstrated that GLP-1R agonists reduce ER stress and decrease inflammation-associated gene and protein expression in macrophages. Liang and colleages showed that exendin-4reduced ER stress and apoptosis in cholesterol-loaded insulin resistant macrophages from Insr-/- or ob/ob-/- mice. Exendin-4activated macrophage Akt and Erk phosphorylation in vitro, mechanisms sensitive to the PKA inhibitor H89. Notably,exendin-4treatment of ob/ob:Ldlr-/- mice significantly reduced macrophage apoptosis, ATF3 and caspase-3 positivity in aortic lesion macrophages. Impaired MEK Signaling and SERCA Expression Promote ER Stress and Apoptosis in Insulin-Resistant Macrophages and Are Reversed by Exenatide Treatment. Diabetes. 2012 Jun 29

Liu and colleagues examined the anti-inflammatory and antimicobial effects of exendin-4 in db/db mice with experimental Listeria monocytogenes infection. Exendin-4treatment improved glucose and lipid levels, reduced lipid content of peritoneal exudate cells (including macrophages) froim infected db/db mice, increased phagocytic activity, and enhanced ex vivo clearance of Listeria, through mechanisms that remain unclear. Exendin-4 improves resistance to Listeria monocytogenes infection in diabetic db/db mice J Vet Sci. 2012 Sep;13(3):245-52

Human studies

Chaudhuri assessed parameters of inflammation in human diabetic subjects treated with a single 5 ug dose of exenatide, followed by exenatide 10 ug twice daily or placebo for 12 weeks. Exenatide therapy was associated with reduction in fasting glucose and A1c (8.6 to 7.4%), and increased levels of plasma insulin, and decreased plasma FFAs. ROS generation and NfkB DNA binding activity, and TNFa, IL-1b, JNK-1, TLR-2 and SOCS-3 expression in mononuclear cells was suppressed after acute or chronic exenatide. Plasma levels of MCP-1, SAA, MMP-9 and IL-6 were also reduced after chronic exenatide. Whether these actions are due to GLP-1 receptor activation, or the acute and chronic glucoregulatory or metabolic actions of exenatide, remain uncertain. Exenatide exerts a potent antiinflammatory effect. J Clin Endocrinol Metab. 2012 Jan;97(1):198-207

Hogan et al studied a series of molecular markers of inflammation in 10 obese subjects with type 2 diabetes (mean BW 128 kg, A1c 8.7%) started on liraglutide for 8 weeks (last 6 weeks at 1.2 mg daily). Liraglutide reduced A1c to 7.9%, reduced body weight by 3 kg, decreased levels of sCD163 (independent of changes in A1c or weight), and decreased peripheral blood mononuclear cell production of TNFa, IL-1b and IL-6, whereas adiponectin was significantly increased and leptin levels trneded lower. Liraglutide directly increased IL-10 and decreased IL-1b in THP-1-derived macrophages in vitro. See Glucagon-like peptide 1 analogue therapy directly modulates innate immune-mediated inflammation in individuals with type 2 diabetes mellitus Diabetologia. 2014 Apr;57(4):781-4

Psoriasis

Hogan and colleagues observed improvements in clinical disease activity in 3 patients with psoriasis who were treated with GLP-1R agonosts. The patients had only modest changes in blood glucose but did experience weight loss. These authors found that GLP-1R activation in iNKT cell lines reduced cytokine secretion, providing a putative mechanistic link for how anti-inflammatory effects of GLP-1 might improve disease activity. See Glucagon-like peptide-1 (GLP-1) and the regulation of human invariant natural killer T cells: lessons from obesity, diabetes and psoriasis Diabetologia. 2011 Jul 9.

A subsequent report described a single patient with a long standing history of psoriasis and T2DM; within days of starting liraglutide, the patient experienced reduced itching and clearling of the skin, before any significant weight loss had been reported. By week 5, a 2 kg weight loss and 2.5% reduction in HbA1c was observed, with further improvements continuing over the next several months. No immunological investigations were carried out in this patient. Improvement in psoriasis after treatment with the glucagon-like peptide-1 receptor agonist liraglutide Acta Diabetol. 2011 Dec 13. A summary of potential mechanisms that might contribute to the improvement in psoriasis in such patients is provided in Glucagon-like peptide-1 (GLP-1) receptor agonists, obesity and psoriasis: diabetes meets dermatology Diabetologia. 2011 Nov;54(11):2741-4.

Preclinical studies

GLP-1 has been used in short term studies to treat subjects with type 1 diabetes, and preclinical studies have examined the efficacy of GLP-1R agonists in models of T1DM such as the NOD mouse. Intriguingly, the results of some of these studies suggest that GLP-1 therapy may have immunomodulatory actions. Initiation of exendin-4 treatment alone after the development of diabetes had little therapeutic benefit in NOD mice. In contrast, exendin-4 together with lysophiline for 28 days, markedly improved glucose control in NOD mice, even 6-14 weeks after cessation of therapy. Furthermore, the combination therapy preserved the number of intact islets, and appeared to reduce the extent of inflammatory cell infiltration in the remaining islets. See Combined treatment with lisofylline and exendin-4 reverses autoimmune diabetes. Biochem Biophys Res Commun. 2006 Jun 9;344(3):1017-22. Similarly, continuous administration of native GLP-1 to 8 week old female NOD mice for 4 or 8 weeks lowered blood glucose, increased formation of new b-cells, suppressed b-cell apoptosis and delayed the onset of diabetes as described in  Continuous stimulation of human glucagon-like peptide-1 (7-36) amide in a mouse model (NOD) delays onset of autoimmune type 1 diabetes. Diabetologia. 2007 Jul 14; [Epub ahead of print]. In contrast, twice daily administration of exendin-4 at two different doses, 100 ng, and 2 ug twice daily, prior to the development of clinical diabetes in NOD mice, produced only modest effects on the numbers of disease-free mice, with detectable but small increased in beta cell mass and reductions in insulitis. Intriguingly, GLP-1 receptor expression was also detected in different immune cell compartments as outlined in Exendin-4 modulates diabetes onset in non obese diabetic mice Endocrinology. 2008 Mar;149(3):1338-49. Kodera and colleagues observed renoprotective effects and reduced inflammation in rats with STZ-induced diabetes treated with exendin-4, 10 ug/kg/day, for 8 weeks. Ex-4 improved blood glucose and renal histology, decreased glomerulat typ IV collagen and urinary albumin excretion, and reduced the expression of pro-inflammatory genes (Cd14, Icam1, Tgfb1) in the renal cortex. Levels of glomerular and urinary 8-OHdG (a marker of oxidative stress), Nox4 gene expression, and NF-kB activity were also reduced in kidneys from Ex-4-treated animals. Expression of the Glp1r was detected in glomerular endothelial cells by IHC, in human THP-1 cells and human monocytes by RT-PCR. High glucose increased cytokine levels in THP-1 cells that were reduced by Ex-4 in an exendin(9-39)-dependent manner. Ex-4 also reduced ICAM1 expression in human glomerular endothelial cells. Glucagon-like peptide-1 receptor agonist ameliorates renal injury through its anti-inflammatory action without lowering blood glucose level in a rat model of type 1 diabetes Diabetologia. 2011 Apr;54(4):965-78.

Sepsis, LPS, IL-6, and GLP-1 secretion

Administration of the cytokine, IL-6, rapidly increases plasma GLP-1 levels in normal and diabetic rodents. Similarly, exercise was associated with increased levels of IL-6 and increased GLP-1 levels detectable within 90 minutes. Remarkably, IL-6 induces GLP-1 synthesis not only in L cells, and in GLUTag cells but also increases proglucagon gene expression, PC1 expression, and GLP-1 production directly from rodent and human islet alpha cells. These changes were mirored by increases in plasma GLP-1 levels and improved glucose tolerance in the setting of IL-6 administration or exercise in mice. Immunoneutralization of IL-6 reduced levels of GLP-1 and deteriorated glucose tolerance. Conversely, alpha cells from IL-6 knockout mice were unable to augment GLP-1 synthesis and secretion in response to high fat feeding. Il-6 failed to improve glucose control in Glp1r-/- mice.   Interleukin-6 enhances insulin secretion by increasing L cell and a cell glucagon-like peptide-1 secretion Nature Medicine 2011 30 October 2011 | doi:10.1038/nm.2513

Nguyen and colleagues demonstrated that injection of LPS enhanced GSIS and improved glycemia in part through stimulation of GLP-1 secretion in mice. The beneficial actions of LPS on insulin secretion and glucose tolerance were blunted by exendin(9-39) and abrogated in Glp1r-/- mice. Lipopolysaccharides-mediated increase in glucose-stimulated insulin secretion: Involvement of the glucagon-like peptide 1 (GLP1) pathway Diabetes. 2014 Feb;63(2):471-82

Kahles examined the factors controlling GLP-1 and insulin secretion under conditions mimicking experimental sepsis in mice, cells and in human subjects. LPS rapidly induced plasma levels of GLP-1 in WT mice, in a dose-dependent manner. The rise in plasma GLP-1 levels was preceded by increased plasma levels of IL-6, and associated with increased insulin and reduced glucose concentrations. Administration of IL-1b or IL-6, but not TNF-a alone also increased circulating levels of GLP-1 and insulin. LPS increased plasma GLP-1 levels in IL-1 receptor knockout mice, but not in IL-6 KO mice. The GLP-1R antagonist Ex-9 blocked the glucoregulatory and insulin stimulatory effects of LPS in vivo. Ex-9 administration also increased plasma glucagon levels in LPS-treated mice, consistent with a role for GLP-1 in controlling glucagon under conditions of endotoxemia. Plasma levels of total GLP-1 were markedly (6.9 x higher) elevated in human subjects with critical illness and inflammation admitted to the ICU, and GLP-1 levels correlated positively with levels of CRP and IL-6 but not with glucose. IL-6, but not other cytokines, directly increased GLP-1 secretion from GLUTag cells GLP-1 Secretion Is Increased by Inflammatory Stimuli in an IL-6-Dependent Manner, Leading to Hyperinsulinemia and Blood Glucose Lowering Diabetes. 2014 Jun 19. pii: DB_140100.

Yanay et al administered a single dose of LPS, with or without exendin-4, to rats and assessed circulating blood cells and cytokines, in 2 month old healthy Wistar rats. Although exendin-4 alone had no effect on neutorphil count 3 hrs after LPS, exendin-4 significantly attenuated and almost completey prevented the neutropenia caused by LPS alone. Furthermore, exendin-4 treatment significantly attenuated levels of multiple plasma cytokines assessed out to 10 hrs after LPS administration. Effects of exendin-4, a glucagon like peptide-1 receptor agonist, on neutrophil count and inflammatory cytokines in a rat model of endotoxemia J Inflamm Res. 2015 Jul 24;8:129-35

                                                                                               Miscellaneous Cell types

Iwai et al demonstrated GLP-1 binding to rat brain glial cells and detected GLP-1 receptor expression in cultured astrocytes, microglia, and neurons by RT-PCR. GLP-1 suppressed the induction of IL-1b, IL-6 and iNOS RNA by LPS and decreased IL-1b secretion into culture medium. The effects of GLP-1 to suppress IL-1b expression were blocked by the adenylate cyclase inhibitor SQ22536 Glucagon-like peptide-1 inhibits LPS-induced IL-1beta production in cultured rat astrocytes Neurosci Res. 2006 Aug;55(4):352-60

                                                                                                   Adipocytes

The GLP-1R agonist exendin-4 also exerted anti-inflammatory actions in 3T3-L1 adipocytes. Incubation of differentiated adipocytes withexendin-4 for 8 hrs increased adiponectin expression and produced a modest increase in adiponectin secretion, effects blocked by exendin(9-39). Exendin-4 reduced the levels of intracellular cAMP and both forskolin and IBMX, and paradoxically, the PKA inhibitor H-89 all blocked the induction of adiponectin expression by exendin-4. Exendin-4 also reduced the expression of IL-6 and MCP1 Exendin-4, a GLP-1 receptor agonist, directly induces adiponectin expression through protein kinase A pathway and prevents inflammatory adipokine expression Biochem Biophys Res Commun. 2009 Dec 18;390(3):613-8. Lee and colleagues also demonstrated thatexendin-4 reduced LPS-stimulated inflammation in 3T3-L1 cells. ob/ob mice treated with rAd-GLP-1 exhibited smaller and more numerous adipocytes, reduced adipose accumulation of macrophages (dcereased M1-ATMs), and reduced cytokine expression in peritoneal macrophages when compared to pair fed controls. Isolated CD11b+ ATMs from epidydimal fat of obese mice cultured for 24 h then exposed to exendin-4 for 1h reduced LPS-stimulated expression of IL6, Tnfa, and Mcp1 and NFkb nuclear translocation. The authors infer a direct anti-inflammatory effect of GLP-1 and exendin-4 independent of weight loss in mouse adipocytes and macrophages Glucagon-like peptide-1 inhibits adipose tissue macrophage infiltration and inflammation in an obese mouse model of diabetes Diabetologia. 2012 Jun 22.

Anti-inflammatory actions of exendin-4 were demonstrated in human islet cultures ex vivo. Treatment of islets with interferon-g induced expression of the chemokine CXCL10 and multiple other chemokines. Treatment of islets with exendin-4 and the PDE inhibitor rolipram suppressed the induction of CXCL9, CXCL10, and CXCL10; the effects of exendin-4 in the absence of rolipram were much more modest, wehereas forskolin and dbcAMP robustly inhibited chemokine expression. Exendin-4 and cyclic AMP also reduced the expression of STAT-1, a mediator of CXCL10 expression in human islets and the anti-inflammatory actions o fexendin-4 were blocked by the adenylate cyclase inhibitor SQ22536, but not by the PKA inhibitor H89, which paradoxically enhanded the supression of CXCL10 expression by cAMP. Anti-inflammatory action of exendin-4 in human islets is enhanced by phosphodiesterase inhibitors: potential therapeutic benefits in diabetic patients Diabetologia. 2010 Nov;53(11):2357-68

                                                                                  Vascular cell types

Dozier et al demonstrated cytoprotective and anti-inflammatory actions of GLP-1 on mesenteric endothelium. Although GLP-1 had no effect on basal microvascular permeability in non-inflamed vessels, LPS increased and GLP-1 decreased permeability in rat mesenteric post-capillary venules. The effects of GLP-1 to reduce inflammation-related permeability were reduced by co-treatment with exendin(9-39) or with the cAMP synthesis inhibitor ddA, whereas rolipram, an inhibitor of PDE4 and cAMP degradation, potentiated the actions of GLP-1 and the PKA inhibitor H-89 blocked the anti-permeability actions of GLP-1. See Glucagon-like peptide-1 protects mesenteric endothelium from injury during inflammation Peptides 2009 Sep;30(9):1735-41.  In contrast, using a different model of endothelial dysfunction, Nathanson and colleagues failed to find any effect of the degradation-resistant GLP-1R agonist exenatide on rat conduit arteries. Incubation of rat femoral artery rings with intralipid impaired acetylcholine-induced vasorelaxation however exenatide had no effect on intralipid-induced endothelial dysfunction. In contrast, significant vasorelaxation was observed following incubation of femoral rings with GLP-1, GLP-1(9-36), ACh and SNP; 23+/-4%, 38+/-4%, 79+/-3% and 97+/-4% relaxation, respectively. These observations suggest that the effect of GLP-1 on endothelial relaxation may be independent of the classical GLP-1R Endothelial dysfunction induced by triglycerides is not restored by exenatide in rat conduit arteries ex vivo Regul Pept. 2009 Oct 9;157(1-3):8-13

Hattori et al examined the ant-inflammatory actions of liraglutide in HUVEC (Human Umbilical Vein Endothelial Cells) cultures. Liraglutide increased NO production in a dose- and time-dependent manner, actions eliminated by the AMPK inhibitor compund C. Consistent with the putative importance of AMPK, liraglutide promoted AMPK phosphorylation and increased eNOS expression and decreased NF-kB activation in HUVECs and SVEC4 cells treated with TNFa. Liraglutide also decreased the expression of cytokines induced by TNFa. A glucagon-like peptide-1 (GLP-1) analogue, liraglutide, upregulates nitric oxide production and exerts anti-inflammatory action in endothelial cells A glucagon-like peptide-1 (GLP-1) analogue, liraglutide, upregulates nitric oxide production and exerts anti-inflammatory action in endothelial cells Diabetologia. 2010 Oct;53(10):2256-63